- Volume 78, Issue 3, 1997

The cervical carcinoma-derived cell line MS751 was examined for human papillomavirus (HPV) DNA and RNA. A genomic fragment containing both viral and cellular sequences was cloned. Sequence analysis showed that MS751 cells contain a partially deleted HPV-45 genome integrated at a single chromosomal site. HPV sequences from the E6-E7 region are expressed as poly(A)+ RNA.

JC virus (JCV) causes the central demyelinating disease progressive multifocal leukoencephalopathy (PML). JCV strains excreted in the urine are distinguishable from those in PML tissue by the configuration of their regulatory region to the right of ori: the archetypal regulatory region, 267 nucleotides long, is rearranged in PML tissue by deletion and duplication. Within the coding region JCV shows variations as a result of virus evolution. Four major genotypes are distinguishable of which Type 1 is based in Europe and Type 2 in Asia. Here, the regulatory region rearrangements and the viral genotypes of 29 JCV strains from PML brain were determined. Rearrangement patterns and genotypes were not associated. In general, deletions occurred before duplications, but exceptions to this rule exist. Each configuration of the 29 rearranged regulatory regions was unique and could be derived directly from the non-rearranged, archetypal form.

The high mobility group (HMG-1) box proteins bind both non-B-DNA conformations and specific nucleotide sequences. They have been implicated in a wide variety of cellular functions involving DNA, such as transcription, replication and recombination. To determine whether HMG-1 box protein T160 plays a role in virus replication, we employed an antisense strategy to inhibit its expression in NIH 3T3 cells. The two T160 clones that expressed levels of T160 50% lower than those expressed by clones transfected with the empty vector (Neo+ clones) were investigated with respect to their permissiveness to the growth of viruses representing three families: Rhabdoviridae, vesicular stomatitis virus (VSV); Picornaviridae, encephalomyocarditis virus (EMCV), and Alpha- and Betaherpesviridae, herpes simplex virus 1 (HSV-1) and mouse cytomegalovirus (MCMV), respectively. They displayed a high degree of resistance to MCMV replication, but were fully permissive to the other viruses. Competitive PCR and probing IE-1 products by Western blot analysis showed that this resistance was not due to depressed levels of virus adsorption during the early phases of infection. We therefore conclude that T160 is involved in replication of the betaherpesvirus MCMV.

The herpes simplex virus type 1 (HSV-1) capsid protein VP24 (encoded by UL26) was expressed as a GST-fusion protein and used to prepare a group of monoclonal antibodies. These were used to characterize the protein in capsids and virus infected cells and demonstrated that it exists as two polypeptide species. The nature of the relationship between these two species was investigated and found to be associated with disulphide bonding. Under non-reducing conditions a species corresponding to dimers of VP24 was identified in preparations of B capsids, the site of action of the proteinase. Biochemical subcellular fractionation studies suggested that only cleaved forms of UL26 and UL26.5 gene products could be detected in the nucleus of the infected cell at early times post-infection.

The vaccinia virus (VV) strain Western Reserve B13R gene encodes a 38.5 kDa intracellular polypeptide that is non-essential for virus replication in vitro and does not affect virus virulence in a murine intranasal model. The protein has 92% amino acid identity with the cowpox virus cytokine response modifier A (crmA) protein which inhibits the interleukin (IL)-1beta converting enzyme (ICE). Here, we show that extracts from THP-1 cells infected with VV strains expressing B13R prevent the cleavage of in vitro transcribed and translated pro-IL-1beta into mature IL-1beta. Similarly, THP-1 cells infected with VVs expressing B13R process pro-IL-1beta into mature IL-1beta inefficiently in situ. Despite its inhibition of ICE, B13R does not prevent fever in infected mice, a systemic effect mediated by IL-1beta. Instead, fever is controlled by the VV IL-1beta receptor, encoded by gene B15R, and deletion of both the B13R and B15R genes did not increase the febrile response compared to deletion of B15R alone. The B13R protein does, however, block apoptosis mediated by anti-Fas antibodies or by tumour necrosis factor (TNF) and cycloheximide. Using DNA fragmentation, chromium release and microscopic analyses it was shown that cells infected with wild-type VV strain WR, or a revertant virus in which the B13R gene had been re-inserted into the B13R deletion mutant, are more resistant than uninfected cells or deletion mutant-infected cells to apoptosis mediated by anti-Fas and TNF.

Protein yields in the baculovirus expression system do not always correlate with the presence of abundant amounts of corresponding mRNAs. Therefore, a novel aspect of the baculovirus expression system was studied: initiation of translation of very late mRNAs of Autographa californica multicapsid nucleopolyhedrovirus. The untranslated regions (UTRs) of the p10 mRNA of this baculovirus were studied by in vitro translation and after transfection into Spodoptera frugiperda insect cells. Lysates from insect cells were optimized for translation of in vitro transcripts containing p10 sequences. The lysates were used to measure the effects of various deletions in either the 5' or 3'UTR on protein synthesis. Transcripts containing the p10 5'UTR were translated efficiently. Large deletions in the 5'UTR severely decreased this efficiency. Deletions in the 3'UTR negatively affected expression of the reporter gene in vivo; however, no effect on translational efficiency in the insect-cell lysates was measured. The translational properties of the p10 transcripts were very similar in lysates made from either uninfected or baculovirus-infected insect cells. Determination of optimal salt conditions for either uncapped or capped transcripts showed that the p10 5'UTR was used very efficiently for translation initiation in vitro, even in the absence of a cap-structure at its 5' end. Addition of cap-analogue to the in vitro translation assays did not inhibit p10 5'UTR-driven translation, while translation of a cap-dependent mRNA was severely inhibited. These data suggest that the very late mRNAs of baculovirus are translated in a cap-independent manner.