TABLE 1.

Large-T-antigen J-domain mutantsa

Mutant typeMutant nameAbility to transformbGrowthInduction of:Ability to:
REF5210T1/2REF/CREFSoft agarcAdvant. MEFsdLow serumdChoroid plexuseLymphomaeHepatocarcinomafBind pRBgOver. pRB G1 arresthAlter p130iDisrupt p130/E2FjTransactivate E2FkIncorp. BrdUlEnhance Tst1mOverride p53nReplicate in celloReplicate in vitrooBind Hsc70pStimulate 70 ATPaseqBacterial/viabilityr
SV40WT++++++++++++++++++++++
H42QNDNDND+NDNDND+NDND+NDNDND
D44N+/−+ND+NDNDND+NDND++/−
L17KNDNDNDNDNDNDNDNDND+NDNDNDNDNDNDNDNDNDNDND
L19F,P28S+/−+/−NDNDNDND++NDNDNDNDNDNDNDNDND+/−NDNDND
P43FNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDND
P43F,T57NDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDND
K45QNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDND
G47ENDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDND
K67PNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDND
    pRB mutantsE107KK1NDNDNDNDNDNDND+ND+ND+
E107,108K3213NDNDNDNDNDNDNDNDNDNDND+ND++ND+
    DeletionsΔ2-82NDNDNDNDNDNDNDND+NDNDNDNDND+/−+ND
Δ17-27NDNDND++NDNDNDNDND+NDND
Δ46-74NDNDNDNDNDNDNDNDND+NDNDNDNDNDNDNDNDNDNDND
Δsb59-64NDNDNDNDNDNDNDND+NDNDNDNDNDNDNDNDNDND
Δ65-70NDND+/−NDNDNDNDNDND+ND+NDNDNDNDND+NDNDNDND
    N fragmentsN121+/−NDNDNDND+++NDNDNDNDNDND+/−NDNDNDND+
N136+/−NDNDNDNDNDNDND+NDNDNDNDNDNDND++
N147ND+/−ND+NDNDNDNDNDNDND+NDNDNDNDNDNDNDNDND+
N136/D44NNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDND
    pRB mutantsN136/KNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDND+
N136/3213NDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDND+
    ChimerasDnaJ2-TNDNDNDNDNDNDNDNDND+ND+NDNDNDNDND+/−ND+NDND
DnaJ2-T HQNDNDNDNDNDNDNDNDND+NDNDNDNDNDNDNDNDND
Hsj1-TNDNDND++/−+NDNDND+++++ND+/−ND+ND+NDND
Hsj1-T HQNDNDND+NDNDND+NDNDNDNDND
SV-JCV+NDNDNDNDNDNDNDNDNDNDND+NDNDNDND+NDNDNDND
SV-YdjNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDND
SV-DnaJNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDND++/−ND
PolyomavirusWTNDNDNDNDNDNDNDNDND+NDNDND++NDND+ND+ND+
V4DNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDND+/−
L13VNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDND+/−NDNDND
32ENDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDND
A33SNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDND
Y34NNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDND
H42NDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDND
P43SNDNDNDNDNDNDNDNDND+NDNDNDNDNDNDNDND
44NNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDND
    RB mutant142VE146QNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDND+ND+
BKVWTNDNDNDNDND+NDNDND+ND+ND+NDNDNDNDNDNDND+
H42QNDNDNDNDNDNDNDND+NDNDNDNDNDNDNDNDNDNDND
    pRB mutantE109KNDNDNDNDND+NDNDNDNDNDNDNDNDNDNDNDNDND+
  • a Mutants in the large T antigens of SV40, polyomavirus and BKV as well as their phenotypes in various assays. See individual footnotes for specifics of each assay. Mutants in the J domain that possess levels of activity near those of the wild-type T antigen are not listed. For comparison purposes, mutant T antigens defective for binding to pRB family members are included.

  • b The ability to transform REF52, C3H10T1/2, REF, or CREF cells was scored on the basis of the appearance of dense foci. Symbols: +, >50% of the foci induced by wild-type T antigen; +/−, ≤50% of the foci induced by wild-type T antigen; −, <3% of the foci induced by wild-type T antigen (175, 178, 219, 223, 224, 236).

  • c Anchorage-independent growth in soft agar or agarose (158, 219, 229).

  • d The ability to convey a growth advantage to primary MEFs in 10% or low serum was scored. The increased number of cells induced by wild-type T antigen was scored as +, mutants that induced an advantage but less than the wild type until late in the experiment (day 10) were scored as +/−, and mutants that induced little advantage throughout the entire experiment were scored as − (88, 229).

  • e Induction of tumors in mice when expression was driven by the lymphotropic polyomavirus promoter was scored. Wild-type T antigen induced choroid plexus papillomas which induced death early in the mice due to hydroencephalopy. Some mice survived and later developed lymphomas (36, 237, 238).

  • f Induction of hepatocarcinomas in mice when expression is driven by the antithrombin III promoter (7).

  • g The ability to bind to pRB family members as determined through coimmunoprecipitation assays (56, 87, 88, 210, 223, 229, 230, 268).

  • h Alleviation of a G1 arrest mediated by transfection with pRB, p107, and p130 in Saos-2 (RB−/−) cells (269).

  • i The ability to alter the phosphorylation state of p107 and p130 correlates with a reduction in the overall p130 steady-state levels (24, 87, 229, 230).

  • j Disruption of p130/E2F complexes as assayed by gel shift analysis of cell culture lysate or immunoprecipitation release assays in vitro (233, 236, 269).

  • k Transactivation of reporter plasmids containing E2F sites (88, 134, 209, 269).

  • l Bromodeoxyuridine incorporation (209; M. Saenz-Robles and J. M. Pipas, unpublished observation).

  • m Ability to enhance the transcriptional activity of the POU protein transcription factor Tst1/Oct6/SCIP. Symbols: −, <30% of the activity of wild type; +/−, ∼50% of the activity of wild type (218).

  • n Ability to override p53-mediated growth arrest induced by a temperature-sensitive allele of p53 in a rat embryo fibroblast cell line. Symbols: −, no activity; +/−, 20 to 50% of the activity of wild-type T antigen (74, 185).

  • o Replication of viral DNA in cell culture or in a cellular lysate in vitro system. Symbols:+/−, between approximately 20 and 60% that of the wild type: −, <5% of that of wild type (24, 111, 134, 137, 151, 175, 195; P. G. Cantalupo and J. M. Pipas, unpublished results).

  • p Ability to coimmunoprecipitate Hsc70 in the context of a cellular lysate or directly as assayed with purified components (24, 198, 209, 234, 236).

  • q Stimulation of the ATPase activity of Hsc70 measured using either steady-state or single-turnover assays. Symbols: − and +/−, <5% and 30 to 60% of wild type, respectively (223, 236).

  • r Ability to substitute as chimeric proteins for the J domain of DnaJ in a hypersensitive E. coli strain at extreme temperatures (9, 118). Note that the pRB-binding mutants and amino-terminal fragments contain a wild-type J domain. Because this is the same domain used in wild-type T-antigen chimeric DnaJ proteins, these mutants were scored as +.