- Volume 63, Issue 1, 1982

The assembly of virion surface glycoproteins by enveloped viruses appears to be both specific and non-specific. It is non-specific in the sense that, during a dual infection by various unrelated viruses, there is very common (if not universal) and efficient mixing of envelope glycoproteins from both genotypes in the progeny virions. However, it seems to be specific in the sense that non-viral, cell surface proteins are, in the main, recognized as alien and are excluded from incorporation into virions during budding. These findings suggest that all enveloped viruses may share an essentially common molecular mechanism for the specific assembly and budding of virus glycoproteins while at the same time incorporating a mechanism for the exclusion of glycoproteins of cellular origin. One of the simplest explanations for this phenomenon may be that virus envelope surface structures all evolved from a single ancestral virus.

The biological activity of a molecularly cloned DNA of a rat endogenous C-type leukaemia helper virus, RHHV, was assessed by intranuclear microinjection into normal rat kidney cells (NRK 153). Release of rat C-type leukaemia helper viruses by the microinjected cells was examined by superinfection of Kirsten-transformed non-producer cells (K-NRK). Immediate release of helper leukaemia viruses at a very low level was observed only in the NRK 153 m3.5/cir cells microinjected with the supercoiled form of RHHV DNA in toto, suggesting that the circular form of the virus DNA might have expedited the replication and expression of virus particles. Genome rescue experiments were also performed by co-cultivating the microinjected NRK 153 m cells carrying various linear RHHV DNAs, in toto or of subgenomic sizes, with K-NRK cells. The results indicated that both the total and the 5.8 to 6.2 kb DNA fragment proximal to the 5′ terminus of the cloned RHHV 8.8 kb DNA were able to rescue successfully a transforming replication-competent pseudotype virus. Subgenomic DNA fragments derived from the centre or the 3′ end of the RHHV DNA were ineffective in the genome rescue experiments.

A Rauscher virus (RV)-transformed erythroid cell line, RA-1, was shown to be a non-producer cell line. RA-1 cells express not only gp51–54 env-related glycoprotein, but also gp70, which is more closely related to gp51–54 coded by a recombinant env gene than to the MuLV gp70. RA-1 cells could be infected by Friend, Moloney and Gross viruses, but not by the homologous Rauscher murine leukaemia virus. Rescue of spleen focus-forming activity was obtained on infection of these cells with MuLV-F or MuLV-Mol, but not with MuLV-Gross. The RNA of the RV complex resembles closely that of Friend virus (FV). It contains a 32S, presumably defective, genome, which most likely is responsible for spleen focus formation, and a 35S helper virus genome. Oligonucleotide fingerprint data suggest that RV has evolved independently of FV. Erythroid early BFU-E cells of mice infected with RV of Friend helper virus-infected RA-1 cells were shown to require no addition of conditioned medium to form large erythroid colonies (BFU-E) in the presence of only small amounts of erythropoietin.

Avian sarcoma viruses (ASV) produced by Japanese quail embryo fibroblasts (QEF) were inactivated to the same degree as ASV produced by chick embryo fibroblasts (CEF), with or without complement, by rabbit antisera to CEF and to membrane antigens of chick embryo and adult chicken erythrocytes, in particular their unique age-specific antigens. ASV produced by duck embryo fibroblasts (DEF) were only inactivated by the antisera to the chicken erythrocyte antigens. Hence, viruses produced by QEF and DEF bear on their envelope antigens related to the chicken antigens picked up by ASV in CEF. These antigens are presumably coded by the quail and duck cells, since antigens related to the chicken antigens were found on QEF and on quail and duck erythrocytes. Antigens related to the chicken antigens have also been found on ASV shed in low amounts by semi-permissive rat sarcoma cells (line 17RBI77) and on the surface of these cells, as well as on that of another semi-permissive cell line (RBH) originating from a hamster sarcoma produced by inoculation of the rat cells. The origin of these antigens, which may play a role in semi-permissiveness, remains to be explained since they do not appear to be normally expressed by rat or hamster cells. It was also found that, contrary to an earlier conclusion and in agreement with a recent report, uninfected CEF bear on their membrane an antigen that is related or identical to the specific antigen of chick embryo erythrocytes. Therefore, only the antigen related to the adult-specific chicken erythrocyte antigen does not pre-exist on CEF.

The ability of adenovirus structural polypeptides to bind nucleic acids was assessed by separating the polypeptides on SDS-polyacrylamide gels, transferring them electrophoretically to nitrocellulose and probing with 32P-labelled nucleic acids. Polypeptides IVa2, V, VI and VII, as well as trace amounts of pVII and a polypeptide of apparent mol. wt. 40 × 103 were able to bind label under these conditions. Labelling was also detected with a smaller polypeptide, possibly related to the cleavage products of pVII and/or pVI. The binding of DNA to polypeptide VI appeared to be more sensitive to detergents than the others. No sequence specificity could be detected in the DNA binding.

Numerous discrete lesions, which we have termed blebs, appeared in the nucleus of BHK cells 10 to 15 h after infection with herpes simplex virus type 1 (HSV-1). They were formed in the inner portion of the nuclear envelope by the apposition of two thickened lamellae overlying a vacuole. As demonstrated by electron microscopic studies, blebs were regular and associated with the peripheral lamina in the nucleus, averaging 3.5 blebs per μm2. They appeared to be associated with an enrichment of the 155K major capsid protein in the nuclear membrane subfractions as compared with the protein composition of nuclei and plasma membrane fractions. We propose that blebs represent the site of assembly of capsid proteins before DNA insertion and eventual envelopment.

In both non-immune and immune mice infected with herpes simplex virus the incidence of latent infection of the trigeminal ganglion was related to the severity of ocular virus infection. During primary infection, virus was shown to travel via the ophthalmic part of the ganglion to reach the brainstem, from where centrifugal spread resulted in latent infection of neurones in the trigeminal ganglion which did not serve the site of inoculation. Primary infection also resulted in latent infection of the superior cervical ganglion. Shedding of virus occurred rarely in the tears of animals which had recovered from primary disease. In immune mice, spread of virus resulted in a much lower incidence of latent infection and that occurred only in ophthalmic neurones.

Two fowl plague virus temperature-sensitive (ts) mutants belonging to different complementation groups were studied. Both were defective in the syntheses of unpolyadenylated complementary RNA [A(-)cRNA] and virus RNA (vRNA) at non-permissive temperature whereas primary transcription was unaffected. In addition, ts 29, in which the ts mutation is in gene 1 coding for polypeptide P3, has a defect in ‘secondary’ synthesis of mRNA at non-permissive temperature whereas inhibition of mRNA synthesis by ts 131, in which the ts mutation is in gene 2 coding for polypeptide P1, appeared to result from a defect in vRNA synthesis. These results indicate, therefore, that different enzymes are responsible for the syntheses of virus mRNAs and A(-)cRNAs, which is consistent with the apparent differences in initiation and termination of transcription in the two reactions. The patterns of synthesis of the various types of virus RNA during infection are discussed.

Twenty-one monoclonal antibodies directed against the measles virus haemagglutinin have recently been obtained. These were known to fall into five groups, each defined by its effects on the biological functions of the H protein. A representative of each group was selected and examined by competitive radioimmunoassay in an attempt to deduce the relationships between antibody-binding sites on the antigen. It was found that these five antibodies formed three binding groups which recognized different but overlapping areas of the molecule. These three areas formed a series of sites which traversed the active region of the H polypeptide. A haemagglutinin-directed monoclonal antibody which displayed a haemolysin-inhibiting activity was also examined. This antibody fitted into the binding group scheme determined here and there was no additional binding site for this molecule on the F protein itself.

The equine-virulent Venezuelan encephalitis virus, Trinidad donkey (TRD), was compared to its vaccine derivative, TC-83 virus, by examining the glycosylation of the two structural envelope glycoproteins (E1 and E2). The number of size classes of glycopeptides on the glycoproteins was determined by P-6 column chromatography following Pronase digestion. The E1 glycoprotein had three glycopeptide size species and the E2 glycoprotein contained four size species ranging in mol. wt. from 1900 to 2700. Both viruses contained similar glycopeptide size species, although the relative amounts on the E2 glycoproteins appeared to be somewhat different. All of the glycopeptide species appeared to be complex, since all were labelled with glucosamine, mannose, galactose and fucose. No mannose-rich species could be detected. The different glycopeptide species appeared to be sialylation isomers of a smaller core glycopeptide with an apparent mol. wt. of 1800 which was the sole product following desialylation of the larger glycopeptides. The number of oligosaccharide attachment sites present on both E1 and E2 of each virus was determined using reverse-phase high pressure liquid chromatography. This analysis indicated that the E1 glycoprotein of both viruses had six or seven similar sugar-labelled peptide fragments following trypsin digestion. However, the E2 glycoprotein of TRD virus contained three oligosaccharide attachment sites, whereas TC-83 E2 glycoprotein had only two.

Nuclei isolated from uninfected HEp-2 cells synthesized RNA for 60 to 90 min. The individual RNA polymerase activities were determined by α-amanitin differential inhibition and the RNA products characterized byelectron microscope (EM) autoradiography and sucrose gradient centrifugation. In nucleiprepared from poliovirus-infected cells, the capacity tosynthesize RNA in vitro decreased with time after infection. RNA polymerase II activity (hnRNA synthesis) was preferentially inhibited more than was the polymerase I activity (rRNA synthesis). Poliovirus-infected cytoplasm (S-30) inhibited in vitro RNA synthesis in uninfected nuclei by selectively affecting the polymerase II activity. Selective inhibition of hnRNA synthesis by the crude extracts could be monitored by EM autoradiography directly. Determinations of individual RNA polymerase activities by differential α-amanitin inhibition were done only after treatment of the infected cytoplasm with micrococcal nuclease to abolish virus RNA replication. Selective inhibition of hnRNA synthesis depended on preincubation of the nuclei together with the infected cytoplasm, indicating that inhibitory substances from the infected cytoplasm entered the nuclei. Isolated nuclei therefore provide a useful system for studying the nature ofthe inhibitor(s) and of host RNA synthesis inhibition by picornaviruses.

Enterovirus type 70 (EV70) agglutinated human ‘O’ erythrocytes at 4 °C as well as 22 °C, but visible agglutination was lost when wanned at 37 °C although the virus remained attached to the surface of the erythrocyte. The receptor sites for the virus were neuraminidase-sensitive. A direct involvement of sialic acid on the cell surface in virus-cell interaction was confirmed by the fact that the presence of fetuin or freeNacetylneuraminic acid inhibited the haemagglutinating activity of EV70. Similar numbers of virus particles were required for 1 haemagglutinating unit (HAU) of EV70 and 1 HAU of mengovirus, whereas 2.6-fold or more of virus particles of echovirus type 7 and type 11 gave the same activity. On the other hand, the number of receptor sites on the cell surface for EV70 was found to be sevenfold more than for mengovirus. Therefore, the erythrocyte receptor for EV70 is different from that for common enteroviruses and similar, though not identical, to the cardiovirus receptor. However, serological tests such as neutralization, complement fixation or haemagglutination inhibition did not reveal any common antigen between EV70 and cardiovirus.

The three serotypes of reovirus differ markedly in their response to a variety of chemical inactivating agents. We used intertypic recombinants containing various combinations of genes derived from the parental serotypes to study the basis of these differences. In addition to recombinants derived from types 1 and 3, and 2 and 3, we were able to isolate recombinants derived from types 1 and 2, suggesting that these two serotypes also undergo unrestricted reassortment. The intertypic recombinants behaved like one parent or the other in the presence of the inactivating agents and allowed us to determine the genes responsible for each difference. Recombinants derived from crosses between wild-type parental serotypes produced straightforward results, while recombinants derived from mutagenized, temperature-sensitive parents often did not. Sensitivity to 2.5 M-guanidine-HCl and pH 11 was determined by the SI gene, sensitivity to 55 °C and 1 % SDS was determined by the S4 gene, and sensitivity to 33% ethanol and to 1 % phenol was determined by the M2 gene. Thus, relatively nonspecific chemical agents appear to have their predominant effect on specific proteins of the reovirus virion.

We have examined the effects of heat and several chemical inactivating agents on the buoyant density, particle-associated polypeptides and ultrastructure of reovirus particles. Treatment at pH 11 removed polypeptide σl from the outer capsid of reovirus type 2 but not from type 1; resultant particles were unchanged in their buoyant density and morphology. Treatment of reovirus types 2 and 3 with 2.5 M-guanidine-HCl produced particles with unchanged polypeptide content but an increased buoyant density, and caused aggregation of type 3 but not type 2. Treatment with 1 % SDS removed polypeptide σ3 from both types 1 and 2 and increased the buoyant density of the virus particles. The outer capsid of SDS-treated virions was greatly altered and often indistinct. Treatment of type 3 with either 1% phenol or 33% ethanol produced particles that had a full complement of polypeptides, were unaltered in buoyant density, but were greatly aggregated. Thus, these inactivating agents affect reovirus particles in specific and distinct ways. The differential effects of such treatments can thus be used to study the structure and function of the reovirus capsid components.

The RNA components of chronic bee-paralysis virus (CPV) and those of chronic bee-paralysis virus associate (CPVA) have been compared. CPV has five singlestranded RNA components, two larger RNAs designated 1 (mol. wt. 1.35 × 106; 4200 nucleotides) and 2 (mol. wt. 0.9 × 106; 2800 nucleotides), and three smaller RNAs designated 3a, 3b and 3c, each with mol. wt. 0.35 × 106 (1100 nucleotides). CPVA has three single-stranded RNA components designated A, B and C, each with mol. wt. 0.35 × 106 (1100 nucleotides). Gel electrophoretic and T1 fingerprint analyses have shown that RNAs 3a, 3b and 3c are very similar to, and probably identical to, RNAs A, B and C respectively, i.e. these three RNAs appear to be encapsidated in both CPV and CPVA particles. A positive correlation was observed between the proportion of RNAs 3a, 3b and 3c in CPV particles and the amount of CPVA (and hence RNAs A, B and C) which had replicated. Fingerprint analysis has shown that RNAs A, B and C are distinct but related and also that about 50% of the sequence of each these three RNAs is homologous with RNA 2. It is therefore possible that, although CPV and CPVA are serologically unrelated, the RNAs of CPVA have evolved from CPV RNA 2.

Highly purified interferon-α (IFN-α) prepared from a human lymphoblastoid line (Namalwa) was analysed by gel filtration and polyacrylamide gel electrophoresis (PAGE). Gel filtration separated the IFN-a into two peaks (A and B). All the compo- nents of peak A were retained by a monoclonal antibody (NK2) column, but some of those from peak B were not retained. The IFN that was not bound was active on mouse cells and could be resolved into two major bands by PAGE. The bound fraction (about 75% of the interferon protein) was purified by means of the monoclonal antibody column, although complete purification of crude interferon was not achieved in one passage.

Tunicamycin alters the biological properties of human interferon-α (HuIFN-α) produced in its presence by changing the mixture of IFN-a subtypes produced. When IFN mRNA extracted from treated cells was microinjected into oocytes the product was also altered. However, treatment of the oocytes with tunicamycin did not alter the properties of the IFN-α produced.

The effects of anti-cytokinetic drugs on virus production, formation of syncytia, cell surface changes and lysosomal damage were examined during mouse hepatitis virus 3 (MHV-3) infection of mouse peritoneal macrophages. Colchicine and vinblastine caused no detectable effect on the infectious process. In the presence of cytochalasin B the acute, highly cytopathogenic interaction that MHV-3 establishes with macrophages was converted into one which persisted for several days. Under these conditions the cell surface changes induced by the infection were maintained unaltered but cell fusion was reduced and no significant lysosomal damage was detectable.