The replication of cytomegalovirus (CMV), Towne strain, in permissively infected cells is characterized by a long eclipse phase and lengthy growth cycle which may reflect the activity of mechanisms which regulate viral gene expression at the transcriptional level. To correlate the study of transcription with other events occurring during CMV replication, experiments were first conducted to determine the time post-infection for the onset of virus maturation and virus DNA synthesis under defined conditions. The onset of virus maturation was between 2 and 3 days post-infection. The hybridization kinetics of labeled CMV DNA with DNA extracted at different times post-infection indicated that the onset of viral DNA synthesis was between 24 and 36 hours post-infection in human foreskin fibroblast cells permissively infected with CMV. The results of hybridizations of stable viral RNA accumulating at various times post-infection, and at early times in the absence of protein synthesis, with labeled CMV DNA, showed that there is temporal regulation of transcription. The transcription of the genome is restricted in the presence of an inhibitor of protein synthesis to 5-6% of the genome (immediate early RNA). There is a rapid switch of immediate early to the early phase of transcription, which extends to at least 24 hours post-infection, and consists of transcripts homologous to approximately 30% of the viral genome throughout the entire phase. After the onset of viral DNA synthesis, the transcription extends into the late phase during which transcripts homologous to approximately 42% of the genome are synthesized. Early and late RNA was analyzed for the presence of symmetric transcripts. Transcription was found to be asymmetric at early times post-infection, with only 5% symmetric transcription during the late phase. The extent to which immediate early, early, and late stable RNA is transcribed from the repeat regions of the CMV genome was determined by hybridizations of stable RNA to labeled XbaI restriction fragments Q and M, which comprise the long repeat regions of the CMV genome. The hybridization of the probes indicated the presence of RNA transcripts at immediate early times that were homologous to 15% of the repeat sequences. Early RNA contained transcripts homologous to 18.5% of the repeat regions, and late RNA was homologous to 34% of the long repeat sequences.

A human isolate of hepatitis A virus (HAV) strain HAS-15 was adapted to rapid growth FRhK-4 cells and a one-step growth curve was determined. Detectable virion production was absent for approximately 20 h post-infection (p.i.) and was followed by a 4-day logarithmic phase of virus production. A maximum intracellular virus titer of 10⁹ radioimmunofocus-forming units (RFU) per milliliter was achieved and remained essentially constant for a period of up to 14 days p.i. An adsorption study with HAV HAS-15 using FRhK-4 cells demonstrated greater than 99.9% of infectious virus adsorbed at 25 C in less than 20 min. Milligram amounts of purified HAV HAS-15 were obtained from persistently-infected RFhK-4 cells. The HAV polypeptides were separated by sodium dodecyl sulfate - polyacrylamide gel electrophoresis and transferred to nitocellulose for detection by an enzyme-linked immunotransfer blot (EITB) procedure. HAV nucleotide-derived amino acid sequence was subjected to computer analysis to identify potential immunogenic regions within the HAV capsid polypeptides. Synthetic peptides corresponding to selected regions of each of the larger putative capsid polypeptides were coupled to keyhole limpet hemocyanin and used to immunize rabbits. EITB reactivities of HAV specific anti-peptide sera have allowed the identification of the gene order for the larger HAV P1 gene products and the determination of the following molecular weights: HAV VP2 or 1B (MW 27,000), HAV VP3 or 1C (MW 29,000), and HAV VP1 or 1D (MW 33,000). The disposition of the HAV capsid polypeptides with respect to the virion external surface was evaluated by EITB reactivity of HAV polypeptides with specific antisera. Hyperimmune rabbit anti-157S HAV and human IgM reacted with VP1, VP2, and VP3, while IgG reacted predominantly with VP1 and VP2. Further evaluation of the HAV virion structure was attempted by examining the relative accessibility of the virion polypeptides to various labeling reagents. Reaction of intact virions with Iodogen resulted in the predominant labeling of VP1 while labeling of VP2 and 3 was barely detectable. Selective labeling of VP1 under controlled conditions, combined with the anti-HAV IgG immunologic reactivity against VP1 and VP2, suggests that these two capsid components are more exposed on the virion surface and may play an important role in the generation of neutralizing antibodies.

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