The adenovirus L4 100-kilodalton protein is necessary for efficient translation of viral late mRNA species.
When screening a number of adenovirus type 5 (Ad5) temperature-sensitive mutants for defects in viral gene expression, we observed that H5ts1-infected 293 cells accumulated reduced levels of newly synthesized viral late proteins. Pulse-labeling and pulse-chase experiments were used to establish that the late proteins synthesized in H5ts1-infected cells under nonpermissive conditions were as stable as those made in Ad5-infected cells. H5ts1-infected cells contained normal levels of viral late mRNAs. Because these observations implied that translation of viral mRNA species was defective in mutant virus-infected cells, the association of viral late mRNAs with polyribosomes was examined during the late phase of infection at a nonpermissive temperature. In Ad5-infected cells, the majority of the viral L2, L3, L4, pIX, and IVa2 late mRNA species were polyribosome bound. By contrast, these same mRNA species were recovered from H5ts1-infected cells in fractions nearer the top of polyribosome gradients, suggesting that initiation of translation was impaired. During the late phase of infection, neither the polyribosome association nor the translation of most viral early mRNA species was affected by the H5ts1 mutation. This lesion, mapped by marker rescue to the L4 100-kilodalton (kDa) nonstructural protein, has been identified as a single base pair substitution that replaces Ser-466 of the Ad5 100-kDa protein with Pro. A set of temperature-independent revertants of H5ts1 was isolated and characterized. Either true reversion of the H5ts1 mutation or second-site mutation of Pro-466 of the H5ts1 100-kDa protein to Thre, Leu, or His restored both temperature-independent growth and the efficient synthesis of viral late proteins. We therefore conclude that the Ad5 L4 100-kDa protein is necessary for efficient initiation of translation of viral late mRNA species during the late phase of infection.
Hayes
B W
BW
Department of Biology, Princeton University, Princeton, New Jersey 08544-1014.
Telling
G C
GC
Myat
M M
MM
Williams
J F
JF
Flint
S J
SJ
D000260:Adenoviruses, Human / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002460:Cell Line; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D007624:KB Cells; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D010766:Phosphorylation; D011132:Polyribosomes / Q000378:metabolism; D011485:Protein Binding; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics* / Q000302:isolation & purification; D012367:RNA, Viral / Q000235:genetics* / Q000302:isolation & purification; D015183:Restriction Mapping; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism
D004279:DNA, Viral; D015852:Eukaryotic Initiation Factor-2; D012333:RNA, Messenger; D012367:RNA, Viral; D014764:Viral Proteins