- Volume 67, Issue 11, 1986

Expression of the early and late genes of herpes simplex virus type 1 (HSV-1) during infection of tissue culture cells requires the prior expression of the immediate early (IE) genes. The requirement for the product of IE gene 3, Vmw175, for the activation of early promoters has been revealed by studies with temperature-sensitive virus mutants. Recent experiments using transfection assays have shown that both Vmw175 and the product of IE gene 1, Vmw110, are involved in the transactivation of a variety of HSV-1 early promoters. This paper describes experiments which compared the activation of two early promoters [those of the glycoprotein gD and thymidine kinase (tk) genes] with that of a member of a later class of genes (the major capsid protein, VP5). Plasmids containing these promoters linked to the chloramphenicol acetyltransferase (CAT) gene were transfected into HeLa cells with plasmids containing one or more HSV-1 IE genes. Promoter activity was estimated by measurement of CAT activity in extracts of transfected cells. The gD and tk promoters were activated by both Vmw175 and Vmw110, and the combination of these two IE gene products resulted in very high levels of activation. Addition of further IE gene products did not result in any significant increase in the activation seen with the combination of Vmw175 and Vmw110. In contrast, the activation of the VP5 promoter brought about by the combination of Vmw175 and Vmw110 was relatively slight, but was increased further when plasmids containing IE gene 2, encoding Vmw63, were included in the transfection. These data suggest that Vmw63, like Vmw175 and Vmw110, is also involved in the activation of transcription from HSV-1 promoters. The effect of Vmw63 may be limited to the activation of a subset of HSV-1 genes.

At times after conjunctival inoculation with bovine herpesvirus type 1 (BHV-1), representing the acute and latent phases of infection, rabbit trigeminal ganglia were examined for the presence of BHV-1 nucleic acids by in situ hybridization using a 3H-labelled BHV-1 DNA probe. During the acute phase of virus infection, both BHV-1 DNA and RNA were detected in ganglionic neurons and occasionally in adjacent satellite cells. However, during the latent phase of infection only viral RNA was detectable in involved neurons. Viral RNA appeared restricted to the nucleus of latently infected cells and was present in varying amounts in individual cells. These results indicate that the BHV-1 genome is transcriptionally active in ganglionic neurons during latent infection.

Oligo-2′,5′-adenylate synthetase activity was investigated in several human lymphoblastoid cell lines of B cell origin treated with reagents having effects on gene expression, 5-azacytosine (5AZct), 5-azacytidine (5AZcd) and 5-iodo-2′-deoxyuridine (IUdR). Enzyme activity did not increase in cells treated with 5AZcd, but the other reagents induced significant levels of activity. Interferon (IFN) activity was not detected in culture fluids of cells treated with 5AZcd or 5AZct. On the other hand, treatment of cells with IUdR led to the production of 5 to 10 units/ml IFN by NC-37 and Raji cells, but not by other human B cell lines. Enzyme induction by IUdR occurred in IFN-producing cells, NC-37 and Raji, exposed to anti-IFN-α sera and also in IFN-non-producing human B cells. The effect of 5AZct on enzyme induction was observed only after cultivation of cells for more than 1 month. This compound could not induce enzyme activity in Epstein-Barr virus (EBV)-negative cells, BJAB, but only in the EBV-positive cell lines NC-37 and Raji. In contrast, treatment of cells with IUdR for only 3 days induced the enzyme in all human lymphoblastoid cell lines of B cell origin except P3HR-1, but not in other human cell lines, FL, HeLa and K562. Oligo-2′,5′-adenylate synthetase activity in P3HR-1 cells, which produce EBV particles, was hardly affected by IUdR or IFN treatment.

Monoclonal antibodies were prepared from mice immunized with an 18-residue synthetic peptide with an amino acid sequence from a major antigenic sequence involved in the neutralization of type 3 poliovirus. Approximately 250 hybridomas secreted antibodies that reacted with the peptide but not the virus, two antibodies reacted with the virus but not the peptide and no antibody reacted with both. Conversely 26 monoclonal antibodies prepared from mice immunized with type 3 poliovirus and known to be directed against the appropriate sequence on the virus, generally failed to react with the peptide. These results might be expected if only a small proportion of the free or coupled peptide molecules adopt molecular conformations which resemble that of the homologous antigenic site in the virus. Antibodies specific for other antigens occasionally reacted well with the synthetic peptides, indicating that antibodies may bind to peptides of inappropriate sequence. The identification of antigenic sites by the use of synthetic peptides therefore requires considerable caution.

Two glycopolypeptides with molecular weights 160000 and 120000 (gp120) are regularly recognized by human immunodeficiency virus (HIV)-specific antisera in lysates of cells persistently infected with HIV. In the present study, gp120 was characterized as the major envelope glycopolypeptide of HIV. Gp120 was identified as the external viral glycoprotein by radiosequencing and by its presence in purified virus. However gp120 was predominantly shed as a soluble protein into the culture fluid. Furthermore gp120 was precipitated by sera from horses infected with equine infectious anaemia virus (EIAV), but not by sera from uninfected animals. This may indicate conserved epitopes common to the envelopes of HIV and EIAV.

The phenomenon known as antibody-dependent enhancement (ADE) has been demonstrated in vitro but its significance in viral pathogenesis is uncertain even though it has been associated with dengue shock syndrome. Here we report for the first time the enhancement of virus virulence in mice using monoclonal antibodies (MAbs) prepared against yellow fever (YF) viruses. Our results show that the average survival time of mice was reduced by up to 33% (i.e. 6.7 to 4.5 days) and that ADE is both antibody dose-dependent and antibody- and virus strain-specific. A total of 12 YF viruses and 11 MAbs were examined and of these only three YF viruses (FNV, Asibi and B11) could be enhanced in vivo by only two MAbs (427 and 126). A particular combination of virus and antibody is required for ADE to take place.

The sequence of the major nucleocapsid protein (NP) mRNA and its encoded protein were deduced by sequencing a cDNA clone representing the complete mRNA. The cDNA sequence was confirmed by dideoxynucleotide sequencing of purified viral genomic RNA by primer extension using synthetic oligonucleotides. The NP mRNA contains 1641 nucleotides exclusive of poly(A) and encodes an NP protein of 515 amino acids. Alignment of the human parainfluenza type 3 virus (PF3) NP protein sequence with that of Sendai virus showed that the two proteins shared considerable sequence identity (58.8%). Additional comparisons provided highly significant statistical evidence that the PF3 NP protein sequence is related to those of measles and canine distemper viruses, but there was no evidence of relatedness with the nucleocapsid proteins of respiratory syncytial virus, influenza B virus, or vesicular stomatitis virus.

The replication of the autonomous parvovirus, minute virus of mice (MVM), requires mitotically active cells and depends on certain factors expressed by cells of particular differentiated phenotype. As an approach to the understanding of these helper functions, we studied the interaction of the fibrotropic [MVM(p)] and the lymphotropic [MVM(i)] strains of MVM with two differentiated cell lines from mouse testicular epithelial origins. The relative support given to viral expression by these cell lines varied extensively. Cells from Sertoli origin (TM4) were permissive to MVM(p) but were mostly restrictive to MVM(i). The other cell line, of Leydig cell origin (TM3), was highly restrictive to both viral strains, but the blocks to their growth in these cells were localized at different stages of their growth cycle, suggesting that the replication of MVM in these cells requires tissue-specific helper functions during at least two stages of viral replication.

Compared with wild-type BK virus DNA having tandem triplication of a 68 base pair (bp) element in its transcriptional control region, a mutant viral DNA with a single copy of the 68 bp element induced remarkably delayed virus production in human embryonic kidney (HEK) cells. We molecularly cloned the DNA of progeny viruses using plasmid vector pAT153. Nucleotide sequence analysis of representative clones revealed that all of the altered viral DNAs examined duplicated various segments extending over origin-distal portions of the 68 bp element and their flanking regions. After transfection of HEK cells, most of these rearranged viral DNAs induced viral growth slightly slower than, or at the same rate as, the wild-type viral DNA. Comparison of the structures of these rearranged viral DNAs suggests that reiteration of a 13 bp sequence, which is located in an origin-distal portion of the 68 bp element, is required for the efficient replication of BK virus.

The nucleotide sequence of a 1587 bp region lying within the HindIII-Q fragment of Autographa californica multiple nucleocapsid nuclear polyhedrosis virus (AcMNPV) DNA has been determined. It begins in the EcoRI-S-EcoRI-X region, continues to the HindIII-P/Q boundary and contains an open reading frame that codes for a polypeptide of 240 amino acids (p26). This open reading frame is also included in the 1100 and 1500 base transcripts previously mapped to this region. The sequence reveals that the 5′ ends of the 1100 and 1500 base transcripts are located 20 bp downstream from the end of a putative TATA box (TAATTAAAT) and 19 bp upstream from the translation start codon (ATG) of the p26 open reading frame. The translation termination codon (TAA) falls in the immediate 5′ flanking region of the major late p10 gene of AcMNPV, 3 bp downstream from the putative TATA box. The probable polyadenylation site for the 1100 base transcript lies 23 bp downstream from the cap site for the 750 and 2500 base transcripts encoding the p10 protein. The 5′ flanking region of the p26 open reading frame contains the EcoRI site-rich region, hr5, whose sequence is included here. The EcoRI site-rich region, hr5, consists of six imperfect tandem repeats of a sequence that includes the EcoRI recognition site. These direct repeats also include many inverted repeats.

The ability of the genome-linked protein (VPg) of cowpea mosaic virus (CPMV) to survive incubation in rabbit reticulocyte lysates was investigated. In contrast to the results obtained with picornavirus RNAs, there was no evidence for the specific removal (‘unlinking’) of the VPg from CPMV RNA during incubation. While linked to RNA, CPMV VPg was protected from proteolytic degradation; if the RNA was first digested with nuclease P1, rapid degradation of the VPg occurred. However if as few as 17 nucleotides were left attached to the VPg, stability was retained.

Assembly of the coat protein subunits of tobacco mosaic virus (TMV) into 20S aggregates (‘disks’) and long helices was monitored by light scattering and electron microscopy. The anti-microtubule agent methyl benzimidazol-2-yl carbamate, which inhibits TMV multiplication in vivo, did not affect assembly of coat protein in vitro. In contrast, the anti-microtubule agents colchicine and vinblastine inhibited disk formation, but stimulated rod elongation from coat protein subunits in vitro. Both agents also disrupted preformed disks. Vinblastine inhibited virus multiplication in leaf tissue, but colchicine did not.