- Volume 68, Issue 8, 1987

Herpes simplex virus (HSV) types 1 and 2 were shown to replicate in a newly described human cell line (Meg) derived from the peripheral blood of a healthy volunteer. The cell line has both megakaryocyte-like and B cell-like properties. Upon infection with HSV-1 or -2, at a m.o.i. between 0·5 and 5, unlike B and T cells, the Meg cells were growth-arrested and this was accompanied by cytopathic effects and virus replication. The HSV proteins and glycoproteins B and D (gB and gD) made in the blood-derived Meg cells were compared to the corresponding proteins made in the nonblood-derived cell lines, Vero (African green monkey kidney cell) and HEp-2 (human epidermoid carcinoma cell). The maximum level of HSV protein synthesis occurred earlier in the Meg cells than in the Vero and HEp-2 cells. The electrophoretic pattern of HSV-1 and -2 proteins made in the Meg cell line was similar to the corresponding proteins made in the Vero and HEp-2 cell lines; however, some qualitative and quantitative differences were evident. There were no apparent differences detected in the migration pattern of gB made in all three cell lines while significant differences were observed with the gD species. However, upon hydrolysis with Staphylococcus aureus V-8 protease of the monoclonal antibody-purified gB and gD, distinct differences were observed in the electrophoretic pattern of the generated peptide fragments of both gB and gD made in the three cell lines. The results demonstrate that a human blood cell can support HSV replication and that species-specific posttranslational modification of gB and gD occurs in HSV-infected Vero cells as compared to HSV-infected human cells.

A hybridoma line was isolated which produced antibody reacting with polypeptides of apparent molecular weights 32000,34000 and 35000 (32K/34K/35K) and 55000 and 57 000 (55K/57K). These were sulphated glycoproteins that have previously been found in the medium of herpes simplex virus type 1 (HSV-1)-infected cells. By tryptic peptide mapping and serological cross-reactions the polypeptides were shown to be related to HSV-1 glycoprotein E (gE-1) but they lacked the Fc binding function. The 32K/34K/35K and 55K/57K polypeptides were not found in the medium of HSV-1 - infected cells incubated in serum-free medium. They could be generated in vitro from purified gE-1 in the presence of serum. It is likely that 32K/34K/35K and 55K/57K are derived from gE-1 by the action of serum proteases.

One-hundred and twenty-three epidemiologically unrelated strains of herpes simplex virus type 2 (HSV-2) isolated in Japan and Sweden (68 Japanese and 55 Swedish isolates) were compared by analysis of their genomes using five restriction endonucleases: BamHI, KpnI EcoRI, HIndIII and BglII. Seven of the 93 restriction sites examined showed statistically significant variation between isolates from the two countries. However, HSV-2 isolates were less variable than the HSV-1 isolates previously analysed from the same countries. Using 12 restriction sites as markers, the HSV-2 isolates were classified into 41 cleavage patterns; 17 were specific for Japanese isolates and 15 were specific for Swedish isolates. Correlation coefficients between some sets of 12 markers were significant, but significant correlations between Japanese and Swedish isolates were distinct for each country. Both Japanese and Swedish isolates were assigned to three major patterns with no significant difference in incidence. In contrast, in two other major patterns, differences in incidence between the isolates were statistically significant. These results suggest that HSV-2 populations in geographically separated countries have distinct cleavage site distributions.

The nucleotide sequence of bovine papillomavirus type 4 (BPV-4) was determined. The viral genome is 7261 base pairs long. Several overlapping open reading frames (ORFs) have been identified both on the basis of amino acid comparison with other papillomaviruses and on their transcriptional pattern. Eight early ORFs (El to 8) were recognized, coding for DNA replication and cell transformation functions, and three late ORFs (L1 to 3), coding for structural proteins. Like the E5 ORF of human papillomavirus type 6 the E5 ORF of BPV-4 is discontinuous. Unlike other papillomaviruses, the non-coding region upstream of the early ORFs (ncr-1) is short (385 base pairs), but there is another non-coding region (ncr-2) of nearly 500 base pairs between the L2 and L1 ORFs. Most of the putative regulatory sites are located in the ncr-1, although potential controlling elements are also found in other parts of the genome. Polyadenylation sites are present at the 3′ end of both the early and the late transcription units. Comparison between the polypeptides of BPV-4 and other papillomaviruses showed that BPV-4 is evolutionarily closer to the epitheliotropic human and rabbit viruses than to BPV-1.

The 26S mRNA and most of the nsP4 encoding regions of the eastern equine encephalomyelitis (EEE) viral genome have been cloned. Excluding the poly(A) tail, the 26S mRNA region was determined to be 4139 nucleotides long and to share the same general organization as that of other alphaviruses. A highly conserved region of 19 nucleotides, the putative transcriptase recognition site for 26S mRNA synthesis, was present at the 26S/42S junction region of the 42S genomic RNA. Translation of the 26S mRNA began at the first AUG (positions 59 to 61) initiation codon and continued with an open reading frame that coded for a polyprotein of 1258 amino acids ending at a UAA ochre termination codon (positions 3776 to 3778). All four putative posttranslational cleavage sites used to generate the capsid, E3, E2, 6K and El proteins were conserved. Transmembrane domains present in the EEE virus structural polyprotein have been identified and their functions discussed. Pairwise comparison of the deduced amino acid sequences of the polyproteins of five alphaviruses (EEE, Venezuelan equine encephalitis, Sindbis, Semliki Forest and Ross River viruses) revealed EEE virus to be more closely related to VEE virus than to the other three viruses.

Infection of mouse L-2 fibroblasts with mouse hepatitis virus (MHV) results in strong inhibition of host cell protein synthesis. Since it has been suggested in other virus systems that translational control is modulated by changes in the intracellular ionic environment, we investigated the possible occurrence of similar changes during MHV infection. Membrane permeability to extracellular sodium ions was measured by culturing MHV-infected cells in the presence of 22Na+. Sodium influx into MHV- infected cells rose dramatically from 4 to 6 h post-infection. This influx correlated chronologically with the expression of MHV-mediated cell fusion. Cell fusion was blocked by the addition of a monoclonal antibody against the MHV E2 glycoprotein. This addition also resulted in a reduction in the normal influx of 22Na+, suggesting that E2 expression was responsible, directly or indirectly, for the increased permeability to sodium ions in infected cells. Cultures of MHV-infected cells were labelled with [35S]methionine in the presence of medium supplemented with sodium chloride at final concentrations ranging from 150 mm to 350 mm. Incorporation of radiolabel into proteins decreased with increasing NaCl concentration; however, the ratio of viral to cellular protein synthesis remained relatively constant. Similarly, alteration of intracellular Na+ and K+ levels by treatment of infected cells with ouabain had little effect on the pattern of viral/cellular protein synthesis. Using monoclonal anti-E2 antibody to inhibit Na+ influx, we demonstrated normal inhibition of host cell protein synthesis. We therefore conclude that MHV-induced shut-off of host translation is not mediated by changes in intracellular Na+ concentrations.

The antigenic characteristics of eight human strains and two bovine strains, one of which is represented by two plaque variants, of parainfluenza virus type 3 were analysed. The strains and variants were compared using 52 monoclonal antibodies against five, two, six and six epitopes of the haemagglutinin-neuraminidase (HN), fusion, nucleocapsid and matrix viral proteins respectively, employing radioimmuno- precipitation and immunofluorescence assays. The human strains, seven of which were isolated over 6 years at different geographical locations and the eighth one representing an older prototype strain, showed very little antigenic variation. Extensive differences were detected in all four proteins examined between the human strains and the two strains of bovine origin. Two bovine variants were less effectively neutralized than the prototype human strain with a series of monoclonal antibodies against the HN protein.

Employing different routes of immunization and different adjuvants, a large number of hybridomas were produced that secreted monoclonal antibodies (MAbs) against the large envelope glycoprotein (GP90) and the fusion glycoprotein (GP70) of respiratory syncytial virus (RSV). The antigenic specificity of the MAbs was established by immunoblot analysis and radioimmunoprecipitation. Three hybridomas secreting anti- GP90 MAbs were established from mice immunized by intraperitoneal injection of purified RSV in Freund’s complete adjuvant. MAbs from these hybridomas did not neutralize viral infectivity. Three other hybridomas established from mice immunized by intraperitoneal inoculation of purified RSV with Bordetella pertussis produced anti- GP90 MAbs that neutralized the virus with or without complement. Similarly three other hybridomas established from mice immunized by infection with RSV via intranasal instillation of the virus produced anti-GP90 MAb that neutralized the virus in the presence and absence of complement. On the other hand, most anti-GP70 MAbs exhibited no neutralizing activity in the absence of complement, although many had low levels of neutralizing activity in the presence of complement, regardless of the type of immunization or the isotype of immunoglobulin. These observations suggest that there may be multiple antigenic sites on GP90, some of which involve a region of the molecule that functions in viral neutralization.

The envelope glycoproteins of two distinct strains of respiratory syncytial virus (RSV) (Long and 18537 strains) were purified by affinity chromatography and characterized by immunological methods. The fusion (F) proteins from the two strains were similar in molecular weight by gel electrophoresis and were very closely related immunologically. Rabbit antisera to either F protein reacted with near equivalent titres with the heterologous F protein by Western blot, enzyme-linked immunoassay (EIA), neutralization and fusion inhibition assays. In contrast to the similarity of the F proteins, the attachment proteins (G) differed significantly. The 18537 G protein had a molecular weight of 78K compared to 84K for the Long G protein. Rabbit antisera to the G proteins clearly reacted preferentially with the homologous protein, although some cross-reactivity was noted by Western blot and EIA. Anti-G serum neutralized the homologous strain of RSV in the presence or absence of complement to much higher titre than the heterologous virus strain. The implications for vaccine development are discussed.

We examined the specificity of BALB/c cytotoxic T (Tc) cells for respiratory syncytial virus (RSV) components, using recombinant vaccinia viruses (VV) coding for several individual RSV proteins. We found that immunization with the different VVs yielded the following Tc memory cell populations: high levels of RSV-specific Tc cells were induced with the fusion protein VV, but low levels were induced with VV coding for the RSV nucleoprotein. Tc cell recognition of attachment glycoprotein, part of the matrix molecule or 1A internal protein was poor. While high levels of fusion protein- specific Tc cells were induced by the fusion protein VV, they showed poor crossreactivity between the A2 and 8/60 RSV strains compared with Tc cells primed by RSV infection.

Ninety analogues of suramin have been examined for their ability to inhibit the exogenous reverse transcriptase (RT) of human immunodeficiency virus type I (HIV- I). Of these compounds, 57 inhibited the poly(rC)oligo(dG)-dependent RT activity. Three classes of dose-response curves could be discriminated. Allocation of a compound to one class did not correspond with obvious structural features. Twenty- four substances were superior to suramin in our RT inhibition assay. The RT- inhibitory activity of these compounds did not correlate with their effect against filariae or trypanosomes. Preliminary antiviral evaluation in susceptible human T cells inoculated with HIV-I demonstrated in vitro therapeutic efficacy for some compounds with lower drug-related cellular toxicity than suramin. Certain structural features relevant for the RT-inhibitory effect of these compounds were recognized. Predictions are made for the design of more effective RT inhibitors. Such compounds will help to understand the molecular mechanism of reverse transcription and might be useful in the therapy of retroviral infections.

The surface protein gp70 of an ecotropic murine retrovirus was followed during entry of [3H]glucosamine-labelled virions into SC-1 mouse fibroblasts. Upon entry, gp70 was cleaved into fragments with molecular weights 35K, 30K and 17K. The 35K and 17K fragments were also observed after trypsin or thermolysin cleavage of the virion, indicating that certain locations on the gp70 molecule are easily accessible from the outside of the virion. The conformation of gp70 on the membrane was shown to have a major effect on the cleavage. This protein is known to be important for early interactions with the cell (binding and membrane fusion). The results indicate that gp70 cleavage may be important for membrane fusion.

Monoclonal antibodies (MAbs) to mouse interferons (MuIFN) have been used to characterize the interferon-like activities spontaneously expressed in mouse peritoneal macrophages freshly explanted from normal pathogen-free mice. Injection of mice with MAbs to MuIFN-α or -β resulted in a significant increase of vesicular stomatitis virus (VSV) multiplication in peritoneal macrophages. Addition of these MAbs to freshly explanted mouse macrophages accelerated the decay of the antiviral state to VSV during the ‘ageing’ in vitro of these macrophage cultures. Furthermore, these MAbs to MuIFN-α or -β markedly inhibited the transfer of the antiviral state from freshly explanted peritoneal cells or macrophages to syngeneic macrophages ‘aged’ in vitro permissive for virus replication. These effects were not observed using a nonneutralizing antibody to MuIFN-α, nor with a MAb to MuIFN-γ. In all experiments sheep polyclonal antibodies to MuIFN-α/β were more effective than the corresponding amount of MAbs to MuIFN-α or -β. A mixture of both these MAbs was more effective than either alone. Interferons produced after stimulation of peritoneal macrophages with Newcastle disease virus (NDV) and of total peritoneal cells with lipopolysacchar- ides (LPS) have also been characterized by means of MAbs to IFNs. The results of neutralization studies with these antibodies indicated that MuIFN-β was the major component of peritoneal cell IFN (induced by both NDV and LPS) and MuIFN-α was a minor component (13 to 17 %). These data indicate that both MuIFN-α and -β, but not MuIFN-γ, are spontaneously present in/on mouse peritoneal macrophages and are produced after stimulation with NDV or LPS.

Intracerebral infection of adult immunocompetent mice with most strains of lymphocytic choriomeningitis virus (LCMV) caused a systemic infection and led to severe meningoencephalitis and death due to the induced T cell immune response. The susceptibility of congenic mice to the two plaque variants Docile and Aggressive of LCMV strain UBC was shown to be mouse strain-dependent. To investigate the possible correlation between acid-stable interferon (IFN) and natural killer (NK) cell responses and the susceptibility to the two UBC LCMV substrains, serum titres of acid- stable antiviral activity, presumably IFN-α,β and NK cell activities were determined in various mouse strains at different times after intracerebral infection. The two viral isolates induced comparable IFN-α,β serum titres and caused similar NK activities in the same mouse strain. Between different mouse strains, marked differences in the kinetics and amount of IFN production were observed, yet there was no correlation with the susceptibility to the two UBC LCMV substrains. Additionally, there was no correlation between the magnitude of the IFN-α,β serum titres and the NK activities induced in the spleen by the viral inocula. Overall, the findings suggest that levels of circulating IFN-α,β are only of minor importance for the development of LCM disease.

Natural killer (NK) cell-depleted or control mice were treated prophylactically with polyinosinic: polycytidylic acid (polyl .polyC) or purified beta interferon (IFN) and then infected with either vaccinia virus or murine cytomegalovirus. NK cell depletion alone enhanced virus titres in the spleen and peritoneal cavity. However, poly I: polyC and IFN inhibited virus replication equally well in control and NK cell-depleted mice. This suggests that prophylactic IFN treatment mediates antiviral effects independently of NK cells.

We have defined the boundaries of the a sequence from human cytomegalovirus (CMV) strain Towne, characterized internal variability and determined the position of the cleavage site used to generate genomic termini. The cleavage site is positioned a fixed distance from two stretches of sequence homology that have been observed near the ends of many herpesvirus genomes. Unlike a comparable region in CMV (AD169), the CMV (Towne) a sequence has a relatively low level of variability within the a sequence and its structure is stable through repeated virus passage.

It has been suggested that herpes simplex virus (HSV) type 1 may induce a virus-specific DNA topoisomerase activity which copurifies with virus-induced DNA polymerase. We have examined DNA topoisomerase (TOPO) I and II activities in HSV-2-infected HeLa S3 cells. Both activities were partially purified using DEAE-cellulose, phosphocellulose and double-stranded DNA cellulose column chromatography. It was found that both activities could be separated from HSV-2-specific DNA polymerase. Throughout the purification TOPO I could be immunologically detected with a monoclonal antibody developed against human TOPO I. Regardless of the source, mock- or HSV-2-infected human cells, both types of topoisomerase were equally tolerant of 200 mm-KCl. There appeared to be no apparent heterogeneity of TOPO I in HeLa S3 cells through the course of the HSV-2 infection. We conclude that host cell topoisomerases are quite stable in HSV-2-infected HeLa S3 cells and that there is no evidence that HSV-2 is capable of inducing HSV-2-specific TOPO I and TOPO II activities.