- Volume 66, Issue 1, 1985

Using immunocytochemical procedures in conjunction with electron microscopy we have examined the distribution of the major DNA-binding protein (DBP) of herpes simplex virus (HSV) in infected nuclei. In embedded specimens, DBP was preferentially associated with fibrillar material of electron-translucent viral inclusions, and to a lesser extent with peripheral (marginated) host chromatin. The latter association was sensitive to a non-ionic detergent (‘Photo flo’). In chromatin spread by the Miller technique, DBP was found to be a component of the 10 nm ‘thick filaments’ previously described in HSV infection.

Supercoiled plasmid molecules containing cloned copies of a DNA fragment which includes a functional herpes simplex virus type 1 origin of DNA replication were cleaved preferentially at two positions within the viral insert by nuclease S1. Plasmids with molecular linker insertions at these sites were constructed, and analysis of two representative plasmids demonstrated the presence of palindromic DNA sequences at the preferred cleavage positions. One of these palindromic sequences occurred within a 90 bp region in which the cis-acting sequences essential for viral origin function had previously been located. Insertion of a linker at this position abolished origin activity, demonstrating an essential role for sequences within the palindrome in the initiation of DNA synthesis. In transfection assays, plasmids containing a functional viral origin of DNA replication markedly interfered with the infectivity of non-defective viral DNA even in the absence of viral encapsidation signals. Inactivation of the origin greatly reduced this effect on DNA infectivity, suggesting that viral interference may be mediated by a mechanism involving the replicative machinery.

The distribution of G + C-rich sequences in the genome of varicella-zoster virus (VZV) was investigated by partial denaturation, equilibrium sedimentation and Southern blot analyses. Portions of the IRS and TRS repeat sequences bounding the US region of the DNA were found to have a G + C content 10 to 20% greater than the overall 47% G + C content of the VZV genome. A stretch of DNA (approx. 1500 base pairs) at the UL-IRS junction and repeated at the terminus of the TRS sequences was found to be about 64% G + C, based on sedimentation equilibrium measurements. We also report the cloning of a novel fragment containing sequences from both the UL and TRS termini of the VZV genome. Our ability to clone this fragment suggests that unusual forms of VZV DNA including closed circular molecules and molecules with an inverted UL region can be packaged into nucleocapsids.

The genome of bovine cytomegalovirus (BCMV) strain 66-P-347 consists of double-stranded, linear DNA with a size of 144 ± 6 kb. It contains polyrepetitive DNA (prDNA) segments like five other BCMV strains. The restriction patterns of the prDNA of all six strains are very similar and indicate that monomeric prDNA units are either 1950 bp (class I and Ia), 2350 bp (class II) or 2750 bp (class III) in size. The complete unit sequence of strain 66-P-347 (class II) was cloned in bacteriophage vector M13mp7 and mapped by the restriction enzymes EcoRI, BamHI, BglI, NaeI, SstII and PstI. From these results the restriction maps of the prDNA of the other five strains were deduced. The 400 bp differences in size between the three prDNA classes are a consequence of the appearance of an internal 200 bp sequence being present one-, three- or fivefold in head-to-tail formation. Hybridization of 35S-labelled recombinant phage DNA to Southern blots with DNA of the different strains leads to the conclusion that prDNA units are present as multimers in tandem formation at both genomic termini in the same orientation. The number of terminal repeat units varies between individual molecules of a strain, but the actual terminal sequences are identical.

The DNA of field isolates and vaccine strains of pseudorabies virus (PRV) was analysed by digestion with the restriction endonuclease BamHI. A number of distinct restriction profiles of the field isolates obtained from different locations within Europe were observed. As for herpes simplex virus, the variations could be classified into two types: first, alterations in the mobility of fragments due to the presence of additional sequences and/or the occurrence of deletions, a phenomenon most apparent in fragments containing part or whole of the repeat sequence of PRV DNA; second, generation of differently sized fragments due to loss and/or gain of restriction endonuclease cleavage sites. By analysis of several strains with BamHI a small number of variable cleavage sites were identified within particular regions of the unique long (UL) segment of the PRV genome. Compared to wild-type PRV, the restriction fragment patterns of vaccine strains showed characteristic alterations, including the absence of bands, which were non-variable in wild-type strains, and/or the presence of new bands some of which were submolar. Some of these characteristics could be explained by a deletion in the unique short (US) region of the genome of most vaccine strains and the occurrence of closely related variants in the uncloned vaccine virus stocks.

When infected in vitro with Friend virus complex, the bone marrow cells of susceptible mice form large colonies (bursts) of erythroblasts after 5 days of culture in semi-solid medium. This virus-induced burst growth occurs without the addition of erythropoietin (EP) which is normally required for erythroid progenitor growth in vitro. Erythroid progenitor cells from C57BL/6 mice infected in vitro with Friend virus are resistant to virus-induced burst growth, while cells from the B6. S mouse strain, which is congenic with C57BL/6 but possesses the ‘Friend virus sensitivity’ alleles at the Fv-2 locus, are susceptible. This susceptibility of the B6. S cells demonstrates that virus-induced burst growth is regulated by the Fv-2 gene. Two mechanisms by which the Fv-2 locus could control virus resistance were analysed. The possible modulation of the erythroproliferative effect of the virus by soluble substances which either promote burst growth in the sensitive strains or inhibit growth in the resistant strain was examined. Also, the possible restriction of virus infection or replication in resistant (Fv-2 rr) haemopoietic cells was investigated. In a variety of experimental conditions designed to test the effects of soluble growth promoters on bone marrow cells infected in vitro, the resistance of C57BL/6 cells to erythroid burst formation could not be overcome. Neither could resistance be transferred to co-cultured sensitive cells by any soluble substances produced in culture by C57BL/6 cells. Use of haemopoietic cells from C57BL/6 animals in various physiological states of haemopoiesis also did not overcome the resistance to virus-induced burst growth. Quantification of several parameters of viral replication in whole marrow cultures or in erythroblasts from bursts of the Fv-2 sensitive and Fv-2 resistant congenic mouse strains showed that haemopoietic cells of both strains support virus growth equally well. These data suggest that Fv-2 rr-mediated resistance to the erythroproliferative effect of Friend virus infection in vitro is an inherent property of an erythroid progenitor target cell and is not determined by external factors. The resistance is also not due to restriction of virus replication.

In vitro proteolytic cleavage of the Gazdar murine sarcoma virus (Gz-MuSV) p65gag polypeptide (Gz-p65gag) was facilitated by detergent-disrupted Moloney murine leukaemia virus (Mo-MuLV). Incubation of radioactively labelled Gz-p65gag in the presence of unlabelled Mo-MuLV under optimal conditions resulted in the cleavage of Gz-p65gag to proteins of 40000 (P40) and 25000 (P25) M r. P40 and P25 appeared to be similar in both mobility and antigenicity to Mo-MuLV intermediates, Pr40gag and Pr25gag, previously found in infected cells. Additional proteins of 30000 (Gz-p30), 15000 (Gz-p12), 12000 (Gz-p15) and 10000 (Gzp10) M r were also generated upon cleavage of Gz-p65gag and contained antigenic determinants of Mo-MuLV structural proteins p30, pp12, p15 and p10, respectively. Both detergent-disrupted Mo-MuLV and Rauscher murine leukaemia virus produced similar cleavage profiles. Trypsin and detergent-disrupted mouse mammary tumour virus generated cleavage patterns very different from that produced by Mo-MuLV. Both visual and quantitative time studies of the reaction indicated that P40 gave rise to Gz-p30 and Gz-p10. Tryptic peptide mapping of Gz-p65gag and its cleavage products supported the results obtained from both immunoprecipitation studies with anti-gag sera and the kinetics of cleavage of Gz-p65gag. Both Mo-MuLV Pr65gag and Gz-p65gag were found to be very similar in primary sequence as judged by peptide mapping. P40 produced tryptic peptides that comigrated with Mo-MuLV p30 peptides; P25 contained tryptic peptides that were also found in Mo-MuLV p15. Gz-p30 and Gz-p15 contained the tryptic peptides of Mo-MuLV p30 and p15, respectively, that were found in P40 and P25. The Gz-p10 fraction contained a tryptic peptide that was also found in P40, but not p30. These results provide good evidence that the protease packaged within Mo-MuLV can cleave, in vitro, the gag-related polyprotein of Gz-MuSV in a manner very similar to the processing of Mo-MuLV Pr65gag in infected cell culture systems.

Mitogen treatment of murine (BALB/c) B-cells induces two different endogenous retroviruses involving two unlinked, presumably proviral, loci Bxv-1 and Bdv-1. To determine the usefulness of these loci as genetic markers for B-cell differentiation their expression was studied under conditions that interfered with B-cell proliferation and differentiation into IgM-secreting plaque-forming cells (p.f.c.). Maximum production of both viruses followed peak DNA synthesis by an interval of about 18 h. Treatments that blocked DNA synthesis or killed proliferating cells inhibited virus production. Addition of BUdR to mitogen-stimulated cultures selectively induced Bxv-1 while inhibiting the generation of p.f.c. Both effects require BUdR incorporation into the DNA of proliferating cells. 5-Azacytidine induced Bxv-1-dependent virus production without inhibiting terminal B-cell differentiation. Pretreatment of mitogen-stimulated B-cells with anti-mouse IgM serum decreased both virus production and generation of p.f.c., but had little effect on DNA synthesis. Experiments using a mitogenic F(ab′)2 preparation of anti-IgM in the presence and absence of lymphokines also suggested that the generation of p.f.c. and Bxv-1-dependent virus production are linked phenomena. The data imply that Bxv-1- and Bdv-1-dependent virus production require DNA synthesis and cell proliferation and, at least for Bxv-1, B-cell differentiation. It is proposed that the induction of these loci reflects the involvement of neighbouring DNA sequences in B-cell proliferation or differentiation.

In the absence of virus-specific antibody, Ross River virus failed to activate either the classical or alternative complement pathways. Instead, it inhibited the cleavage of C3 via both pathways. The virus did not appear to act by disrupting C3bBb complexes or by preventing cleavage of factor B by factor D. Instead Ross River virus was found to interfere with the actual cleavage of C3 by activated factor B (C3bBb) of the alternative pathway and C4b2a of the classical pathway.

We were able to initiate a persistent infection (PI) in HeLa cells with a temperature-sensitive (ts −) mutant of rhinovirus type 2 (TS-1), but not with the corresponding wild-type (wt) virus. The ability to initiate a PI may be related to the multiplicity of infection. Persistence was established at 37°C but not at 32°C and the virus isolated from the PI was no longer temperature-sensitive. Infectious virus was continually produced at low levels throughout the course of the PI and cell cultures underwent multiple episodes of partial destruction (crisis) and subsequent recovery. PI virus and the initiating virus were neutralized to the same extent by hyperimmune polyclonal TS-1 antiserum indicating that no significant change had occurred with respect to serological type. The presence of either interferon or virus-related interfering activity could not be demonstrated in the PI cultures. Superinfection experiments in cells that were ‘cured’ of PI virus indicated the selection of a cell population during persistence that could no longer support the growth of homologous-type virus. This effect became less pronounced upon further passage of the cured cells. When compared with the wt and ts viruses, the PI virus yielded comparable amounts of infectious virus in HeLa cells but with decreased synthesis of RNA.

The replication of Dugbe (DUG) virus, a member of the Nairovirus genus of the Bunyaviridae, has been investigated. During the infection of BS-C-1 cells a virus-specific c.p.e. was initially observed followed by recovery of the cell monolayer but with continued production of infectious virus. Six DUG virus-induced polypeptides were identified with apparent molecular weights, determined by gel electrophoresis, of 92000 (p92), 82000 (p82), 77000 (p77), 52000 (p52), 48000 (p48) and 34000 (p34). The polypeptides p77 and p34 were detected in purified DUG virions but not in extracts of virus-infected cells pulse-labelled with [3H]leucine. Polypeptides p48 and p52 were found in both purified virus preparations and in extracts of infected cells. p82 and p92 were found only in lysates of infected cells. When two-dimensional gel electrophoresis was used to analyse infected cells, p48 was found to have a net positive charge.

The replication of canine distemper virus (CDV) in Vero cells was found to require certain amino acids such as arginine, methionine and valine. The deprivation of methionine caused the most marked reduction in virus yield. In cells cultured in medium deprived of methionine, the early processes of viral replication such as adsorption, penetration and uncoating of virus occurred at normal rates, but the syntheses of viral RNA and protein were markedly reduced. The addition of S-adenosylmethionine to methionine-free medium resulted in the growth of CDV to the level obtained in cells with complete medium. Moreover, cycloleucine, which is known to reduce the methylation of mRNA by inhibiting the synthesis of S-adenosylmethionine, also inhibited the growth of CDV, and the addition of methionine or S-adenosylmethionine reversed the inhibitory effect of cycloleucine. The possibility of an inhibition of methylation of mRNA in methionine-deprived cells is discussed.

Three BHK-21 cell lines persistently infected with the CVS strain of rabies virus were passaged for 2.5 years at 37°C. These cells contained relatively stable amounts of N, M1 and L proteins. Comparison of tryptic peptide maps of proteins from persistently infected (PI) cells and from purified virions did not reveal any changes in the N protein, whereas M1 had undergone several modifications. Phosphorylation of M1 seemed to be greater in PI cells than in acutely infected cells, whereas phosphorylation of N was equivalent. Amounts of viral G and M2 proteins in PI cells were less than 10% of those present in acutely infected cells incubated for 24 h. Stability of the N protein, limited genetic evolution of the M1 protein probably implicated in transcription and replication of the virus, and reduction of the quantity of the membrane proteins G and M2 were general characteristics of our three PI cell lines. Decrease of the quantity of virus proteins associated with external membranes could explain why cells survive to the persistent infection. The composition of viral material released from PI cells was different from that of the virions produced from acutely infected cells. Substantial reduction of L, G and M2, and/or M1 was observed and the particles were of low infectivity.

The late region of bovine papillomavirus type 2 (BPV-2) DNA has been identified. The complete nucleotide sequence of the region was determined and revealed two large open reading frames. The DNA sequence results and the predicted amino acid sequence of putative polypeptides encoded by this region are presented. Comparative analysis of the BPV-2 late region and the corresponding area of BPV-1 was performed. This study demonstrates that identical genetic organization and considerable nucleotide sequence conservation exists between these two serotypes.

The entire genome of hepatitis B virus (subtype adr) has been cloned into pBR322. The clones could be classified into at least seven groups by their restriction endonuclease cleavage maps. The nucleotide sequences of the hepatitis B surface antigen (HBsAg)-coding regions for five clones were determined and compared with published sequences of the HBsAg gene including those of adw, ayw, adyw and adr. The 13 available versions of the amino acid sequence of the polypeptide, predicted from the nucleotide sequences, were analysed in terms of the established specificities of the HBsAg, including the four subtypes. The analysis indicated that a relatively hydrophilic region of the HBsAg protein, spanning amino acid residues 110 to 160, specifies the major (w) and (r) subtype system. The (w/r) subtype appears to depend on changes in one or more variable amino acids at positions 47, 110, 113, 126 and 160 of the HBsAg polypeptide.

Tunicamycin treatment of radioactively labelled infected Vero cells followed by electrophoresis in polyacrylamide gels showed that the mol. wt. of the putative polypeptide backbones of GP59(E1) and GP43(E2), the intracellular counterparts to the envelope proteins E1 and E2 of rubella virus, were 53000 and 34000, respectively. Two possible intermediates in the glycosylation of GP43(E2) were also identified. [3H]Mannose-labelled E1, E2, GP59(E1) and GP43(E2) were digested with Pronase and the glycopeptides separated by gel filtration. GP59(E1) contained glycopeptides in two size classes, designated R1.5 and R2.1; E1 contained these and an additional size class, R2.7. GP43(E2) contained glycopeptides in three size classes, R1.5, R2.1 and R2.7; E2 contained these and size class R3.3. The glycopeptides derived from GP59(E1) and GP43(E2) were all sensitive to endoglycosidase H treatment whereas the glycopeptides of E1 and E2 contained both sensitive and resistant components.

By combining the results of Fourier transform calculations from digitized electron micrographs and molecular volume calculations based on the amino acid composition and RNA content of narcissus mosaic virus particles, firm evidence that there are 8.8 protein subunits per turn of the helix in the virus particles was obtained.