- Volume 68, Issue 11, 1987

Hybridomas secreting monoclonal antibodies to simian virus 5 (SV5) were obtained following immunization of mice with purified preparations of a human isolate (LN) of SV5. Immune precipitation studies showed that these monoclonal antibodies had specificities for the haemagglutinin-neuraminidase (HN), fusion (F), nucleo-, matrix and phospho- (P) proteins of SV5. By use of a radioimmune competition assay the monoclonal antibodies to the HN protein were assigned to four groups, members of which recognized different antigenic sites on the protein. All the anti-HN antibodies and the anti-F antibody neutralized virus infectivity. The 54 monoclonal antibodies obtained were used to determine whether there were antigenic differences between five human, two canine and one simian isolate of SV5. Although most of the monoclonal antibodies reacted with all isolates, a few did reveal antigenic differences in the HN, F and P proteins. Furthermore, analysis by SDS-PAGE showed that while the electrophoretic mobilities of most of the virus polypeptides of these isolates were similar some differences could be detected. In particular the P protein showed the most marked mobility differences between the human, canine and simian isolates. Slight differences in the mobility of the F1 glycoprotein could also be visualized.

Western blotting and immunoperoxidase staining with monoclonal antibodies were employed to analyse epitopic and polypepide molecular weight variation among isolates of respiratory syncytial (RS) virus collected in Newcastle between 1965 and 1986. One group of isolates resembled the A2 and Long prototype subgroup A strains of RS virus in possessing a P protein of M r 34000. Isolates in this subgroup showed two patterns of reactivity with subgroup A-specific monoclonal antibodies to the G glycoprotein and 22K protein. Isolates with both reactivity patterns were isolated throughout the period studied. Isolates in the second group resembled the 8/60 subgroup B prototype strain in their lack of reactivity to subgroup A-specific monoclonal antibodies but were heterogeneous in P protein molecular weight. The earliest isolate only, made in 1965, possessed a P protein of Mr 31000 resembling the prototype strain. All subsequent subgroup B isolates possessed a higher M r, 33000, P protein. Overall, subgroup A viruses were isolated most frequently although subgroup B strains may have predominated in some epidemics.

The nucleotide sequences of the adjacent genes coding for the F protein and 22K protein of the RSS-2 strain of human respiratory syncytial (RS) virus were determined from cDNA clones of genomic RNA. Comparison of these sequences and the inferred amino acid sequences of the F and 22K proteins with the corresponding published sequences of another subgroup A virus, the A2 strain of RS virus, reveals extensive overall homology (>95%) at both the nucleotide and amino acid levels, even though these two viruses were isolated 15 years apart in different continents. The intergenic region and the hydrophobic amino-terminal signal sequence of the F proteins of the two viruses, however, are much less conserved.

DNA from human parvovirus B19 was prepared from infected serum and examined by electron microscopy. Double-stranded molecules were seen, often with characteristic ‘fold-back’ ends that were assumed to be due to the inverted terminal repeats of the genome DNA. This double-stranded DNA was mapped with 13 restriction enzymes. More than 40 isolates, including the virus from the original B19 serum, were compared. Although isolates could be grouped by this method, there was no correlation between a particular restriction endonuclease map and any of the several disease presentations of the virus.

Five of five human teratocarcinomas cultured in vitro could be induced to synthesize retrovirus-like particles, albeit in extremely low amounts. The accumulation and purification of the human teratocarcinoma-derived retrovirus (HTDV) from one of these cell lines allowed characterization of its genomic material as high mol. wt. (60S) RNA. Partial purification of HTDV-associated RNA-dependent DNA polymerase (reverse transcriptase) has also been achieved. HTDVs are easily distinguishable from the exogenous human T-lymphotropic viruses and human immunodeficiency viruses by morphological, biological and immunological criteria.

The experiments described in the present work were designed to study the function of the N-terminal end of thymidine kinase (TK) encoded by herpes simplex virus type 1. Specifically we were interested to know whether this end was involved in binding of the enzyme to other molecules, had any influence on its subcellular localization or affected one or more of the activities associated with the enzyme. A parental enzyme and a deletion mutant, lacking the 45 N-terminal amino acids, derived from this strain, were used. Thymidine kinase from the parental virus bound to DNA-Sepharose, but the truncated enzyme did not. This was apparently not due to a specific ability to bind to DNA, since immunofluorescence studies indicated that both the normal and the deleted TK were mainly located in the cytoplasm, preferentially in the perinuclear region. Phosphorylation of thymidine as well as the amounts of TK polypeptides were markedly reduced at late times after infection with the mutant, but not to the same extent after infection with the wild-type. The deleted TK gene was efficiently transcribed as shown by hybridization of RNA to a probe specific for the gene, and this RNA directed the synthesis in vitro of TK polypeptides. Deletion of the 5′ end of the gene seems to affect the stability of either the enzyme or TK-specific mRNA, or both.

The TMP phosphorylating activity seems to be particularly destabilized relative to the thymidine phosphorylating activity.

We studied the effect of an analogue of polyinosinic acid: polycytidylic acid, the mismatched poly(rI).poly(rC12U), on herpes simplex virus type 2 (HSV-2)-induced cutaneous disease in the guinea-pig. Recurrence patterns and HSV-2-induced immune responses were also defined. Intranasal administration (l·5 μg/g body weight, five doses at 48 h intervals) of poly(rl). poly(rC 12U) during initial HSV-2 infection caused a significant (P < 0·05) reduction in virus titres in the skin and decreased (P < 0·01) the duration and severity of the primary cutaneous lesions. The incidence and frequency of subsequent recurrent episodes were also significantly (P < 0·01) reduced. Titres of serum neutralizing antibody were identical in treated and untreated animals. Interferon (IFN) activity was detectable in the sera from poly(rl). poly(rC12U)-treated animals. Peripheral blood mononuclear (PBL) and spleen cells from treated animals had enhanced cytotoxic activity for HSV-2-infected and uninfected target cells. The cytotoxic activity of the PBL was enhanced by treatment in vitro with poly(rl). poly- (rC12U) or IFN.

Rat-9G cells carry several stably integrated copies of the major immediate early (IE) transcription unit of the human cytomegalovirus (HCMV). In these cells IE expression is repressed but inducible. In this report we describe the DNA methylation status of HpaII, HhaI and AhaII sites within the IE gene, determined at different passage levels. Most, if not all, of the resident IE genes were progressively methylated in a similar fashion. This resulted in DNA methylation patterns in which sites surrounding the IE upstream region were preferentially methylated to a high degree. In contrast, sites within the 19 bp IE enhancer elements were markedly under-methylated. This particular DNA methylation pattern probably resulted from differences in DNA methylation rates, sites within the IE enhancer being methylated at only a very low rate. Methylation of the IE genes did not affect their inducibility, which might be related to the very low methylation level of the IE enhancer.

All Epstein-Barr virus (EBV) isolates can be classified as type A or type B depending upon the identity of their EBV nuclear antigen (EBNA) 2 protein. The great majority of isolates examined to date encode an EBNA 2A protein like that of the reference type A strain B95-8. Type B virus strains, encoding an antigenically distinct EBNA 2B protein, have as yet only been rescued from rare Burkitt’s lymphoma (BL) cell lines of African origin (Jijoye, AG876). Our recent finding that type B isolates are less efficient than type A in in vitro transformation assays prompted us to determine (i) the relative contribution the two types of virus make to the incidence of BL in endemic areas of Africa (Kenya) and New Guinea and (ii) the relative incidence of infection with these two types in the normal population in these same areas. On the first point, EBNA 2 gene typing using specific DNA probes showed that four of ten recently established Kenyan BL cell lines and two of four BL cell lines from New Guinea carried type B virus isolates. To address the second point, spontaneous lymphoblastoid cell lines were established from the blood of normal virus carriers and typed for EBNA 2 at the protein level; a significant proportion (> 20 %) of the normal population in both the above BL- endemic areas were infected with type B isolates. This is the first indication of the widespread nature of type B virus infection in any community and the first isolation of such viruses from a non-BL source. The reproducible size of the EBNA 2B protein encoded by all type B isolates irrespective of their geographical origin, and of the EBNA 1 protein encoded by all type B isolates from one area, contrasted markedly with the extreme variability in the size both of EBNA 2A and of EBNA 1 seen generally among type A isolates. This suggests that the number of type B virus strains in existence worldwide could be quite limited. Most importantly, the data suggest that type B viruses, despite their relatively poor performance in in vitro transformation assays, can contribute at least as efficiently as can type A viruses to the pathogenesis of BL.

The Quebec isolate of bovine coronavirus (BCV) was found to contain four unique major structural proteins. These proteins consisted of the peplomeric protein (gp190/E2, gp100/E2), the nucleocapsid protein (p53/N) and its apparent trimer (p160/N), a family of small matrix glycoproteins (gp26/El, gp25/El and p23/El) and the putative haemagglutinin (gpl24/E3). Pulse-chase experiments utilizing polyclonal antiserum and monoclonal antibodies indicated that the unique BCV E3 protein had as its primary precursor an A-linked glycoprotein with an M r of 59000 (gp59) which underwent rapid dimerization by disulphide bond formation to yield gp118. Further glycosylation of gp118 produced gp124/E3 which incorporated fucose. Thus gp124/E3 was probably a homodimer. The processing of the E2 and E1 proteins of BCV was similar to that shown previously for mouse hepatitis virus. A large AM inked precursor glycoprotein, gpl70, underwent further glycosylation to yield gp190/E2 before subsequent proteolytic cleavage to yield gp100/E2. The glycosylated El (gp26, gp25) proteins arose as a result of O-linked glycosylation of p23/El as indicated by the resistance of these species to tunicamycin.

The replication of influenza virus RNA was monitored by RNA-RNA hybridization with subsequent analysis of hybrid duplexes, as well as by immunosorbtion of viral nucleocapsids from extracts of [3H]uridine-labelled cells followed by the isolation and characterization of nucleocapsid-associated RNA. The nucleocapsid-associated RNA preparations contained mostly negative-strand genomic RNA. Electrophoresis of the hybrid RNA duplexes or single-stranded nucleocapsid-associated RNA in polyacrylamide gel revealed an ‘early’ replication pattern, with a predominance of the NP and NS gene segments, in cells labelled from 0 to 1 h post-infection. At later stages of infection the pattern changed to the ‘late’ one, with the M gene segment in excess of NS, and the NP gene no longer predominant. Cycloheximide added as late as 2 or 3 h post-infection suppressed RNA replication. Moderate concentrations of cycloheximide inhibited the replication of NS and NP gene segments to a lesser degree than the replication of the other RNA segments, thus restoring the ‘early’ replication pattern. Cycloheximide treatment resulted in a slight increase in the percentage of positive strands in nucleocapsid-associated RNA. The role of protein synthesis in the transition from the ‘early’ to the ‘late’ pattern of influenza virus RNA synthesis is discussed.

To understand better the action of guanidine and its antagonists on poliovirus replication, guanidine uptake by HeLa cells was studied. It was discovered that guanidine entered HeLa cells by at least two different mechanisms. At low concentrations (< 2 mm), it was transported mostly by a carrier-mediated, saturable mechanism with an apparent affinity constant of 1·26 mm-guanidine. About a third of uptake by this mechanism was sensitive to valinomycin and possibly dependent on membrane potential difference. At higher concentrations (5 to 10 mm), transport was predominantly by a non-saturable, low affinity process. The carrier-mediated transport mechanism was similar to the organic cation H+ exchanger-mediated excretion of organic cations from the kidney, because it was inhibited by organic cations and by high [Li+]. Physiological [Na+] caused less but significant inhibition of guanidine uptake by HeLa cells. Approximately 20% of total uptake could not be inhibited with organic cations and was probably due to diffusion. The antiguanidine agents choline, dimethylethanolamine and tetraethylammonium, but not methionine, inhibited guanidine uptake by HeLa cells. The inhibition caused by these agents depended on their concentration and more or less paralleled their reported ability to block guanidine inhibition of poliovirus replication. Even though choline inhibited guanidine uptake it appeared to reverse this inhibition of virus replication primarily by blocking the intracellular action of guanidine. Some unexplained previous observations on the action of guanidine and its antagonists were discussed in view of the results of this study.

Cytotoxic T lymphocytes (CTLs) induce rapid, extensive internal disintegration in target cells and this is unique among immune lytic mechanisms studied. This raises the question of whether CTLs are uniquely capable of halting virus infections by inducing damage within the target cell causing inactivation of intracellular virus. Reovirus infection of mouse P815 cells provided a suitable system for evaluating this question. An increase in cell-associated infectious virions began 8 h after infection and increased until 20 h post-infection, at which time the titre levelled off at about 100- to 1000-fold higher than the initial value. The infectious activity was compared between host cells killed by CTLs and those killed by sonication at various points in the infection cycle. The presence of reovirus within the target cell did not inhibit the usual internal disintegration events associated with the death of a target killed by CTLs. Nevertheless, the results indicated that CTLs were incapable of inactivating intracellular reovirus at any point in the life cycle of the virus: CTL-induced cytolysis simply released the infectious virions into the medium. Thus, at least in the case of reovirus, the utility of direct killing by CTLs would appear to be limited to reduction of the virus yield by lysis of the host cell before virus replication and assembly is completed.

A collection of Mov mouse strains, each carrying in their germ line a Moloney murine leukaemia virus (M-MuLV) proviral genome at a different chromosomal location, was used to study expression of endogenous retroviruses. No M-MuLV - specific RNA was detected in the non-viraemic Mov strains studied, indicating that less than two copies of RNA are transcribed per cell. Virus expression was seen in three viraemic Mov strains. In Mov-3 mice the provirus was activated shortly after birth, whereas proviruses in Mov-9 and Mov-14 were activated at different stages of embryogenesis. The results suggest that the chromosomal position influences proviral expression during development. The first appearance of virus particles was hot accompanied by detectable amounts of viral transcripts, suggesting that viraemia is a consequence of provirus activation in a small, as yet unidentified, population of cells, followed by virus spread and infection of susceptible cells.

In Rat-9G cells several copies of the major immediate early (IE) transcription unit (regions 1 and 2) of the human cytomegalovirus (HCMV) are stably integrated. The cells show a repressed phenotype for IE expression but can be induced by inhibition of protein synthesis. In this report we present evidence that the repressed phenotype is due to the absence of IE transcription and that heat-shock and sodium arsenite treatments each result in the transcriptional activation of the repressed IE transcription unit. Either treatment resulted in the induction of HCMV IE transcripts and IE nuclear antigen expression. An octameric DNA sequence present in three of the 18 bp IE enhancer elements (GGACTTTC) resembles the cellular heat-shock element core consensus sequence and may therefore be involved in the heat-shock response.

A number of human papillomavirus (HPV) type 16 proteins have recently been identified in human cervical carcinoma cell lines using polyclonal antisera against papillomavirus gene products expressed in Escherichia coli. E7 protein has been found to be the most abundant papillomavirus protein in these cells. Here we describe a panel of monoclonal antibodies recognizing a 15K non-glycosylated cytoplasmic HPV-16 E7 protein. One of the antibodies cross-reacted with HPV-18 E7 protein.

Amino acid sequences of fusion (F) proteins of two trypsin-resistant mutants of Sendai virus, TR-2 and TR-5, were deduced from nucleotide analysis of cDNA encoding the F gene and were compared with that of the trypsin-sensitive wild-type Sendai virus. In both mutants, amino acid substitutions were found at residues 116 (Arg → He), the cleavage site of the F protein, and 109 (Asn → Asp). Two trypsin- sensitive revertants, TSrev-52 and TSrev-58, derived from TR-5 were both activated by trypsin similarly to the wild-type virus and had a single amino acid reversion from lie to Arg at residue 116, leaving Asp as before at residue 109. These results indicate that the trypsin sensitivity of Sendai virus can be changed by a single amino acid substitution at the cleavage site of the F protein and a mutation from Arg to lie is responsible for the acquisition of resistance to trypsin.