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Microbiology and Molecular Biology Reviews, June 2000, p. 239-280, Vol. 64, No. 2
1092-2172/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Translational Control of Viral Gene Expression in Eukaryotes

Michael Gale Jr.,1,* Seng-Lai Tan,2 and Michael G. Katze2

University of Texas Southwestern Medical Center, Dallas, Texas,1 and University of Washington, Seattle, Washington2

SUMMARY
INTRODUCTION
    Overview of Eukaryotic mRNA Translation and Sites of Viral Regulation
        Viral translational programming.
        Translation initiation.
        Cap-binding reaction.
        Ribosome scanning and AUG site selection.
        Elongation.
        Termination.
        Improving translation efficiency: the closed-loop model of mRNA translation.
TRANSLATIONAL CONTROL OF VIRAL GENE EXPRESSION
        Advantages and liabilities of cap-dependent host translation.
    Host Shutoff and Selective Translation of Viral mRNA
    Mechanisms and Control of Viral mRNA Translation
        Internal ribosome entry.
        Ribosome shunt.
        Leaky scanning.
        Frameshifting.
        Control of termination and reinitiation.
        Functional recoding.
    Coupling the Virus Life Cycle to Translational Control
        The herpesviruses.
        HSV and the shutoff of host cell protein synthesis.
        Selective repression of mRNA translation initiation during HSV infection.
        Selective translation of HSV mRNA.
        Inhibition of eIF2alpha phosphorylation during HSV infection.
        Implications of viral modulation of translation in HSV pathogenesis and disease treatment.
    Recruitment of Host Factors for the Efficient Translation of Viral mRNA
        IRES binding proteins and proteins that bind the viral 3' UTR.
        Influenza virus.
        Selective translation of influenza virus mRNAs.
        Contribution of influenza virus mRNA structure upon selective translation.
        Influenza virus recruitment of Grsf-1.
        Temporal regulation of influenza virus mRNA translation.
        Maintenance of translation in influenza virus-infected cells.
        Recruitment of P58IPK and inhibition of eIF2alpha phosphorylation during influenza virus infection.
        Role of the influenza virus NS1 protein in viral mRNA translation.
VIRAL MODIFICATION OF CELLULAR FACTORS
        Inactivation of eIF4E and modulation of the eIF4E-binding proteins.
        Cleavage of eIF4G.
        Cleavage of PABP: disruption of the closed-loop translation complex.
        Modification of EF-1.
        Disruption of eIF2alpha phosphorylation.
        (i) PKR structure and function.
        (ii) Mechanisms of PKR inhibition by eukaryotic viruses.
        Disruption of the IFN-induced cellular antiviral response through inhibition of PKR.
        (i) Viral inhibition of PKR: HCV.
VIRAL PERSISTENCE AND TRANSLATIONAL CONTROL
        Translational programming and maintenance of viral persistence.
        Translational control, persistent infection, and regulation of host apoptosis.
        Cell growth control, eIF2alpha phosphorylation, and oncogenic transformation.
MRNA TRANSLATION AS A TARGET FOR ANTIVIRAL THERAPY
CONCLUSIONS AND PERSPECTIVES
ACKNOWLEDGMENTS
REFERENCES


SUMMARY
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As obligate intracellular parasites, viruses rely exclusively on the translational machinery of the host cell for the synthesis of viral proteins. This relationship has imposed numerous challenges on both the infecting virus and the host cell. Importantly, viruses must compete with the endogenous transcripts of the host cell for the translation of viral mRNA. Eukaryotic viruses have thus evolved diverse mechanisms to ensure translational efficiency of viral mRNA above and beyond that of cellular mRNA. Mechanisms that facilitate the efficient and selective translation of viral mRNA may be inherent in the structure of the viral nucleic acid itself and can involve the recruitment and/or modification of specific host factors. These processes serve to redirect the translation apparatus to favor viral transcripts, and they often come at the expense of the host cell. Accordingly, eukaryotic cells have developed antiviral countermeasures to target the translational machinery and disrupt protein synthesis during the course of virus infection. Not to be outdone, many viruses have answered these countermeasures with their own mechanisms to disrupt cellular antiviral pathways, thereby ensuring the uncompromised translation of virion proteins. Here we review the varied and complex translational programs employed by eukaryotic viruses. We discuss how these translational strategies have been incorporated into the virus life cycle and examine how such programming contributes to the pathogenesis of the host cell.


INTRODUCTION
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Perhaps nowhere in nature is a parasitic relationship as well defined as that which occurs between a virus and its host cell. Viruses rely on the host cell for propagation, utilizing cellular machinery for the replication and assembly of viral components and the release of progeny virions. Whether possessing a DNA or RNA genome, the eukaryotic virus exhibits a general life cycle that is initiated through interaction with its cognate receptor(s) on the surface of the host cell (117) (Fig. 1). After virion adsorption and internalization, uncoating exposes the viral genome and associated proteins to the host milieu, whereupon genome replication and transcription take place. The translation of viral RNA is followed by the assembly of structural proteins, packaging of the viral genome, and eventual release of progeny virions. While some viruses encode or carry the enzymatic machinery required for autonomous genome replication and/or transcription, others recruit host polymerases to carryout this task (117). In contrast, viruses do not encode or carry the machinery for mRNA translation. Thus, the ensuing stage of viral protein synthesis is completely dependent on the translational machinery of the host cell (Fig. 1). Not surprisingly, viruses have devoted much attention to this dependency and have evolved strategies that reduce the impact of translational dependence on viral replication. As discussed in this review, these strategies are themselves limited by the nature of the viral RNA, the cellular translation machinery, and the translation regulatory pathways of the host cell.


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FIG. 1.   General model of eukaryotic viral replication. Viral particles are shown in black. Viruses recognize their target cell through interaction with specific receptors and/or other components on the cell membrane. Interaction with the host cell induces cell membrane penetration and virion internalization. Virion uncoating releases the viral genome, whereupon it is available for transcription and translation. Poxviruses and the RNA viruses (with the exception of retroviruses) replicate in the cytoplasm. Transcription of all other DNA viruses takes place in the nucleus. Transcription and genome replication are followed by the cytoplasmic stages of mRNA translation and virion assembly. Release of mature virions may include membrane lysis and death of the host cell. Adapted with modification from reference 322.

This treatise presents an overview of translation strategies used by viruses that infect the cells of higher eukaryotes. Where appropriate, we have focused on specific virus systems to present examples of the diverse mechanisms by which viruses overcome the problems of translational dependence. For complementary material on mRNA translation, virus-host interactions, host antiviral pathways, and the virus-host interactions of lower eukaryotes and bacteria, we direct the reader to several fine texts and reviews (1, 10, 117, 137, 179, 199, 303, 324, 353, 415, 416, 428, 455, 480, 484). We begin this review with a brief overview of the current models for eukaryotic mRNA translation, including points of translational control, and effects on host translation due to virus infection. This is followed by examples of translation strategies that are dependent on the structure of the viral mRNA and those that are directed at recruitment and modification of the translation machinery and other host factors. Given the recent emphasis in the translational control field on identifying and characterizing cellular signaling pathways that govern mRNA translation (43, 120, 433), we have included discussion of how viruses might exploit these pathways to facilitate completion of their translational programs. Attention is directed to the ways in which disruption of host translational control pathways may contribute to viral pathogenesis and disease progression. Finally, we conclude with a section describing the prospects of targeting viral mRNA translation for antiviral therapy, as well as perspectives for future research in the increasingly overlapping disciplines of virology, viral pathogenesis, and translation control.

Overview of Eukaryotic mRNA Translation and Sites of Viral Regulation

Translation in eukaryotes is a complex multistep, multiprotein process (198, 335). As with most complex biochemical reactions, it is subject to strict regulatory controls, and is extremely sensitive to both the intracellular and extracellular environments (43, 163, 232, 281, 324, 433). In general, the translation of a given mRNA can be modulated in response to nutrient availability, mitogenic stimulation and cell cycle regulation, stress, and, as described herein, viral infection (reviewed in detail in reference 199). It is also increasingly clear from research spanning the past several years that regulation of mRNA translation is critical for maintaining control of cell growth (96, 287, 429). As presented in the "Viral persistence and translational control" section (below), disruption of the major translation checkpoints and signaling cascades renders cells unable to respond to translation-modulatory signals and may constitute a mechanism of oncogenic transformation (67). The following sections provide a general overview of viral translational programming and eukaryotic mRNA translation. Major sites for virus regulation of translation are noted, and they are discussed in detail in this review.

Viral translational programming. Viruses face enormous pressures to maintain a "functional" genome size, which greatly influences the rate and efficiency of viral replication. Thus, host translational dependence may in part reflect the limitations placed on viral replication due to the enormous genome capacity that would be needed to encode the components for autonomous viral protein synthesis (335). This idea is supported by the highly specialized nature of the protein synthetic machinery, which encompasses well over 30 different gene products and yet remains highly conserved between the prokaryotic and eukaryotic kingdoms (335, 470). Eukaryotic viruses have evolved effective means of exploiting their innate translational dependence through mechanisms of translational programming. This is the process in which eukaryotic viruses (i) redirect the host translation machinery to favor viral protein synthesis and (ii) control the expression of their own gene products. The latter is especially important for the RNA viruses, which have limited transcriptional control and rely heavily on translational control strategies to modulate viral gene expression.

Translational programming, such as the use of regulatory upstream open reading frame(s) (uORFs), overlapping reading frames, multicistronic transcripts, and termination control, allows viruses to conserve the functional genome size by making efficient use of genome coding capacity. In general, the mechanisms of translational programming are intrinsic to the structure of the viral mRNA itself. As summarized in Table 1, structural elements within a viral mRNA that affect translational efficiency or impart translational control include the length and structural complexity of the 5' and 3' untranslated regions (UTR), the position and context of the initiator AUG codon, the stability and accessibility of the of the m7G cap and the cap-binding complex, and the presence of uORF(s) preceding the major cistron (137, 148, 149, 269, 270, 322, 329, 430, 431). In addition, cis-acting sequence elements that recruit or bind trans-acting factors can impart an additional level of translational control to viral mRNA by facilitating translational selectivity (23, 76, 187, 244, 361, 362, 394, 411). As described in the following sections, virus translational programming affects all levels of the translation process, including translation initiation, elongation, termination, and host translational control signaling pathways.

                              
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TABLE 1.   mRNA structural features that confer translational control.

Translation initiation. The majority of control over cellular mRNA translation occurs during initiation. Translation initiation is the process in which the mRNA assembles into a macromolecular complex with the components required for protein synthesis, including the eukaryotic initiation factors (eIF) and elongation factors (EF). Figure 2 shows the major steps in the cap-dependent translation initiation process and important sites of virus regulation (for comprehensive reviews of translation initiation, the reader is referred to references 163, 221, 335, and 359). Initiation begins with the binding of initiator methionyl-tRNA (Met-tRNAi) to the 40S ribosomal subunit. This step is facilitated through the formation of an eIF2-GTP-Met-tRNAi ternary complex (462). The recent discovery and functional analyses of eukaryotic homologues of prokaryotic initiation factor 2 (IF2) indicates that Met-tRNAi delivery also proceeds via a more general, universally conserved mechanism (60, 291). In this case, IF2 does not participate in the formation of a ternary complex but, rather, may bind directly to the ribosomal A site and facilitate binding of the Met-tRNAi to the ribosomal P site during translation initiation. IF2 activity is not subject to direct regulation and therefore may not contribute to the control of mRNA translation. In contrast, formation of the eIF2-dependent ternary complex and its delivery of Met-tRNAi to the 40S ribosomal subunit can constitute a rate-limiting step when the alpha subunit of eIF2 (eIF2alpha ) is phosphorylated by specific protein kinases (see below) (65, 335). Phosphorylation of eIF2alpha thus represents a major point of control over the translation initiation process. eIF2alpha phosphorylation dramatically alters the efficiency and rate of mRNA translation and is a critical component of antiviral and cell growth control pathways (243, 321, 322, 335). eIF2 directs the ternary complex to the 40S ribosomal subunit to form a 43S pre-initiation complex that includes eIF3 (Fig. 2). eIF3 facilitates binding of the 43S pre-initiation complex to the mRNA via the cap-binding complex, eIF4F, that has been assembled around the mRNA m7G cap structure (335, 359).


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FIG. 2.   Schematic illustration of eukaryotic mRNA translation and major sites of viral regulation. Details of the translation process are described in the text. Translation initiation factors are shown by their letter and number designation (335). 40S and 80S denote the small ribosomal subunit and the elongating ribosome, respectively. 1, Ternary-complex formation and assembly of the 43S pre-initiation complex; 2, assembly of the cap-binding complex and ribosome loading onto the mRNA; 3, Ribosome scanning to the first AUG codon, recycling of eIF2-GDP, and joining of the 60S ribosomal subunit. TER denotes a translation termination codon. Major sites for viral control of translation and mechanisms of translation control are shown in the surrounding boxes. Not shown is the mRNA 3' UTR, which can also influence translational efficiency. aa, amino acid.

Cap-binding reaction. Assembly of the eIF4F complex on the mRNA is dependent on the eIF4E component of this complex, which recognizes and binds the m7G cap (173). The affinity of eIF4E for the m7G cap constitutes a second major control point in the translation initiation pathway and is subject to variation through eIF4E phosphorylation (237, 315, 433). In addition, the cap-binding activity of eIF4E can be blocked through the formation of an eIF4E-eIF4E binding protein (4E-BP) complex, resulting in inhibition of cap-dependent translation (367, 433). Formation of the eIF4E/4E-BP complex itself is subject to regulation through 4E-BP phosphorylation and dramatically affects cell growth control by altering the efficiency and selectivity of mRNA translation (reviewed by Sonenberg and Gingras [433]). As discussed in detail below, these regulatory steps are targeted by a group of viruses, which are best defined by the family of picornaviruses and includes poliovirus and encephalomyocarditis virus (EMCV) (155, 174, 322). These viruses initiate translation through a cap-independent mechanism that involves internal ribosome entry through use of the internal ribosome entry site (IRES); virus-mediated cleavage of the 220-kDa cap-binding protein, eIF4G (161, 174, 283); and dephosphorylation of the 4E-BPs to sequester eIF4E in an inactive eIF4E/4E-BP complex (Fig. 2) (155). IRES-mediated translation requires specific cis-acting sequences within the viral RNA that mediate the interaction with trans-acting host factors. Thus, the global process of IRES-mediated translation essentially eliminates the competition for host factors from cap-dependent cellular mRNA translation, favoring the translation of viral mRNA.

Ribosome scanning and AUG site selection. Following association with the mRNA, the 43S preinitiation complex begins scanning from the 5' end of the mRNA or the site of ribosome entry (as in the case of cap-independent translation) and continues scanning until the Met-tRNAi interacts with the initiator AUG codon. Ribosomal scanning is not always compatible with mRNAs that possess a long and/or structured 5' UTR. As described in "Mechanism and control of viral mRNA translation" (below), viral mechanisms to overcome the inefficiency of scanning the 5' UTR and to bypass the host shutoff phenomenon include the use of internal ribosomal entry on the mRNA and the ribosomal shunt (93, 223, 494). These mechanisms allow the preinitiation complex to effectively avoid a large part of the 5' UTR and begin scanning within the region of the initiator AUG codon on the viral mRNA (Fig. 2).

Once the Met-tRNAi associates with the initiator AUG codon, GTP is hydrolyzed from the ternary complex, bound initiation factors are released, and the 60S ribosomal subunit joins the preinitiation complex. The resulting 80S initiation complex then mediates the elongation phase of translation. In this model, initiation begins at the 5'-proximal AUG codon (221, 335). However, the AUG site selection for translation initiation is dependent in part on the context of the AUG codon, where the canonical accAUGg sequence (initiation codon in capitals) exerts the highest preference for initiation (221, 270). Departure from this sequence is associated with leaky scanning, in which the preinitiation complex will recognize a noncanonical or weak AUG only at a low frequency and scans past to initiate translation at a downstream codon more closely matching the canonical initiator AUG (Fig. 2) (221). Leaky-scanning initiation of translation is popular among viruses, and in retroviruses it can provide a mechanism for achieving defined stoichiometric ratios of translation products (412, 413).

Elongation. During the elongation phase of translation, the mRNA is associated with multiple 80S ribosomes, or polyribosomes, as amino acid residues are sequentially placed on the carboxyl end of the growing peptide chain. In many virus systems the replicative cycle is demarked by early- and late-stage events that can be distinguished by the differential recruitment of viral mRNA into polyribosomal complexes at specific times after infection. As with herpes simplex virus type 1 (HSV-1), this often coincides with the synthesis of latency factors and determinants of virulence (114, 159, 279). The process of translation elongation itself is subject to viral regulatory control (Fig. 2). Elongation control mechanisms include ribosomal frameshifting (108), functional recoding (151), and virus-directed modification of EF-1 (257); the first of these is prevalent in retroviruses and reveals otherwise cryptic ORFs within the viral mRNA (78, 108, 109, 151).

Termination. The process of translation termination occurs when the translating 80S ribosome encounters an in-frame termination codon within the template mRNA. The termination codon is recognized by a release factor, which mediates the hydrolysis of the peptide chain from the bound tRNA (335, 501). This results in the release of the nascent polypeptide from the 80S ribosome and leads to the eventual dissociation of the ribosomal subunits. Once termination has occurred, the 40S subunit is free to continue scanning the mRNA (Fig. 2). In multicistronic transcripts, termination can be followed by reinitiation at the downstream cistron, subject to ternary-complex availability (221). However, reinitiation is usually very inefficient, and the presence of a uORF can confer limitations to the translational efficiency of the major, downstream ORF. As described in "Frameshifting" (below), this termination-reinitiation translational control mechanism is prevalent among viruses and is used to control the synthesis of specific viral gene products (148).

Improving translation efficiency: the closed-loop model of mRNA translation. Since the discovery of 3' polyadenylation in eukaryotic mRNA, it has become quite clear that the poly(A) tail imparts stimulation of mRNA translation in eukaryotes (reviewed by Jacobson [225]). More recent analyses indicated that the translation-stimulatory function of the poly(A) tail was due, in part, to the actions of the poly(A)-binding protein (PABP). In mammalian cells, PABP interacts with elements of the cap-binding complex assembled on the 5' end of the mRNA, thus rendering a "closed-loop" translation complex (Fig. 3) (138, 225, 401). PABP promotes the closed loop by binding to eIF4G and to PABP-interacting protein 1 (Paip-1) (75). Paip-1 interacts with components of the mRNA cap-binding complex, including eIF4G and the eIF4A helicase (306). Analyses of translation initiation in yeast and plants indicate that the interaction between PABP and eIF4G stimulates mRNA translation (289, 452, 476). The proximity of the mRNA ends provided by the closed-loop translation complex is thought to contribute to the stability of the mRNA and the 5' cap complex and to provide for the efficient recruitment and recycling of ribosomal subunits (225). Thus, the overall effect of the closed loop is to increase translation efficiency. Viruses exploit the closed-loop translation complex as a means of redirecting the host translation machinery to favor viral mRNA translation. As described below, viruses accomplish this by targeting PABP and disrupting the interaction of the mRNA ends, resulting in attenuation of host mRNA translation (233, 259, 376).


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FIG. 3.   Model of the closed-loop mRNA translation complex. The mRNA-bound eIF4F initiation complex interacts with the 3' end of the mRNA via PABP. Poly(A) sequences within the 3' UTR direct PABP binding to the mRNA. PABP mediates interaction with the cap-binding complex either directly through eIF4G (4G) (452) or indirectly through an eIF4G, eIF4A (4A)-dependent interaction with Paip-1 (75). Assembly of the closed-loop complex may stabilize the interaction of the 40S ribosomal subunit with the mRNA (225, 401).


TRANSLATIONAL CONTROL OF VIRAL GENE EXPRESSION
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Translational dependence has driven viruses to adopt translational programming that maximizes efficiency and facilitates the selective translation of viral mRNA over the endogenous host transcripts. Viral translation strategies have evolved to utilize both the advantages and the limitations inherent within the cap-dependent host translation process. These range from cap-dependent translation competition strategies to cap-independent strategies of IRES-mediated translation initiation. As discussed below, such strategies allow viral mRNA translation to persist, even under the extreme conditions imposed by the host shutoff phenomenon, which severely limits cellular metabolism. This section describes the various translation strategies utilized by eukaryotic viruses to overcome the problems associated with translational dependence and concludes with a discussion of how viral translational programming may present novel targets for the development of anti-viral therapeutics.

Advantages and liabilities of cap-dependent host translation. The majority of mRNA translation within eukaryotic cells is dependent on the m7G cap, a unique structure present at the 5' terminus of the mRNA (335). The 5' cap promotes mRNA stability and nuclear export and provides for various levels of control over the translation initiation process. Cap-dependent control of mRNA translation confers several advantages to the cell. First, and perhaps most importantly, cap dependency allows the cell an immediate mechanism through which to control gene expression by modulating the assembly and activity of cap-binding complex components. Second, cap-dependency provides selectivity of translation by combining the translational regulatory properties inherent within a specific mRNA with those due to modification of the cap-binding complex. Translational control thereby allows the cell to fine-tune gene expression by stimulating or repressing the translation of specific mRNAs, usually through the reversible phosphorylation of translation factors (335).

While cap-dependent translation clearly affords several advantages to the host cell, it also presents liabilities that are effectively exploited by viruses. Cap dependency requires an intact pool of specific initiation factors, namely, the components of the eIF4F cap-binding complex (100, 292, 335, 457, 468). Moreover, it necessitates the capping and nuclear export of mRNAs. Viruses have learned to disrupt these processes in order to reprogram the host cell toward the synthesis of viral proteins. Viral disruption of cap-dependent host translation contributes to the host shutoff that is often observed during productive infections (10).

Host Shutoff and Selective Translation of Viral mRNA

Host shutoff is the process in which cellular macromolecular synthesis is suppressed due to viral domination of host metabolism that occurs during infection (reviewed in reference 10). Host shutoff is not an absolute; not all virus infections exhibit host shutoff, and shutoff is not always required to facilitate viral replication. Within the many viral systems in which host shutoff is known to occur, shutoff ultimately favors the translation of viral mRNA over endogenous host transcripts, although host shutoff itself may not be directly attributed to viral disruption of host mRNA translation (1, 10). The selective translation of viral mRNA during the host shutoff is clearly a multicomponent process that has been attributed to a variety of factors. These include viral perturbation of intracellular ion concentration (144) and nucleotide metabolism (215, 244, 279), alterations in RNA stability, processing, and export (119, 245, 311, 352, 497), and the recruitment of specific host factors (249, 294). From a simpler perspective, the selective translation of viral mRNA during host shutoff may reflect a general competition between viral and host mRNA for the translational machinery. For example, host shutoff in cells infected with vesicular stomatitis virus (VSV) is coupled to the selective translation of viral mRNA (Fig. 4, lanes 3 and 4). Interestingly, however, the abundance and stability of cellular mRNAs and their efficiency of translation initiation remain unaltered (54). Examination of VSV-infected cells revealed that the preferential translation of viral mRNAs was a result of ribosome competition from an overwhelming abundance of viral mRNA (309). At the other end of the spectrum is the host shutoff that occurs during picornavirus infection. In this case, the shutoff of host protein synthesis and selectivity for viral mRNA translation is clearly a virus-directed event mediated, in part, through cleavage of eIF4G by the virus-encoded 2A protease (2A-pro) (174, 273, 468). Cleavage of eIF4G by 2A-pro disrupts cap-dependent translation initiation to favor the IRES-mediated translation of the picornavirus mRNA (Fig. 4; also see Fig. 14) (23). Similarly, the host shutoff in cells infected with influenza virus features a strong selection for viral mRNA translation (Fig. 4, lanes 5 and 6). However, unlike the picornaviruses, influenza virus mRNA translation is cap dependent (251). In this case, the predominance of viral protein synthesis is facilitated, in part, by virus-mediated endonucleolytic cleavage of the host mRNA m7G cap, subsequent mRNA destabilization, and the dephosphorylation of eIF4E (244). The selectivity of viral mRNA translation is then mediated through the recruitment of the cellular G-rich sequence factor 1 (GRSF-1) protein and other host factors to the 5' leader sequence of the influenza virus mRNAs (361, 362).


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FIG. 4.   Virus-induced shutoff of host cell protein synthesis. Murine NIH 3T3 cells (lanes 1 to 4) or Madin-Darby bovine kidney cells (lanes 5 and 6) were mock infected (U) or infected (I), respectively, with EMCV (lanes 1 and 2), VSV (lanes 3 and 4), or influenza virus (lanes 5 and 6). To visualize the virus-induced host shutoff of protein synthesis and the concomitant shift to viral protein synthesis, proteins were biosynthetically labeled by the addition of [35S]methionine to the culture medium. Protein equivalents from mock-infected and virus-infected cells were separated by gel electrophoresis and visualized by autoradiography of the dried gel. Arrows denote the positions of viral proteins. The positions of molecular mass standards are indicated in kilodaltons.

Host shutoff can be seen as beneficial for viruses, since it places cellular resources largely at their disposal. However, the shutoff phenomenon also presents several challenges to the virus, not least of which is maintaining the integrity of the host cell long enough to complete the virus replicative cycle. This is especially important from the standpoints of translational dependence and viral persistence, in which the virus must ensure that the host translation machinery remains competent for the synthesis of viral proteins. Problematically, however, the metabolic repression and stress of host shutoff are potent inducers of cellular apoptosis and translational control programs that function within the cellular antiviral response to block viral infection (179). Viruses have taken a two-pronged approach to these problems of host shutoff, and they encode mechanisms to (i) disrupt host apoptotic programs (353) and (ii) control the antiviral translational response imposed through the phosphorylation of eIF2alpha (65, 66). As described in detail in "Viral modification of cellular factors" (below), eIF2alpha phosphorylation by the cellular serine/threonine protein kinase (PKR) presents a translational blockade to viral replication (68, 131). Disruption of host apoptosis and the phosphorylation of eIF2alpha therefore facilitates viral replication by maintaining host cell integrity and ensuring translational competence during host shutoff.

The relationship between translational control, apoptosis, and viral infection has been an intense area of study in recent years. The emerging picture now suggests that translational suppression through eIF2alpha phosphorylation is an important component of apoptotic programming (450). Thus, disruption of eIF2alpha phosphorylation may serve the dual purpose of maintaining the translational competence of the host cell and preventing apoptosis during host shutoff. This idea is supported by the many studies of vaccinia virus replication in which the viral K3L and E3L gene products have been implicated in disrupting eIF2alpha phosphorylation and blocking apoptosis (52, 55, 82, 83, 135, 255, 420). Moreover, studies by Roizman and colleagues have demonstrated that disruption of eIF2alpha phosphorylation by the HSV-1 gamma 134.5 gene product was a requisite for sustained translational competence and viral persistence during the host shutoff induced by HSV-1 infection (188, 190). Influenza virus similarly ensures that eIF2alpha phosphorylation is blocked and translational competence is maintained during host shutoff (244, 294). However, rather than preventing shutoff-induced apoptosis, influenza virus may delay or reprogram apoptosis to facilitate cell lysis and virion release during late-stage infection (116, 445, 446). In closing this section, it is important to note that maintenance of translational competence during host shutoff may ultimately contribute of viral pathogenesis. As described in "Viral persistence and translational control" (below), the ability to suppress mRNA translation is a key component for the control of cell growth. In persistent viral infections, such as those by hepatitis C virus (HCV) or the DNA tumor viruses, constitutive modulation of host translational control pathways and release of translational suppression may make important contributions to viral oncogenesis (125, 134, 248, 444).

Mechanisms and Control of Viral mRNA Translation

Viruses utilize the canonical translation factors and machinery of the host cell to facilitate completion of their translational programming. Figure 5 depicts various means by which viruses implement their translational programming toward the common end of synthesizing viral proteins and completing the virus life cycle. Reflecting the nature of the virus-host relationship itself, the host cell has evolved countermeasures that impose blockades upon viral protein synthesis. As described below, viral translational programming often includes mechanisms to manipulate the host translational machinery and overcome these antiviral blockades.


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FIG. 5.   Viral mechanisms of translational programming. The top diagram shows structural features of a representative mRNA containing a m7G cap and consisting of a series of overlapping and nonoverlapping reading frames (denoted by rectangles). The first reading frame is indicated by an AUG initiation codon and is preceded by a 5' UTR. Upright arrows indicate translation initiation of the corresponding reading frame(s), resulting from the mechanisms listed at left. Specific viruses examples presented within the text are listed at right. Depiction of the IRES and ribosome shunt includes the relevant stem-loop structures within the 5' UTR. Arrow shows ribosome bypass, or shunting, around the stem-loop. Figure adapted with modification from reference 322.

Internal ribosome entry. Translation initiation, mediated through the internal entry of ribosomes onto the substrate mRNA, was first found in 1988 during studies of poliovirus and EMCV replication (227, 370). Examination of the nucleotide sequence of the picornavirus 5' UTR has revealed a region of significant secondary structure spanning approximately 500 nucleotides (nt) and punctuated by multiple AUG codons (222, 228). This region was initially known as the ribosome landing pad and later termed the internal ribosome entry site (IRES) (for detailed reviews of the IRES, see references 193, 223, and 329). Translation studies performed in vitro and in vivo demonstrated that the IRES could confer internal ribosome entry to a downstream ORF when placed between the cistrons of a multicistronic mRNA (226, 227, 370). Moreover, incorporation of the IRES to precede the ORF of a circular mRNA facilitated ribosome entry and translation of the circular cistron (57). These studies concluded that the IRES is a genetic element that facilitates internal ribosome entry and mRNA translation independent of the m7cap structure. Since then, the observation of IRES-mediated translation has been extended to include other virus families, most notably the other picornaviruses and the members of the genera Pestivirus and Hepacivirus of the family Flaviviridae (which include bovine diarrhea virus and HCV, respectively [Table 2]) (223). We also note that IRES-mediated translation of certain cellular mRNAs has also been identified and may constitute a minor proportion of total cellular mRNA translation within the cell (223, 234, 235, 347).

                              
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TABLE 2.   Representative viruses that utilize IRES-mediated translation

Picornaviruses, pestiviruses, and hepaciviruses carry one copy of an uncapped, single-stranded RNA of positive polarity that functions directly as the viral mRNA and substrate for translation (117). These virus families translate their genomic RNA as a single large polyprotein that is posttranslationally processed into distinct structural and nonstructural polypeptide products through a series of proteolytic cleavage events (for reviews of the replication of picornaviruses, pestiviruses, and hepaciviruses, see references 23, 33, 117, 390, and 398). Picornavirus infections generally exhibit an intense host shutoff (with the exception of hepatitis A virus [HAV]) that is characterized by IRES-mediated selective translation of the viral mRNA (Fig. 4, lanes 1 and 2) and rapid lysis of the host cell within 6 to 12 h after infection (398). In contrast, the prototypic hepacivirus, HCV, mediates persistent infection in which the effects, if any, on host shutoff are less well understood. What, then, is the role of the IRES element in the contrasting life cycles of these two virus families?

The functional role of the IRES is perhaps best understood by considering IRES structure, the context of the IRES within the 5' UTR, and the advantages conferred by IRES-mediated translation. IRES secondary structure has been concisely modeled from several different viruses, and the prototypic IRES structures in poliovirus, EMCV, HAV, and HCV are depicted in Fig. 6. The extensive secondary structure of the IRES makes the long viral 5' UTR (which ranges from 341 nt in HCV to over 1,400 nt in various picornaviruses) incompatible with standard 5' ribosome entry, scanning, and AUG site selection. Among picornaviruses, the IRES itself spans approximately 450 nt and begins a variable distance from 5' terminus of the RNA (101). The enteroviruses and rhinoviruses comprise a structurally conserved IRES group that is distinct from a second IRES group represented by the cardioviruses and aphthoviruses (193, 223). A third and structurally distinct picornavirus IRES group is represented by HAV (156).


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FIG. 6.   IRES structure. Structural representation of the 5' UTR from EMCV (top left), poliovirus (top right), HAV (bottom left), and HCV (bottom right). The major stem-loops are labeled according to previous designations (42, 193, 206, 483). The region encompassing the IRES is underlined. Pyrimidine-rich sequence elements are shown as solid rectangles. AUG denotes the position of the translation initiation codon. The box on the HCV IRES denotes the core protein-coding region.

The IRES groups also diverge in relation to the position of the authentic initiator AUG codon. Both the cardiovirus-aphthovirus IRES group and HAV initiate translation from the AUG codon at the immediate 3' boundary of the IRES (193). Thus, the site of ribosome entry actually corresponds to the initiation codon. In contrast, the enteroviruses and rhinoviruses initiate translation from an AUG codon located approximately 40 and 160 nt downstream, respectively, from the 3' IRES boundary (23, 223). In this case, the ribosome scans the mRNA from the point of ribosome entry to the authentic AUG codon, in accordance with the conventional scanning model. The prototypic HCV IRES diverges in both length and structure from the three picornavirus IRES groups (Fig. 6). The actual boundaries of the HCV IRES have yet to be precisely defined, but initial studies suggest that the IRES begins approximately 20 nt from the RNA 5' terminus and extends at least 30 to 40 nt into the actual coding region of the viral core protein (211, 391). Ribosome entry on the HCV RNA takes place within the IRES itself rather than at the 3' IRES boundary. This coincides with the actual site of translation initiation, which resides approximately 40 nt internal to the 3' IRES boundary (391).

Structural conservation between picornavirus IRES groups is limited to a 10- to 15-nt pyrimidine-rich sequence element located near the 3' IRES boundary (101) and, to a lesser extent, a common 3' structural core related to the group I intron (290). The significance of the latter is not clear, although it may reflect common structural features of RNA required for the assembly of mRNP complexes. Several independent studies have identified the pyrimidine-rich sequence element as a conserved IRES feature, indicative of a role for this element in IRES-mediated translation. Indeed, partial deletion or purine substitution of the pyrimidine-rich sequence abolished the function of the poliovirus IRES and, to a lesser extent, limited the translation efficiency of the EMCV IRES (192, 351). Together, these experiments demonstrated that the number of bases residing between the 3' end of the pyrimidine-rich sequence and the actual initiator AUG codon was an important variable influencing the site of ribosome entry and AUG codon selection. These results suggested that (i) the length of the pyrimidine-rich sequence element and its proximity to the initiator AUG codon may influence translation start site selection and (ii) this sequence element may function in the process of IRES-mediated recruitment of the translation initiation complex to the site of initiation. The latter notion is supported by the findings that several host proteins, including the cellular poly(rC)-binding protein 2 (PCBP2) and the pyrimidine tract-binding protein (PTB), may interact with the pyrimidine-rich sequence element and affect the translation efficiency of the picornaviral IRES (34, 35, 238, 264, 394).

Interestingly, the HCV genome contains a pyrimidine-rich sequence element located just outside the IRES, at the 3' end of the core-coding region (206) (Fig. 6). Recent analyses indicate that the HCV core region pyrimidine-rich sequence functions in cis to regulate translation from the HCV IRES. In these studies, Ito and Lai found that the core region pyrimidine-rich sequence had a suppressive effect on IRES-mediated translation, most likely induced through interaction with PTB (217, 218). This translational suppression was relieved by an interaction of PTB with the highly conserved 98 nt of the HCV 3' UTR. These results are consistent with the idea that sequences outside the HCV IRES impart control over the translation initiation process and that this may involve (i) the recruitment of a host factor(s) to the HCV RNA, and (ii) cross talk between the viral 3' UTR, internal elements in the HCV RNA, and the IRES itself. Such a model may provide a mechanism allowing the virus to modulate polyprotein synthesis in response to changing conditions within the host cell through specific protein interactions with the viral RNA.

IRES-mediated translation avoids the potential limitations posed by cap dependency and provides important advantages for viral replication. By mediating internal ribosome entry, the IRES allows the viral mRNA to bypass rate limitations on translation imposed by the cap-binding reaction. This can be seen as critical for the picornaviruses, which can reach an astounding rate of 5 × 106 virion particles/cell over a 6-h period (398), and HCV (1012 virion particles/day/ml of infected blood examined [348]). Cap-independent translation bypasses the requirement for a virion-encoded mRNA-capping enzyme or alleviates the requirement for host capping enzymes and nuclear localization of viral mRNA synthesis and replication. The picornaviruses have taken this a step further by encoding mechanisms to disrupt cap-dependent translation in favor of viral protein synthesis. As described in "Viral modification of cellular factors" (below), these involve cleaving the eIF4G component of the cap-binding complex (174, 468), regulating the activity of eIF4E (155), and cleaving PABP (233, 259). Perhaps most importantly, the IRES allows the 43S preinitiation complex to avoid scanning the long 5' UTR of the viral mRNA. Thus, IRES entry of ribosomes avoids the pitfalls of scanning through highly structured regions within the viral mRNA and bypasses any initiation interference from upstream AUG codons and uORFs (Fig. 5). This also ensures that the translation machinery avoids interfering with the genome replication signals embedded within the 5' UTR of the viral RNA (37).

As suggested by the IRES-mediated translation model (223), IRES function is determined, in part, through interactions with host proteins in addition to the translational machinery itself. IRES-host protein interactions may contribute to host range specificity and virulence phenotype. This was suggested by early experiments that measured the in vitro translation efficiencies of the poliovirus and EMCV IRES elements within a rabbit reticulocyte lysate translation system (41, 99). These experiments found that the cardiovirus and aphthovirus RNAs were efficiently translated in vitro whereas, in contrast, the enterovirus and rhinovirus RNAs were translated with low fidelity and inefficiency. Supplementation of the translation mixture with HeLa cell extract significantly increased IRES efficiency and restored the accuracy of translation (371). These studies immediately suggested that viral host range might directly reflect the requirements for specific host factors, in addition to the canonical translation factors, to facilitate IRES function and viral protein synthesis. Indeed, IRES-binding host factors have now been identified that play a functional role in IRES-mediated translation (23, 372, 394) (see below). The identification of such factors, and their cognate binding sites within the IRES, should lead to a better understanding of the impact of translational control on host range restriction and viral pathogenesis.

The concept that the translational efficiency of viral mRNA affects pathogenesis is supported by the results of analyses of poliovirus Sabin vaccine strains (400). A subset of attenuating mutations within the Sabin strains were mapped to the major stem-loop V (Fig. 6) of the poliovirus IRES (reviewed by Ehrenfeld [101]). These mutations resulted in reduced translation efficiency of the viral mRNA, resulting in the attenuated vaccine phenotype. These studies suggested that the stem-loop structures within the IRES were critical determinants of translational efficiency and, in poliovirus, were important elements of neurovirulence. Analyses of the neuropathogenic phenotype of rhinovirus/poliovirus chimeras support this notion. Studies of poliovirus neurovirulence in which the poliovirus IRES was replaced with the structurally related IRES from human rhinovirus type 2, conducted by Wimmer and colleagues (167), demonstrated that the neurovirulent phenotype of poliovirus was dependent on the authentic poliovirus IRES. Interestingly, the chimeric virus retained its ability to replicate in nonneuronal tissues, indicating that the IRES itself is an important determinant of host range specificity. More recent structure-function analyses have identified stem-loops V and VI as the poliovirus genetic elements responsible for the neurovirulent phenotype (168). Together, these results suggest that poliovirus IRES stem-loops V and VI provide the capacity for viral replication within the central nervous system. It is therefore possible that these genetic determinants may function to recruit tissue-specific host factors to the poliovirus IRES. Future experiments to test this idea should aid in our understanding of the relationship between IRES structure and viral pathogenesis.

Substantial progress is now being made toward understanding how elements within the HCV IRES may affect viral replication and pathogenesis. An initial comparison of IRES structure and IRES-dependent translation from representative isolates of the various HCV genotypes has revealed some interesting features. First, by measuring the relative translation rates of chloramphenicol acetyltransferase (CAT) reporter constructs placed under control of HCV IRES sequences, Buratti et al. demonstrated a lower translation efficiency for HCV genotype 3 compared to genotypes 1 and 2 (47). Moreover, these investigators found that conservation of both the secondary structure and the sequence within linear domains of HCV IRES stem-loop III was important for maintaining IRES function. A broader analyses of HCV IRES translational efficiency supported and extended these results, revealing a lower efficiency of translation mediated by the IRES elements of HCV genotypes 3 to 6 (71). It is interesting that the level of polymorphism between the IRES elements used in the latter study was limited to a mere total 17 nucleotide positions. Most of these differences could be mapped to within the regions on the major stem-loop III and, to a lesser extent, stem-loop IV. Thus, it appears that subtle differences in nucleotide sequence can confer sufficient alterations in RNA secondary structure to affect the function of the HCV IRES and the efficiency of viral mRNA translation. In accordance with this idea, Honda et al. have revealed that major alterations in stem-loops III and IV of the HCV IRES resulted in loss of function, presumably due to the induction of gross aberrations in IRES structure (204-206). However, these studies also identified differences in translation efficiencies between HCV genotypes 1A and 1B that were not attributed to major structural differences between the respective IRES sequences (206). In follow-up studies, it was found that the reduced translation efficiency of the HCV 1B RNA was attributed to the presence of an AG dinucleotide sequence (HCV nt 34 and 35) present within a single-stranded region that preceded the IRES (207). Mutation of the AG dinucleotide to GA (present in HCV 1A) increased the translation efficiency of HCV 1B RNA, demonstrating that the wild-type (wt) AG dinucleotide sequence had an inhibitory effect on translation. Remarkably, translation inhibition was attributed to an RNA-RNA interaction mediated between the AG dinucleotide sequence and a region far downstream within the viral core coding sequence (207). These results suggest that the long-range RNA-RNA interaction between the viral 5' UTR and the core-coding region functions cooperatively to influence the activity of the HCV IRES. Such an interaction may sufficiently alter the structure of the IRES to affect the efficiency of viral RNA translation. An intriguing question is whether the correlation between HCV genotype and IRES translation efficiency in vitro extends to the phenotypic differences exhibited by these viral genotypes during the course of HCV infection.

Molecular epidemiological studies have identified an association between HCV genotype, response to the current anti-HCV interferon (IFN) therapy, and severity of infection. In these studies, patients who were infected with HCV genotype 1 or 2 consistently presented a more severe pathology than did patients infected with HCV genotype 3, 4, 5, or 6 (7, 45). Moreover, analyses of the virus load in serum revealed an association between increased viral titer, resistance to therapy, and a poor prognosis, especially in patient groups infected with HCV genotype 1 (103, 115). It has been proposed that these biological differences between HCV genotypes may be due, in part, to the variations in translation efficiency conferred by the subtle polymorphisms between the respective genotype-specific IRES. Secondary-structure analyses of the HCV IRES isolated from healthy patients with low viral titers who responded to anti-HCV therapy and from those who did not respond and maintained high viral loads revealed that a significant level of IRES structural variation was associated with a low viral titer and complete response to therapy (439). In stark contrast, conservation of IRES structure was consistently associated with maintenance of a high virus load and resistance to therapy. Taken together, these studies suggest that IRES structure and the corresponding translation efficiency of a given HCV strain are critical factors affecting the virulence of HCV and the sensitivity to antiviral therapy. Such results have identified the HCV IRES as a potential target for therapy of HCV infection (31).

Ribosome shunt. The processes of cap-mediated ribosome entry, 5' scanning, and internal initiation are combined features of the ribosome shunt mechanism of translation. Ribosome shunting allows the small ribosomal subunit to avoid the problems of scanning a long and complex linear sequence preceding the major ORF. Unlike IRES-mediated translation, shunting is dependent on the 5' cap and the cap-binding complex for ribosome entry onto the mRNA (410) (Fig. 5). The best evidence for ribosome shunting, and the general ribosome shunt model, comes from studies of the pararetrovirus cauliflower mosaic virus (CaMV) (94, 410), although shunting also takes place in other viral systems (reviewed in reference 203). Upon engaging the mRNA, the ribosome scans until it reaches a cis-acting shunting element that promotes ribosomal translocation to a downstream receiving element(s) (94). By this process, scanning becomes nonlinear and the ribosome can bypass a large portion of the 5' UTR to initiate translation at the downstream cistron (Fig. 5 and 7).


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FIG. 7.   The CaMV ribosome shunt. Specific details are described in the text. The relative positions of sORFA, the major stem-loop, and the gene VII ORF of the 35S RNA are shown. Ribosome shunting (denoted by the lower arrow) occurs around the major stem-loop encompassing nt 70 to 550 (196). After terminating sORFA translation, the 40S ribosomal subunit is shunted around the major stem-loop structure and resumes scanning to initiate translation at the ORF VII AUG codon (upper arrow).

CaMV encodes two major RNAs, a monocistronic 19S RNA that encodes a translational transactivator and the 35S pregenomic RNA (123). The 35S RNA spans the complete CaMV genome, serves as the template for virus-mediated reverse transcription, and functions as the major viral mRNA for the synthesis of at least seven viral proteins. Translation from the 35S RNA is cap dependent and is under the control of the 600-nt multifunctional RNA leader (196). Structural analyses of the CaMV leader revealed that it contains several short uORFs and assumes a complex stem-loop structure (123, 195). Although these features of the CaMV leader are incompatible with the 5'-scanning model, translation from downstream ORFs still occurs with reasonable efficiency in vitro and in vivo (410).

Previous studies of CaMV translation demonstrated that ribosome shunting could occur in trans using separate RNA molecules (124). These experiments involved RNAs that were designed to restore the secondary structure of the 5' leader by annealing and adopting the native structure of the 35S RNA. The results identified the 35S 5'-UTR stem-loop structure as a requisite element in the ribosome shunting process and implicated the immediate flanking sequences as the shunt donor and shunt acceptor sites. Subsequent structure-function analyses of the CaMV stem-loop demonstrated that translational control of the CaMV 35S RNA was dependent on the presence of an elongated hairpin comprising nt 70 to 550 of the 5' leader (195, 196). Moreover, experiments examining the influence of the 35S uORFs (sORFA to sORFF) on CaMV translation found that the integrity of sORFA was required for translation of the major downstream cistrons (196). sORFA is the 5'-proximal 35S uORF and is followed closely by the elongated hairpin structure (123). These studies demonstrated that mutation of the sORFA initiation codon impaired translation past the elongated hairpin on the 35S RNA. Together, these results are consistent with the model that CaMV translation initiates at sORFA, allowing assembly of the 80S ribosomal complex onto the 35S RNA (Fig. 7). Perhaps concomitantly while encountering the termination codon of sORFA, the 80S ribosomal complex encounters the shunt donor element at the base of the elongated hairpin structure. sORFA translation then terminates, with the shunt donor accepting the 40S subunit and promoting a shunt around the elongated hairpin to the site of the shunt acceptor. Shunting is then followed by resumption of mRNA scanning by the 40S ribosomal subunit. The 80S ribosomal complex then reassembles at the downstream ORFVII AUG codon, and CaMV translation begins. Alternatively, the shunt donor may facilitate passage of the intact 80S ribosomal complex around the elongated hairpin structure without a requirement for complex disassembly, although this would require the shunt donor to deposit the ribosomal complex directly at the ORFVII initiation codon (Fig. 7). The exact sequence elements responsible for ribosome shunting in CaMV are not yet known. Similar to the IRES mechanism of translation, shunting may involve assembly of host factors at and round the shunt donor and shunt acceptor sites. Recent evidence indicates that shunting, at least in CaMV and related viruses, may be host independent (410). Thus, the ribosome shunt may represent a general mechanism of translational adaptability among viruses. Future studies to determine the nature of the host "shunting factors" will certainly aid in understanding the molecular mechanisms of ribosomal shunting.

What advantages does the ribosome shunt mechanism of translation confer to viral replication? The shunt has clearly evolved as a mechanism to avoid the problems associated with scanning a highly structured 5' UTR. In this respect, shunting confers translational efficiency to the viral mRNA. More specifically, however, ribosomal shunting may preclude or reduce the requirement for the host eIF4F helicase activity to melt the secondary structures within an mRNA that impede the normal scanning and the AUG site selection process. This would also relieve the competition with cellular mRNAs for the limited amounts of eIF4F present within the host cell. Thus, the ribosome shunt may facilitate the selective translation of viral mRNA, especially under cellular conditions in which the pool of functional eIF4F becomes limiting. Schneider and colleagues have investigated this idea by examining the mechanisms of selective translation of late adenovirus mRNAs within infected cells (reviewed in reference 411). Adenovirus induces the dephosphorylation of eIF4E and concomitant reduction in the functional pool of eIF4F during late-stage infection, resulting in inhibition of host cell protein synthesis (210, 499) (see below). However, the translation of late adenovirus mRNAs remain uncompromised, due to the function of the conserved tripartite leader.

The adenovirus late mRNA tripartite leader sequence is composed of a linear 5' end followed by regions of stable secondary structure (91). In uninfected cells, the tripartite leader can direct the translation of a downstream cistron by both conventional 5' scanning and ribosome shunting. However, during late-stage adenovirus infection, the translation of late viral mRNAs is mediated exclusively by the ribosome shunt (494). Structure-function analysis of the tripartite leader has shown that (i) the linear 5' end is essential for the efficient recruitment of ribosomes when eIF4F is limiting, (ii) the stem-loop structural elements inhibit ribosome scanning during late-stage adenovirus infection, and (iii) the selective translation of late adenovirus mRNAs is influenced by the actions of one or more viral gene products. These results, taken together, suggest that limitations in eIF4F select for expression of the late adenovirus gene products through the function of the late mRNA tripartite leader. Stem-loop elements within the tripartite leader, along with virus-encoded transacting factors, then direct the selective translation of late mRNAs through a process of ribosome shunting, independent of eIF4F helicase activity. Thus, it appears that the ribosome shunt has evolved the dual functions of allowing the ribosome to avoid the impediments on translation imposed by regions of mRNA secondary structure and the limitations in the quantity and quality of the host translation machinery.

Leaky scanning. Translation initiation site selection is determined, in part, by the context of the nucleotide sequence surrounding the first AUG codon encountered by the scanning ribosomal subunit. Departure from the canonical accAUGg sequence (the initiation codon is shown in bold letters) often results in the scanning ribosome initiating translation from this weak AUG at a low frequency or bypassing it completely in favor of a stronger downstream AUG start site (221). This AUG selectivity is referred to as leaky scanning. Leaky scanning allows the translation of multiple ORFs from a common mRNA substrate (Fig. 5). The versatility of leaky scanning is quite evident when one considers that each ORF need not be in the same reading frame. Thus, the process of leaky scanning allows the virus to maximize its genome coding capacity and encode functionally distinct proteins from a common mRNA.

Leaky scanning is widely used by viruses and is perhaps best defined from studies on retrovirus replication. Human immunodeficiency virus (HIV) encodes a heterogeneous class of mRNAs that include several multicistronic species. Among these are the bicistronic mRNAs encoding the viral Vpu and Env proteins (368). The ORFs for Vpu and Env are tandemly arranged such that the Vpu coding region precedes the Env ORF (Fig. 8A). Synthesis of Env is essential for viral replication and takes place through a mechanism of leaky scanning from the upstream Vpu ORF (368, 412). Env synthesis requires a weak Vpu translation initiation codon. Mutation of the weak Vpu start site to a sequence more closely matching the canonical start site sequence resulted in suppressed Env translation from the bicistronic Vpu-Env mRNA (412). Thus, the weak context of the Vpu initiation codon allows the ribosome to scan pass the Vpu ORF and to initiate translation at the downstream Env AUG codon (Fig. 8A). Analyses of HIV mutants in which the vpu gene was deleted or lacked the Vpu initiation codon revealed a stimulation of Env synthesis (413). Accordingly, vpu mutant viruses exhibited defects in virus release and showed increased syncytium formation in vitro (368). These results support a model for the coordinate expression of Vpu and Env during HIV infection, which is dependent on the presence of the vpu ORF. By this model, the synthesis of Vpu occurs inefficiently via a weak initiation codon, perhaps allowing Vpu to coordinately accumulate during infection to levels sufficient for its function in late-stage HIV replication. In accordance with the leaky-scanning mechanism, the synthesis of Vpu itself may coordinate Env production by impeding ribosome scanning to the downstream Env ORF. This model remains to be directly examined by analyzing the polyribosome distribution of the relevant mutant Vpu-Env mRNAs. However, it suggests that the limitations placed on Env translation by the upstream vpu ORF may allow Env expression to coincide with late-stage replication events, including virion assembly, and release. Thus, while ensuring that both viral proteins will be produced during HIV infection, translational control by leaky scanning provides for the coordinate expression of Vpu and Env during viral replication.


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FIG. 8.   Leaky scanning and translational frameshifting during retroviral mRNA translation. (A) Leaky scanning during HIV mRNA translation accounts for synthesis of the Vpu and Env proteins. The HIV gene structure (upper) consists of several overlapping cistrons encoded within a heterogeneous array of mRNAs (438). Synthesis of the Vpu and Env proteins (lower diagram) of HIV proceeds via a leaky-scanning mechanism in which the scanning ribosome bypasses the weak vpu AUG codon (shown) to initiate translation from the env AUG codon (bent arrow). Translation initiation occurs at the vpu AUG codon at a low frequency and may account for the stoichiometric ratios of Vpu and Env protein accumulation during HIV infection (413). (B) Translational frameshifting at the gag-pro junction during MMTV mRNA translation. An RNA pseudoknot near the 3' end of the gag gene causes the elongating ribosome to pause at the gag-pro slippery sequence element. As a result, the mRNA slips backward by 1 nt and the ribosome-bound tRNAs mediate new anticodon base pairing in the -1 reading frame (12). Frameshifting is favored by weak codon-tRNA anticodon base pairing in the original reading frame and strong base pairing in the new reading frame. Synthesis of the entire Gag-Pro-Pol polyprotein is facilitated by a second frameshift at the downstream pro-pol slippery site. Model adapted from reference 12.

Frameshifting. As in the example provided by the HIV genome, retroviral genomes exhibit overlapping gene arrangements (Fig. 8A). The mouse mammary tumor virus (MMTV) genome exhibits an overlapping gag, pro, and pol gene arrangement (72). Translation of these gene products involves the process of frameshifting, in which the translating ribosome shifts position by +1 or -1 nt, resulting in a change of reading frame (reviewed by Gesteland and Atkins [151]) (Fig. 5). The process of frameshifting was first described from studies of Rous sarcoma virus replication (220) and has been extensively defined in the MMTV and infectious bronchitis virus (IBV) (a coronavirus)] systems, although it occurs widely throughout other eukaryotic RNA viruses (151). Frameshift sites within the viral mRNA correspond to heptanucleotide sequences in which the mRNA slips 1 base with respect to the tRNAs in the A and P sites on the translating ribosome (40, 108). The frameshift site, also known as the slippery site, allows the tRNA to move along the mRNA template by 1 base (forward or back) and reestablish codon-anticodon pairing, resulting in a stable +1 or -1 reading frame shift.

Frameshifting is stimulated by the presence of an RNA pseudoknot structure located 2 to 4 nt downstream of the slippery site (59, 219, 319). A general model for retroviral frameshifting has been proposed (108), in which the elongating ribosome pauses on the mRNA upon encountering the pseudoknot structure (Fig. 8B). Ribosomal pausing facilitates the realignment of the slippery-sequence-decoding tRNAs in the -1 reading frame. The heptanucleotide sequence that comprises the slippery site typically conforms to the motif XXXYYYN. Frameshifting occurs at this site through the slipping of two ribosome-bound tRNAs that are translocated from the current reading frame of X-XXY-YYN, to the -1 reading frame of XXX-YYY. In MMTV, translation initiation of the gag-pro mRNA begins at the 5' end of the gag gene (Fig. 8B). The translating ribosomes encounter a slippery site and a pseudoknot structure near the 3' end of the gag gene. The majority of the ribosomes read through this region, but approximately 25% hesitate at the heptanucleotide site, where the mRNA will slip backward by 1 nt. This event is stabilized by the new pairing with the two tRNAs in the -1 reading frame. Meanwhile, most of the translating ribosomes will terminate to make Gag-Pro but another 10% will slip again at the pro-pol site to make the requisite Gag-Pro-Pol polyprotein (108).

What are the molecular mechanisms by which the tRNAs and pseudoknot contribute to frameshifting during mRNA translation? Evidence has accumulated to indicate that the actual frameshift occurs at the second (underlined) codon of the tandem slippery codon pair, XXXYYYN, corresponding to the ribosome aminoacyl (A) site. Slippery A sites within eukaryotic viruses correspond to the codon sequence of AAC, AAU, UUA, UUC, and UUU (108). Interestingly, these codons are decoded by tRNAs with a highly modified base in the anticodon loop (185). Thus, it has been suggested that hypomodified variants of these tRNAs may function to promote shifting by being less bulky and therefore more easily moved within the slippery site (186). However, this idea remains controversial, since other researchers have proposed that frameshifting is mediated by standard cellular tRNAs and is simply dependent on the strength of the codon-anticodon tRNA interaction (40, 465). In either case, frameshifting requires a pseudoknot structure near the slippery site to stimulate the frameshifting events.

Recent evidence indicates that the actual secondary structure of the pseudoknot is important for stimulating frameshifting. Analysis of the IBV frameshifting signals clearly demonstrated that the pseudoknot causes ribosome pausing. Replacement of the IBV pseudoknot with a simple stem-loop structure of equivalent base pairs did induce ribosome pausing but, remarkably, did not stimulate frameshifting (427). These results suggested that ribosome pausing was necessary but not sufficient for frameshifting to occur and support the hypotheses that (i) conservation of pseudoknot structure is essential for frameshifting and (ii) pseudoknot interactions with specific trans-acting factors may promote the frameshift events. Atomic modeling of the MMTV gag-pro pseudoknot supports the former hypothesis, in that this pseudoknot does not have coaxially stacked helices but, rather, assumes a wedge conformation induced by an A nucleotide between the helices (418). Structure-function analyses of the MMTV pseudoknot revealed that this A nucleotide was essential for stimulating frameshifting activity. Structural analyses of other viral pseudoknots should provide further insight into the contribution of pseudoknot sequence and structure in ribosome frameshifting.

Control of termination and reinitiation. Translational control by reinitiation involves two or more tandemly arranged ORFs on a common mRNA. In the simplest model of reinitiation, a short uORF controls the translation of the major downstream ORF by impeding ribosome scanning (reviewed by Geballe [146]). In this sense, the uORF commonly asserts a suppressive effect upon translation of the downstream ORF. However, and as in the CaMV 35S RNA translation, exceptions to this rule do apply. As described above, the sORFA uORF of CaMV actually plays a stimulatory role in translation of downstream ORFs within the 35S RNA (195). An example of viral use of reinitiation to negatively control translation from a downstream cistron comes from studies of cytomegalovirus replication. During cytomegalovirus infection, expression of the gp48 product of the polycistronic viral gpUL4 mRNA is coordinately controlled to reach peak levels during late-stage viral replication. gp48 is translated from the third of three cistrons within the gpUL4 mRNA (146, 313). Gelballe and colleagues have determined that coordinate control of gp48 expression is mediated through the actions of the second gpUL4 uORF (uORF2) (147). Remarkably, the uORF2 inhibitory effect on gp48 translation is dependent upon the sequence of uORF2 (48, 85); introduction of uORF2 missense mutations severely diminished the inhibitory signal upon gp48 translation, while introduction of mutations that preserved the coding content of uORF2 led to retention of gp48 translational inhibition. In vitro and in vivo expression studies revealed that the translational control actions of uORF2 (i) function exclusively in cis to repress gp48 synthesis through ribosome stalling at the uORF2 termination codon and (ii) are mediated through interference of uORF2 translation termination by the uORF2 peptide product itself. Analysis of uORF2 translation revealed that the 20-kDa peptide product remained bound to the ribosome complex as a peptidyl-tRNA covalently linked to tRNApro, which decodes the uORF2 carboxyl-terminal codon (49). Recent studies have now demonstrated that the uORF2 peptidyl-tRNApro blocks its own hydrolysis and ribosome release to remain stably bound to the ribosomal complex (50). These results suggest that inhibition of uORF2 peptidyl-tRNApro hydrolysis blocks the translation of gp48 by creating a barrier that obstructs ribosome scanning to the downstream gp48 ORF (Fig. 9).


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FIG. 9.   CMV control of gp48 synthesis by inefficient termination of uORF2. During CMV infection the viral gp48 glycoprotein is translated from the third of three cistrons (rectangles) within the gpUL4 mRNA. 1, gp48 synthesis is facilitated by a leaky-scanning mechanism in which the scanning ribosome bypasses the weak upstream AUG codons within the gpUL4 mRNA to initiate translation at the gp48 AUG (indicated by arrow); 2, translation initiation at the uORF2 AUG occurs at a low frequency and results in control of gp48 translation. Synthesis of the uORF2 peptide produces a stable peptide-tRNApro-ribosome complex that prevents peptide release and stalls the elongating ribosome at the uORF2 termination codon. As a result, ribosome scanning and reinitiation at the downstream gp48 AUG codon is blocked.

If uORF2 blocks gp48 translation, what facilitates synthesis of the gp48 protein during CMV infection? Sequence analyses has shown that the initiator AUG codon of uORF2 is presented within a "weak" context for optimal translation initiation (147). Accordingly, it was found that the uORF2 AUG codon is recognized by the ribosomal complex only at a low frequency and is actually bypassed by a leaky-scanning mechanism in favor of initiating translation from the gp48 start codon. Alteration of the uORF2 initiation codon to within an optimal context for translation initiation results in nearly a complete block in gp48 synthesis (48). Together, these results support a bipartite model for the control of gp48 translation by uORF2 (Fig. 9). It is not clear how uORF2 peptidyl-tRNApro blocks its own hydrolysis and release from the uORF2 termination site. Possible explanations may be found by examining the influence of the uORF2 product on the function of translation elongation and release factors.

Another example of reinitiation control comes from studies of reovirus translation. Reoviruses are double-stranded RNA (dsRNA) viruses with a segmented genome. The S1 mRNA of reoviruses is bicistronic and encodes the sigma 1 capsid protein and the sigma 1 nonstructural protein (409, 417). Initial analyses of S1 mRNA translation revealed that it was translated inefficiently compared to other