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.

View larger version (35K): [in this window] [in a new window]   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.

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.

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-tRNA_i) to the 40S ribosomal subunit. This step is facilitated through the formation of an eIF2-GTP-Met-tRNA_i ternary complex (462). The recent discovery and functional analyses of eukaryotic homologues of prokaryotic initiation factor 2 (IF2) indicates that Met-tRNA_i 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-tRNA_i 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-tRNA_i to the 40S ribosomal subunit can constitute a rate-limiting step when the alpha subunit of eIF2 (eIF2) is phosphorylated by specific protein kinases (see below) (65, 335). Phosphorylation of eIF2 thus represents a major point of control over the translation initiation process. eIF2 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).

View larger version (32K): [in this window] [in a new window]   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-tRNA_i 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).

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).

View larger version (24K): [in this window] [in a new window]   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).

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).

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).

View larger version (79K): [in this window] [in a new window]   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 eIF2 (65, 66). As described in detail in "Viral modification of cellular factors" (below), eIF2 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 eIF2 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 eIF2 phosphorylation is an important component of apoptotic programming (450). Thus, disruption of eIF2 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 eIF2 phosphorylation and blocking apoptosis (52, 55, 82, 83, 135, 255, 420). Moreover, studies by Roizman and colleagues have demonstrated that disruption of eIF2 phosphorylation by the HSV-1 ₁34.5 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 eIF2 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).

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.

View larger version (21K): [in this window] [in a new window]   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).

View larger version (22K): [in this window] [in a new window]   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.

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).

View larger version (7K): [in this window] [in a new window]   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).

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.

View larger version (26K): [in this window] [in a new window]   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.

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 tRNA_pro, which decodes the uORF2 carboxyl-terminal codon (49). Recent studies have now demonstrated that the uORF2 peptidyl-tRNA_pro blocks its own hydrolysis and ribosome release to remain stably bound to the ribosomal complex (50). These results suggest that inhibition of uORF2 peptidyl-tRNA_pro hydrolysis blocks the translation of gp48 by creating a barrier that obstructs ribosome scanning to the downstream gp48 ORF (Fig. 9).

View larger version (18K): [in this window] [in a new window]   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.