- Volume 64, Issue 5, 1983

The 5′ ends of two early herpes simplex virus type 1 mRNAs have been identified by nuclease S1 and exonuclease VII analysis using cloned virus DNA probes. These mRNAs (5.0 kb and 1.2 kb), located within the genome region between map coordinates 0.56 and 0.60, are unspliced and share a 3′ terminus. Genomic DNA at the 5′ ends has been sequenced and the 5′ termini have been located on the virus DNA sequence. The DNA sequence has revealed signals involved in the initiation of transcription of both mRNAs, and the 5′ end of the 1.2 kb mRNA is encoded within the internal sequences of the 5.0 kb mRNA. The probable translational initiation codons for the polypeptides specified by these mRNAs have been identified, and the results indicate that the coding regions of the two mRNAs do not overlap.

Pre- and postnatally acquired protection against herpes simplex virus type 2(HSV-2) infection mediated by maternal antibody was investigated in the newborn mouse. Newborn mice, 2 days old, were inoculated with HSV-2 intraperitoneally after maternal immunization with live or inactivated virus. The survival rates improved in proportion to the maternal neutralizing antibody titres. Ninety-three percent of animals delivered by Caesarean section from immune mothers and suckled by non-immune mothers survived viral infection, whereas 7% of control animals survived. The same was true with animals born to non-immune mothers and nursed by immune mothers. In foetal sera and milk of immunized mice, anti-HSV activity was associated primarily with antibody of the IgG class as measured by enzyme-linked immunosorbent assay (ELISA). In addition, oral administration of antibody conferred protection on newborn mice. These studies indicate that maternal IgG, acquired not only postnatally but also prenatally, plays an important role in protecting newborn mice against HSV infection.

Phosphonoacetic acid (PAA) effectively inhibited herpesvirus saimiri (HVS) replication and the onset of virus DNA synthesis. A PAA-resistant (Pr) mutant of HVS was isolated which plaqued efficiently in the presence of concentrations of PAA sufficient to reduce the growth of wild-type virus to < 0.02% of control values. In contrast, virus growth and DNA synthesis in cells infected with unselected strains of herpesvirus saimiri was highly resistant to concentrations of aphidicolin, an inhibitor of α-type polymerases, which completely inhibited the growth and DNA replication of uninfected cells. An increased level of DNA polymerase activity was induced in cells infected with herpesvirus saimiri. This HVS-induced DNA polymerase was more sensitive to PAA but more resistant to aphidicolin in vitro than the uninfected cell activity and could also be distinguished on the basis of its response to ionic strength (40 to 50 mm-ammonium sulphate for optimal activity versus 20 nm for the uninfected cell activity). Under suitable in vitro assay conditions, an increase in the PAA-resistance of the DNA polymerase induced by the HVS(Pr) mutant was demonstrated. Comparison of the effects of aphidicolin and PAA demonstrated that the former was a much more effective and rapid inhibitor of susceptible cell DNA synthesis in vivo. Taken together, these results demonstrate novel properties of a DNA polymerase activity in cells infected with herpesvirus saimiri and suggest that aphidicolin should provide a useful reagent to analyse the functions of this enzyme in productive and non-productive infections with the virus.

A herpesvirus isolated from several organs of an American bison affected with malignant catarrhal fever was cultured in bovine foetal spleen cells and studied by electron microscopy. The fine structural features of the mature virion and the mode of virus morphogenesis were found to be similar to herpesviruses classified in the subgroup cytomegalovirus. The capsids were granular, hexagonal in shape and contained pleomorphic cores in thin sections. Envelopment of the capsids occurred primarily by budding on cytoplasmic membranes which appeared to be formed as extended vesicles of the Golgi apparatus; budding on nuclear membranes was only rarely observed. Cytoplasmic inclusions consisting of granular threads and amorphous electron-dense material were found in association with virions during the late stages of infection. The formation of cytoplasmic inclusions, the morphogenesis and ultrastructure of the virus are all consistent with classification of this virus as a cytomegalovirus.

The DNAs of 17 isolates of varicella-zoster virus (VZV) were analysed by restriction endonuclease cleavage and agarose gel electrophoresis. By comparing gel patterns of DNAs cleaved with only a few enzymes, all epidemiologically distinct isolates were shown to be unique. Two isolates recovered from members of a family infected in a common-source outbreak were identical to each other (4/4 enzymes) but distinct from the other strains. In addition, three isolates recovered at different times during the course of a single episode of zoster in another individual were identical by endonuclease analysis (4/4 enzymes) but once again were distinct from all other isolates. The differences that have been recognized in cleavage profiles of all VZV strains reported thus far map into four regions of the viral genome. Two of these variable regions lie within the long unique sequences while the other differences appear to map in each of the inverted repeat sequences.

The development of an in vitro system prepared by lysis of LuIII virus-infected cells with Brij-58 has enabled the study of the assembly pathway of a parvovirus. Under optimal conditions, radioactive precursors are incorporated both into viral replicative form double-stranded DNA and into progeny viral DNA (vDNA) during pulses as short as 30 s. Labelled 110S particles can be isolated at the end of such pulses. Therefore, synthesis and encapsidation of progeny viral DNA into pre-existing empty viral capsids appear to be closely related processes. Up to about 10 min after incorporation of vDNA, the 110S particles band at 1.44 g/ml and are relatively unstable in 3.5 m-CsCl. Moreover, newly synthesized vDNA molecules show an abnormal electrophoretic behaviour probably due to the presence of a covalently linked terminal protein. This so far uncharacterized alkali-stable polypeptide is lost (cleaved off?) concomitant with the maturation of the 110S virus particles. Maturation is reflected by a change in the virus stability in CsCl and a shift in density from 1.44 to 1.41 g/ml around 10 to 15 min after encapsidation of progeny vDNA.

Nucleoprotein (NP) intermediates in the assembly pathway of parvovirus LuIII were isolated. These structures consist of replicating viral DNA and of preformed viral capsids into which progeny viral DNA is encapsidated concomitant with synthesis. The NP complexes sediment at between 70S and 100S. They are unstable in CsCl and dissociate in the presence of 1 m-NaCl. After fixation with glutaraldehyde, however, they accumulate at a density of 1.37 g/ml in CsCl gradients. Electron microscopy of fixed complexes revealed structures consisting of DNA threads associated with capsid-like particles. A considerable proportion of the DNA labelled during short pulses in such complexes, in a subcellular in vitro system derived from infected nuclei, can be effectively chased into 110S virions. The resulting 110S particles apparently have to undergo a sequence of morphogenic events to acquire the physicochemical properties of the mature infectious parvovirus.

The 58000 dalton (58K) protein coded for by early region 1B of human adenovirus type 5 (Ad5) was found to be phosphorylated. At least three major tryptic phosphopeptides were identified and the average number of phosphates per 58K molecule was estimated to be between two and three. Thus, it was possible that each phosphopeptide contained just one phosphate group. The ratio of phosphoserine to phosphothreonine was about 2 to 1 on average and essentially no phosphotyrosine was detected. No evidence was found to suggest that cAMP-dependent protein kinase was involved in the phosphorylation of 58K. Previous studies have shown that 58K was phosphorylated when immunoprecipitates containing Ad5 early region 1 proteins were incubated in vitro with [γ-32P]ATP. Analysis of the phosphopeptides of 58K labelled under these conditions indicated a large number of phosphorylation sites which differed from those found in vivo. Thus, the action of kinases in the in vitro phosphorylation of 58K in immunoprecipitates did not mimic the enzymic activity responsible for 58K phosphorylation in vivo.

Adenovirus type 5 (Ad5) mRNAs present in cells transformed with left-terminal Ad5 DNA fragments (XhoI-C, 0 to 15.5%; HindIII-G, 0 to 7.7%; HpaI-E, 0 to 4.3% were characterized by ‘Northern blotting’ and S1 nuclease analysis. They were compared with the mRNAs transcribed from the Ad5 E1 region in the early and late stages of lytic infection. It is shown that in XhoI-C-transformed cells the same mRNAs were transcribed as early during lytic infection: two co-terminal mRNAs from region E1a, differing only in their splicing, and one major E1b transcript. In HindIII-G-transformed cells additional E1a mRNAs were detected with a novel 5′ terminus, but with the normal splicing pattern. Instead of the normal E1b mRNA, HindIII-G-transformed cells were found to contain mRNAs consisting of a viral E1b segment and a non-viral segment. This E1b-encoded segment was shown not to be involved in RNA splicing. The mRNAs in cells transformed with Ad5 HpaI-E were similar to the E1a mRNAs found in XhoI-C- and HindIII-G-transformed, and in lytically infected cells but had aberrant 3′ termini. These results are discussed in the light of the Ad5 E1 DNA and RNA sequences, and protein mapping data.

Scanning electron microscopy and immunofluorescence techniques show that the attachment of adenovirus type 5 to HeLa cells is followed by a temperature-dependent redistribution of virus particles on the cell surface. Metabolic inhibitors and cytochalasin B, a drug that impairs microfilament function, blocked this redistribution. Transmission electron microscopy studies demonstrated that inhibition of redistribution was paralleled by an inhibition of virus internalization. In further experiments virus and cells were incubated at 4 °C and then treated with adenovirus ‘soluble’ antigens to block unoccupied virus receptors. On warming these preparations to 37 °C, the internalization of attached virions was found to be impaired. It is proposed that energy-dependent redistribution of attached adenovirus particles on the cell surface facilitates entry by bringing virions into contact with sufficient receptors to allow internalization.

The Lincoln strain of bovine rotavirus, cytocidal for bovine AU-BEK cells, can establish in the same cell cultures in the presence of foetal calf serum (FCS) a persistent infection that depends on selection of highly resistant cells. In fact, after the induction of the carrier state only a small fraction of the cell population was infected. The parental and the carried viruses are not demonstrably different, the cultures are resistant to superinfection by the homologous virus, the cultures can be cured by antiviral serum in the medium and uninfected resistant cell clones can be selected. The presence of FCS was essential during induction and maintenance of persistence. Its effects appear to be exerted not on the control of virus replication in fully sensitive cells but on the proliferation and selection of the resistant cells.

The virulent Venezuelan equine encephalitis (VEE) virus strain, Trinidad donkey (TRD) and its vaccine derivative strain TC-83, demonstrated biological and biochemical differences in their replication. These two viruses had similarly shaped growth curves; however, TRD virus-infected cells produced significantly more infectious virus than did the TC-83 virus-infected cells during the very early period of the replication cycle. TRD virus inhibited host cell protein synthesis in Vero cells earlier than did TC-83 virus as measured by the incorporation of [35S]methionine into cellular proteins. Virus-specified proteins were detected 1 to 2 h earlier in TRD virus-infected cells than in the TC-83 virus-infected cells; however, pulse-chase studies failed to show differences in the processing of the viral structural proteins in cells infected by either of the viruses. TRD virus-infected cells produced more virus RNA than did the TC-83 virus-infected cells, and analysis of the intracellular viral RNA species showed an increased synthesis of 26S RNA in the TRD virus-infected cells. The difference in amounts of 26S virus RNA produced was most pronounced early during the infection and may explain why TRD virus infection resulted in an earlier production of viral proteins and inhibition of cellular protein synthesis than was observed in the cells infected with the avirulent vaccine strain of VEE virus.

A monoclonal antibody (30.2) raised against Sindbis virus is able to precipitate both E1 and PE2 from [35S]methionine-labelled infected cells solubilized with non-ionic detergent. Addition of SDS to the lysate abolishes the precipitation of PE2 without affecting that of E1, thus demonstrating that the antibody is specific for E1. Other Sindbis E1-specific monoclonal antibodies (30.11 and 30.12) precipitate only E1, even from lysates containing only non-ionic detergent, and their presence in such a lysate prevents precipitation of PE2 by antibody 30.2. These data indicate that E1-PE2 complexes stable in the presence of non-ionic detergent can be precipitated as such by one antibody, but that binding of the other antibodies induces dissociation of E1 and PE2. Competition experiments using 125I-labelled antibodies indicate that all three antibodies bind to distinct antigenic sites on the E1 molecule. Antibodies 30.11 and 30.12 stimulate each other′s binding in such experiments, which suggests that binding of either of these antibodies alters the conformation of E1 in such a way as to increase its affinity for the other, and at the same time to release PE2. Antibody 30.2 also enhances binding of the other two antibodies, but this stimulation is only weakly reciprocated.

Lassa virus was purified from culture fluids of infected CV-1 monkey kidney cells and its structural proteins analysed by polyacrylamide gel electrophoresis. Stained gels showed a typical arenavirus profile, with a prominent protein of molecular weight 60000, corresponding to the nucleocapsid protein N, and two faint broad bands with molecular weights of 45000 and 38000, the envelope glycoproteins G1 and G2. G1 and G2 were shown to be glycosylated by their ability to bind concanavalin A to nitrocellulose transfers of the separated proteins (‘Western blots’). N and G2 bound antibody from guinea-pig or human convalescent sera but G1 was inactive, presumably as a result of denaturation. This technique also revealed other apparently virus-specific minor bands with molecular weights of 76000 and 68000. When Western blots of proteins of infected cells which had been lysed in SDS were probed with anti-Lassa virus serum or stained for glycoproteins, four virus-specific bands were apparent: the N, G1 and G2 proteins seen in purified virus, and a glycoprotein of molecular weight 72000 which probably corresponds to the envelope protein precursor (GPC) seen in other arenavirus systems. Immunoprecipitates from infected CV-1 cells labelled with [35S]methionine contained three major virus-specific proteins: the nucleocapsid protein N and proteins of 36000 and 24000 molecular weight (designated fN1 and fN2). Similar immunoprecipitates from Vero cells contained fN1 and fN2 and only very low levels of N. The polypeptides fN1 and fN2 are most probably fragments of N, since Western blots probed with anti-Lassa virus serum showed that lysis of cells in non-ionic detergent rather than SDS results in the appearance of fN2 with concomitant reduction or disappearance of N. These fragments do not exist in the intact cell, but are found as a consequence of rather specific proteolysis upon disruption under non-denaturing conditions. The proteolytic activity responsible was refractory to inhibition by phenylmethylsulphonyl fluoride, aprotinin, pepstatin A or sodium bisulphite, and was more active in Vero than in CV-1 cells.

Encephalomyocarditis and influenza viruses attach to human erythrocytes causing haemagglutination. The receptor for both viruses on these cells is the major membrane sialoglycoprotein, glycophorin, solubilized preparations of which inhibit haemagglutination by either virus. We show here that glycophorin preparations inhibited haemagglutination of both viruses, even after the preparations were digested with chymotrypsin. To determine which component(s) in the digest exhibited activity, peptides separated by gel filtration were assayed for haemagglutination inhibition; one peptide only, CH-0, was active. A tentative structure was deduced for CH-0 from amino acid and sialic acid analyses. It was already known that neuraminidase treatment of erythrocytes or glycophorin prevents interaction with either virus, suggesting that sialic acid may form part of the active binding site in the receptor. However, receptor activity requires more than the presence of a particular arrangement of sialic acid since the arrangement in CH-0 was identical to that in two other inactive chymotryptic peptides. Examination by gel filtration, sucrose density gradient centrifugation and SDS-polyacrylamide gel electrophoresis demonstrated that CH-0 readily aggregated, unlike the inactive peptides. It was proposed that the CH-0 chymotryptic peptide showed receptor-like activity (inhibited haemagglutination) because its tendency to aggregate allowed strong multivalent binding with virus particles.

Exposure of influenza B virus-infected MDCK cells to chloroquine at the time of infection resulted in significant inhibition of infection. The appearance of input virus in the intracellular vesicles was not affected in the presence of the drug, but primary transcription of the virus genome did not occur. Chloroquine caused a rapid rise in the pH inside the lysosomes of MDCK cells, to 6.5 from the physiological pH 5.6. In contrast, exposure of infected cells incubated in acidic medium (pH 6.0) to chloroquine did not cause an increase in lysosomal pH and this low pH treatment during the chloroquine-sensitive phase was followed by virus production. Influenza B virus induced haemolysis of chick erythrocytes at low pH values (5.0 to 5.9) which was associated with cell-cell membrane fusion. It is likely that chloroquine prevents the uncoating of influenza B virus by increasing the lysosomal pH above the critical value required for inducing fusion between the virus envelope and the lysosomal membrane.

In neonatal or congenital lymphocytic choriomeningitis (LCM) virus carrier mice, low numbers of T lymphocytes were always infected, but attempts to infect resting or stimulated T lymphocytes in previously uninfected adult mice have consistently failed. Only T cells in newborn mice were susceptible to LCM virus and their infection persisted when the animals grew older. Infectibility declined with increasing age of the mice. Initially, the majority was demonstrated among thymocytes, but later more splenic cells were infected. In contrast to neonatal carrier mice, in drug-induced carriers (in which persistent infection had been established later in life by immuno-suppression after infection) spleens and thymi contained initially no or a few infectious T lymphocytes; their numbers increased with the age of the mice but remained relatively low and erratic. We follow the hypothesis that LCM virus-specific immunological tolerance of carrier mice is due to age-dependent virus susceptibility of T lymphocytes and propose that murine T lymphocytes in general lack viral receptors for LCM virus, but that the few that carry LCM virus-specific immunological receptors bind the virus. If this occurs during an early stage of cellular development, infection results and functional inactivation is the consequence.

Herpes simplex virus type 2 wild-type and 13 temperature-sensitive mutants have been examined for their ability to be recovered from latently infected Biozzi mice. After inoculation into the rear footpad, virus could be recovered from both the dorsal root ganglia (DRG) and the footpad (FP) at the site of inoculation (wt, ts 1, ts 2, ts 4, ts 6, ts 7 and ts 8), or from the DRG only (ts 11 and ts 13), or from FP only (ts 3, ts 9, ts 12). Two mutants (ts 5 and ts 10) have not been recovered from either site. Both DNA-positive and DNA-negative mutants have been shown to be capable of establishing latent infection.

L(O)cl 3 and L(K)cl 1 cells, biochemically transformed by varicella-zoster virus (VZV), were labelled with l-[35S]methionine and [32P]orthophosphate. Cell extracts were immunoprecipitated with anti-VZV monkey serum and analysed by SDS-polyacrylamide gel electrophoresis. Four polypeptides of apparent mol. wt. 135000 (135K), 48K, 44K and 35K were detected in the l-[35S]methionine-labelled extracts, and, of these, the 35K band showed marked intensity. However, this band was not detected in extracts from cells infected with a VZV tk − strain (Kanno strain). Also, the 35K polypeptide showed very low intensity when immunoprecipitated from extracts of transformed cells grown in non-selective (NS) medium, i.e. cells that had a very low thymidine kinase (tk) activity. In the case of [32P]orthophosphate-labelled cells, polypeptides of apparent mol. wt. 180K, 81K, 48K, 44K and 37K were obtained. In both instances, the 44K polypeptide was not immunoprecipitated from L(K)cl 1 cell extracts. From our data it is postulated that the expression of the 35K polypeptide is correlated with the VZV-specific tk activity of the cells.