Restriction of vaccinia virus replication in CHO cells occurs at the stage of viral intermediate protein synthesis.
Vaccina virus (VV) and cowpox virus (CPV) differ in their abilities to replicate in Chinese hamster ovary (CHO) cells because VV has a disrupted host range (hr) gene. To facilitate an examination of the molecular events associated with abortive infection of CHO cells with VV, we constructed two sets of recombinant viruses that contain a viral early promoter regulating the cat gene encoding chloramphenicol acetyltransferase and viral intermediate or late promoters regulating the lacZ gene encoding beta-galactosidase. The first set has the disrupted hr gene and the second set has the intact CPV homolog, allowing replication in CHO cells. Reporter chloramphenicol acetyltransferase and beta-galactosidase assays demonstrated that early gene expression was unperturbed, whereas intermediate and late gene expression were severely inhibited under abortive conditions. Metabolic labeling studies confirmed the absence of viral late protein synthesis. The accumulation of viral DNA under abortive conditions was consistent with the synthesis of viral early proteins and established that inhibition of late protein synthesis was not primarily due to a replicative block. Analysis of steady state levels of viral mRNAs revealed substantial quantities of early and intermediate species but only very small amounts of late mRNAs under nonpermissive conditions. Despite the presence of viral intermediate mRNAs, the corresponding intermediate proteins, which function as late transcription factors, were not detected by immunoprecipitation of lysates from metabolically labeled infected CHO cells. Furthermore, when expression of lacZ was regulated by an intermediate promoter, no beta-galactosidase was detected even though lacZ transcripts were present. Thus, the abortive phenotype in CHO cells can be explained by a block to translation of intermediate mRNAs which prevents the synthesis of late transcription factors.
Ramsey-Ewing
A
A
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Moss
B
B
D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D002522:Chlorocebus aethiops; D015608:Cowpox virus / Q000502:physiology; D006224:Cricetinae; D004261:DNA Replication; D005814:Genes, Viral; D007700:Kinetics; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000096:biosynthesis; D012367:RNA, Viral / Q000096:biosynthesis*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D015183:Restriction Mapping; D013997:Time Factors; D014162:Transfection; D014616:Vaccinia virus / Q000235:genetics / Q000502:physiology*; D014764:Viral Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D014779:Virus Replication; D001616:beta-Galactosidase / Q000096:biosynthesis
D012333:RNA, Messenger; D012367:RNA, Viral; D011994:Recombinant Proteins; D014764:Viral Proteins; D015500:Chloramphenicol O-Acetyltransferase; D001616:beta-Galactosidase