- Volume 66, Issue 5, 1985

Apparent interferon-mediated persistent infection of rabies virus (HEP-Flury strain) was established in a human neuroblastoma SYM-I (clone K-104) cell line, which had the ability to produce interferon. This infection produced variable but small amounts of progeny virus and interferon (up to 100 IU/ml), and resisted superinfection with vesicular stomatitis virus (VSV) and Sindbis virus as well as homologous rabies virus. The treatment of this infection with anti-interferon antibody stimulated virus replication and extensive c.p.e. However, some cells survived and grew rapidly without any sign of c.p.e. These produced increased amounts (100 to 1000 times) of infectious and DI particles in the presence of anti-interferon antibody, becoming susceptible to superinfection with VSV but remaining resistant to the original rabies virus. Small plaque mutants appeared and replaced the original virus during the long-term cultivation of the persistent infection. Several mutants tested were all identified as Sdi (DI-resistant) mutants, suggesting that the persisting viruses were endowed by the Sdi mutation with a selective advantage over the original virus even in interferon-mediated persistent infections.

Rinderpest virus (RV) grew readily in cultures of purified bovine peripheral blood lymphocytes and udder macrophages. The growth of three strains of RV was compared and there appeared to be a relationship between increasing virulence and increased ability to infect lymphocytes and macrophages. The proportion of infected cells as determined by the presence of virus antigens was a better indicator of affinity between a strain and cell type than production of new infectious virus. RV grew better in populations of predominantly T lymphocytes than in T-depleted cultures. Although RV could infect 100% of cells in macrophage monolayers, it did not appear to infect more than about 30% of cells in lymphocyte cultures. Virulent RV grew more readily in bovine than caprine or ovine lymphocytes, whereas virulent peste des petits ruminants virus (PPRV) grew better in lymphocytes from sheep and goats. There was no marked difference in the growth of either virus in lymphocytes from uninfected or recently convalescent animals.

cDNA clones of the mumps virus N and P messenger RNAs were isolated from an infected cell cDNA library. The N and P clones selected the two predominant polyadenylated RNAs found in mumps virus-infected cells with mol. wt. of 0.69 × 106 and 0.51 × 106, respectively. In addition, clones of the P gene hybridized to and selected mRNAs of higher mol. wt. probably representing polycistronic transcripts of the mumps genome. Hybrid-select translation experiments confirmed the specificity of the clones as representing the nucleocapsid (N) and nucleocapsid-associated protein (P) genes.

The fate of the haemagglutinin-neuraminidase glycoprotein (HN) of Sendai virus in three types of infection was studied by measuring its sensitivity to endoglycosidase H and its rate of appearance and turnover at the cell surface. HN behaved differently in the three types of infection. When highly expressed at the surface, as in a lytic standard virus infection, HN accumulated at the surface in a stable form (half-life of disappearance from the surface ⪢ 10 h). When moderately expressed, as in a non-lytic standard virus plus defective interfering virus infection, HN reached the membrane normally, but turned over rapidly (half-life about 2 h) and was re-internalized. When poorly expressed, as in long-term persistent infection, HN did not reach the cell surface and appeared to be degraded before reaching it. In contrast to HN, the other viral glycoprotein, F0, exhibited a similar turnover rate at the cell surface in the three situations. However, when compared to surface expression in standard virus-infected cells under standardized conditions, F0 surface expression in persistently infected cells was reduced. This reduction correlates with a decreased maturation rate in these cells.

The proteins and RNAs of St. Abb’s Head virus have been analysed, and were found to be characteristic of the Uukuvirus genus of the family Bunyaviridae. Two glycoproteins, G1 and G2 (mol. wt. 62K and 75K), and a nucleocapsid protein, N (mol. wt. 25K), were detected in infected cells by immunoprecipitation; the synthesis of N preceded the synthesis of G1 and G2. The glycoproteins were relatively cysteine-rich compared to the N protein, and the unglycosylated forms of G1 and G2 (using the inhibitor tunicamycin) had a mol. wt. of about 58K. Translation in vitro of mRNA from infected cells gave two immunoprecipitable products which are thought to be equivalent to N and G1/G2. Three RNA species were found in St. Abb’s Head virus nucleocapsids, and were estimated to be 8500 bases (L), 3600 bases (M) and 1900 bases (S) in length. At least one additional virus-specific RNA species was detected in infected cells. The similarity between the proteins and RNAs of St. Abb’s Head virus and Uukuniemi virus (the prototype of the genus) is discussed.

The roles of the L and NS polypeptides in transcription by vesicular stomatitis virus New Jersey were studied using a mutant, tsE1, which contains a temperature-sensitive transcriptase and an altered NS polypeptide, both phenotypic changes being the consequence of the ts mutation. Mutant tsE1, its revertant (tsE1/R1) and the wild-type virus were dissociated into sub-viral fractions and, after reconstitution of these fractions in all combinations, the transcriptase was assayed in vitro at the permissive (31 °C) and restrictive (39 °C) temperatures. Reconstitution of the pellet fractions (containing polypeptide N complexed with the virion RNA) and the supernatant fractions (containing polypeptides L and NS) restored transcriptase activity at 31 °C in all combinations, but at 39 °C transcription was observed only in the presence of the supernatant fractions of wild-type and revertant viruses but not in the presence of the supernatant fractions of tsE1. When the pellet fractions and the L fractions were reconstituted, the transcriptase activity was restored in all combinations both at 31 °C and 39 °C. However, in vitro transcription at 39 °C by reconstituted pellet and L fractions was strongly inhibited when the NS fraction of tsE1 was also added, while addition of the NS fractions of wild-type and revertant viruses had no effect. Since only traces of polypeptide NS were present in the L fractions and none in the pellet fractions, the results strongly suggest that polypeptide L is the transcriptase itself while polypeptide NS exerts some control over transcription.

The structural proteins L and NS of vesicular stomatitis virus were obtained from purified viral ribonucleoprotein complex followed by phosphocellulose column chromatography and assayed for protein kinase activity using [γ-32P]ATP as the phosphate donor. The fractions containing purified L protein phosphorylated NS protein in vitro. 8-Azido-ATP, a photoreactive analogue of ATP, was also used as the phosphate donor for phosphorylation of NS protein by the L protein. In the presence of ultraviolet light, only L protein was specifically cross-linked with 8-azido-[γ-32P]ATP. In the absence of u.v. light 8-azido ATP did no inhibit RNA transcription in a reconstituted reaction or substitute ATP for RNA synthesis in vitro. The above results, taken together, suggest that 8-azido-ATP was bound to the kinase site and phosphorylation of NS protein was mediated by th L protein. Exogenous phosphate acceptor proteins such as phosvitin and casein were also phosphorylated by the L protein fraction. However, addition of an excess of phosvitin failed to compete with the phosphorylation of NS by L, indicating that the protein kinase activity possessed higher affinity for NS. The phosphorylation of NS was strongly inhibited by photoreaction of L protein with 8-azido-ATP with concomitant inhibition of transcription in vitro. These results suggest that phosphorylation of NS protein by L may have a role in the regulation of the virus genome transcription in vitro.

Antisera raised against isolated structural polypeptides VP1, VP2 and VP3 of poliovirus type 1, strain Mahoney, reveal a differential reaction against mature virus and its precursor particles. During virus morphogenesis antigenic sites recognized by VP1 and VP2 antisera are lost stepwise from the surface of precursor particles. These sites are cross-reacting between serotypes and are also lost from precursor particles of type 2 (MEF-1) and type 3 (Saukett). They are absent on the surface of mature virus of all three serotypes. In contrast, the VP3 antiserum recognizes sites expressed maximally on the surface of infectious virus of type 1 (Mahoney). This antiserum did not show significant intertypic cross-reactions with virus particles, empty capsids or 14S particles of poliovirus types 2 and 3. However, it does recognize intertypic crossreacting sites, like the VP1 and VP2 antisera, on denatured polypeptides and 5S particles of each serotype.

When present during the whole infective cycle, the lysosomotropic drug, chloroquine, inhibited cytopathic changes and production of African swine fever virus (ASFV) in Vero cells. This inhibition decreased when the drug was added from 1 h to 4 h after infection. Chloroquine had no direct effect on the virus nor on viral adsorption and internalization. Electron microscopy showed that, in the presence of the drug, the virions were retained in large vacuoles having a lysosomal appearance. This inhibition was fully reversible, even when the drug was removed as late as 72 h after infection. The results support the hypothesis that ASFV enters the cells by adsorptive endocytosis and not by fusion with the plasma membrane.

The morphologically normal rat fibroblast cell line Rat-2 was used as a target cell type to test the transforming ability of a human papillomavirus (HPV-1a). To this end, molecularly cloned HPV-1a genomes were introduced into cultured Rat-2 cells in cotransfection experiments using a cloned herpes simplex virus thymidine kinase gene as a selectable phenotypic marker. In each of 13 HPV-1a-positive cell clones examined the papillomavirus DNA sequences were associated with the high molecular weight fraction of the cellular DNA, and restriction endonuclease plus Southern blotting analyses revealed patterns of hybridization which were consistent with integration of the viral genomes. Even Rat-2 clones containing multiple copies of the entire HPV-1a genome retained the normal, i.e. flat, cell morphology and were unable to grow in soft agar. De novo methylation of the HPV-1a sequences in many Rat-2 cell clones was evidenced by resistance of the viral DNA to complete cleavage with the HpaII endonuclease. Two out of three cell lines harbouring multiple copies of the HPV-1a genome contained detectable levels of HPV-1a transcripts, whereas no transcripts were detected in the third such cell line in which the viral HpaII sites were methylated virtually to completion. These results are consistent with the notion that HPV-1a genes are expressed inefficiently in Rat-2 cells; consequently integration of the viral DNA occurs, and there is no effect of the virus on the growth properties of this cell type. It is possible that methylation of the HPV-1a sequences is responsible for the low levels of expression of the viral genome.

Monoclonal antibodies which react with herpes simplex virus type 1 (HSV-1) glycoproteins, but do not neutralize infectivity, were prepared. The protein to which each monoclonal antibody was directed was determined by various techniques including their reaction with polypeptides from glycoprotein-deficient mutants after protein blotting, and also tests using passive haemagglutination. Monoclonal antibodies A7 and αC3 were directed against a type-specific determinant on gC and a type-common determinant on gB respectively. In addition, A7 reacted only with the HFEM strain of HSV-1 and did not react with any of the 20 low-passage human isolates also tested. The monoclonal antibodies were used in immunoadsorption chromatography to purify individual glycoproteins from detergent extracts of HSV-1-infected cells. The ability of the monoclonal antibodies or purified glycoproteins to protect mice against a lethal encephalitis induced by intracerebral inoculation of HSV-1 was investigated. Passive immunization was not very effective; however, purified gC or a mixture of gB and its precursor pgB induced good levels of neutralizing antibody which persisted for at least 9 weeks and mice survived virus challenge.

Nude mice have been shown to be as resistant to intraperitoneal infection with herpes simplex virus type 1 (HSV) as their heterozygous littermates. Here we document that both activation of natural killer (NK) cells and interferon induction were normal in nu/nu mice after injection of HSV. Injection of silica caused increased mortality by HSV in C57BL/6 mice. Silica, in addition, led to a significant reduction of NK cell activity but had no effect on the interferon response. Treatment of C57BL/6 mice with anti-asialo GM1 (an antiserum with a predominant effect on NK cells) caused complete abolition of the NK cell response, but had no effect on interferon induction or virus-induced mortality. In further studies a monoclonal anti-thy-1.2 antibody was utilized which possessed high activity in vivo in depleting T cell responses in mice. Injection of anti-thy-1.2 decreased NK cell activation but was without effect on the interferon response. Unexpectedly, in view of the data in nu/nu mice, this antibody increased HSV-induced mortality in C57BL/6 mice. Similar data were obtained when anti-thy-1.2 was injected into nu/nu mice. Our results are compatible with the hypothesis that T cell precursors sensitive to anti-thy-1.2 present in homozygous nude mice play a role in resistance against HSV. Furthermore, the data in the euthymic mice may indicate a role of T cells in the primary resistance of mice against HSV.

In BHK-21 cells infected with herpes simplex virus type 1 many virus-induced proteins were found attached to the nuclear matrix. To understand the role of this cell fraction during virogenesis, matrix-associated proteins were analysed at different stages of infection. All the immediate-early protein species were bound to the nuclear matrix and their association with this structure was stable. During the first few hours of infection, the pattern of virus-induced proteins attached to the nuclear matrix remained identical, indicating that polypeptides from the early group are not associated with this cell fraction. Among the late proteins, which are generally structural proteins, 60% of the nuclear proteins were tightly bound to the nuclear matrix. This suggests that the nuclear matrix is involved in at least two different events during virogenesis, regulation of viral infection and assembly of viral capsids.

Comparison of the induction of plasma interferon (IFN) by murine cytomegalovirus (MCMV) in BALB/c and CBA mice demonstrated similar kinetics of induction with maximum titres at 6 and 48 h after infection. Although neutralized by antibody to type I (α and β) IFN, the IFN was predominantly acid-labile. The 6 h titre was markedly dependent upon the dose and method of preparation of MCMV and also the strain of mouse used. Higher plasma IFN titres were found in CBA than in BALB/c mice following both intraperitoneal or intravenous infection but this strain variation was not apparent in IFN levels in the spleen and liver. IFN induced by MCMV in CBA and BALB/c mice was equally effective at inhibiting MCMV replication in vitro. Cultured embryo fibroblasts derived from CBA and BALB/c mice produced similar levels of IFN, but it was not detected until 24 h after infection. Use of irradiation chimeras prepared from H-2-compatible high IFN producers (CBA) and low producers (B10.BR) showed that the level of IFN produced at 6 h was dependent upon bone marrow-derived cells.

The protein immunoblot technique was used to identify Epstein-Barr virus-specific antigens present in sodium butyrate-induced P3HR-1 cells. Using sera from patients with either nasopharyngeal carcinoma or arthritis, 16 polypeptides were detected ranging in molecular weight from 22K to 140K. Each of the anti-EA-, anti-VCA-positive sera were found to contain antibodies to different subsets of the antigens. A 72K protein was identified which was consistent with the nuclear antigen (EBNA), and culturing cells in the presence of disodium phosphonoacetate allowed identification of 140K and 22K antigens as late viral products. Treatment of cells with sodium butyrate revealed that expression of some antigens increased in parallel with the time of incubation of the cells in butyrate while other antigens either appeared early and then decreased in intensity or were only present after a number of days of butyrate treatment. One of the antigens which decreased with the time cells were treated with butyrate was EBNA.

The mechanism of resistance of murine macrophages (Mϕ) to infection by herpes simplex virus type 1 (HSV-1) was examined. Infection of bone marrow-derived Mϕ (BMDMϕ) and resident peritoneal Mϕ (Res-Mϕ) was compared with infection of permissive Vero cells. In contrast to HSV-1 infection in Vero cells, no infectious virus was produced from either Mϕ cell type. However, marked cytopathic effect (c.p.e.) was evident in BMDMϕ at 48 h post-infection, while there was no c.p.e. at any time post-infection in the Res-Mϕ. Cloned EcoRI subgenomic fragments representing the entire HSV-1 genome were used as probes in DNA:DNA hybridization experiments to determine the viral genome content in the infected cell types. In Res-Mϕ, HSV-1 DNA was present at early times post-infection but declined rapidly. In BMDMϕ, the virus genome was always detected and increased with time after infection. The results suggest that Res-Mϕ restrict HSV-1 production at a point prior to viral DNA synthesis, whereas the block in HSV production in BMDMϕ occurs at a later stage in the viral replicative cycle.

The steps from the precursor to the processed forms of a secreted glycoprotein (gA) of Marek′s disease virus (MDV) and of herpesvirus of turkeys (HVT) were examined by two-dimensional gel electrophoresis of immunoprecipitates from the cell lysate and medium of infected cultures with monoclonal antibodies. Differences between MDV-and HVT-gA were particularly observed in isoelectric points of the glycosylated or unglycosylated precursor forms.

Precipitin lines formed between serum from a goat infected with caprine arthritis-encephalitis virus (CAEV) and radiolabelled viral proteins in polyethylene glycolconcentrated culture medium were excised from immunodiffusion (ID) plates and analysed by polyacrylamide gel electrophoresis. The two major precipitin lines contained the 135000 mol. wt. glycoprotein (gp135) and the internal 28000 mol. wt. structural protein (p28). This method obviates the use of purified proteins or monospecific antisera to positively determine viral constituents in ID precipitin lines formed between a crude antigen preparation and antiserum against whole virus.