- Volume 73, Issue 12, 1992

The molecular biology of the live attenuated Sabin vaccine strains of poliovirus has been studied extensively, and surprisingly few mutations are required to account for the greater part of the attenuated phenotype. The viruses are clearly capable of extremely rapid, extensive and precise variation in the vaccinee to adapt from the attenuated form to a form able to grow successfully in the host, yet despite this they cause almost no disease. The high degree of genetic variation in the face of general phenotypic stability in the wild-type suggests that polioviruses are extremely well adapted to their hosts and that vaccines exploit some aspects of the virus host ecology to be safe and effective. The precise mechanisms by which they do so raise possibilities of improving vaccine production and testing methods, and designing better vaccine strains.

Two immature T cell lines (FT1 and FT4) were established after in vitro cloning of peripheral blood lymphocytes (PBLs) from an asymptomatic human immunodeficiency virus type 1 (HIV-1) seropositive, human T cell-lymphotropic virus type 1 seronegative homosexual subject. Although derived from a limiting dilution cell cloning assay, these cell lines were not recloned for this study. Their growth was independent of exogenous interleukin-2. Both cell lines were able to form colonies when cloned in agar, but failed to form solid tumours when injected into nude mice. FT lines belong to the very immature T cell lineage as they exhibit rearranged TCR genes but no expression of T cell membrane antigens, including CD2, CD3, CD4, CD6, CD7 and CD8. They also contain an HIV-1 genome that was detected only in an extra-chromosomal DNA form, even after several passages in vitro. The presence of unintegrated viral DNA was also detected by polymerase chain reaction analysis in the same sample of fresh uncultured PBLs. Furthermore, despite the absence of CD4 expression, both T cell lines were susceptible to CD4-independent HIV-1 superinfection (lack of superinfection inhibition in the presence of OKT4A monoclonal antibodies).

In the present study we investigated to what extent the peripheral carbohydrate structure of N-linked glycans influences the antigenic properties of human immunodeficiency virus type 1 glycoprotein 120 (gp120). Recombinant gp120 was purified from GMK cells infected with a recombinant vaccinia virus expressing gp120. Purified gp120 was then coated onto 96-well ELISA microplates and subjected to sequential removal of peripheral monosaccharide units. Modified or unmodified gp120 was then incubated with monoclonal antibodies recognizing specific epitopes of gp120 and with a reporter lectin to determine the extent of carbohydrate elimination. Antibody and lectin binding was quantified in an enzyme-linked system. We found that the carbohydrate structure NeuAc-Galβ(1-4) of N-linked glycans, defined both by lectin reactivity and by specific glycosidases, is involved in modulating the binding of antibody to a number of epitopes of peptide nature. The binding of antibody to one class of epitopes, situated in a region between amino acids 200 and 230, was strongly increased by removal of NeuAc-Galβ(1-4). whereas the binding to epitopes in the V3 region was decreased and the binding to epitopes in the far N-terminal region was not altered by the treatment. These results suggested that peripheral structures of N-glycans are involved in modulating the overall conformation of gp120.

To determine whether selection of genome segments in coinfections is strain-specific, chicken embryo fibroblasts were coinfected with avian reovirus strain 883 and one of three other avian reovirus strains (176, S1133 and 81-5). Viral progeny from each coinfection (883 × 176, 883 × S1133 or 883 × 81-5) was serially passaged at a low m.o.i. The electropherotypes of the coinfection progeny and those of the plaque-derived clones obtained from passages 1 and 20 were analysed. Two 883 segments (M2 and S2) were found to be selected in the 883 × 176 coinfection, three 883 segments (M2, M3 and S2) in the 883 × S1133 coinfection, and only one 883 segment (M3) in the 883 × 81-5 coinfection, i.e. different 883 genome segments were selected in the three coinfections. It was, therefore, concluded that selection of genome segments in a coinfection of a given cell line is virus strain-specific. The selection of genome segments in coinfections was shown to be due to enhanced infectivity of the reassortants that were formed in the coinfections. In addition, defective interfering particles that lack the S1 segment were identified in the 883 × 81-5 coinfection progeny following serial passage. Selection of genome segment(s) in coinfections as described herein may have potential importance on the effect and production of divalent or multivalent vaccines.

The 12 nucleotide conserved sequence at the 3′ end of influenza A virion RNA is sufficient to function as a promoter in vitro. By introducing point mutations in all 12 positions of this promoter in model RNA templates and studying the efficiency of RNA synthesis in vitro, we show that only three nucleotides, residues 9, 10 and 11, are crucial for activity, although other nucleotides play a significant but less important role. Additions or deletions within the promoter are tolerated, resulting in either an increase or a decrease in promoter activity, depending on the mutation introduced; in some cases premature termination is caused. Taking these observations into account, a model for RNA polymerase binding and copying of the promoter is discussed.

Vesicular stomatitis virus (VSV) fails to replicate in mouse T lymphocytes unless the cells have been mitogenically stimulated with concanavalin A (Con A). We have examined the possibility that the failure of VSV to replicate in unstimulated T lymphocytes can be attributed to a deficiency in a host protein kinase which activates the viral P protein by phosphorylation, thus rendering it transcriptionally competent. Soluble extracts were prepared from purified mouse T lymphocytes, with or without prior treatment with Con A. The ability of these extracts to phosphorylate bacterially synthesized P protein of two VSV serotypes was measured in vitro. Activity of the protein kinase on the P proteins of the Indiana or New Jersey serotypes of VSV increased, on average 2.4- and 2.1-fold respectively, after treatment of the cells with 3 µg/ml Con A. Higher concentrations of Con A induced proportional increases (up to 10-fold) in the activity of the host protein kinase. Activities of the kinase phosphorylating the P protein in separate populations of CD4- and CD8-containing murine T lymphocytes increased similarly on mitogenic activation. No biochemical or immunological differences were observed between the T cell protein kinase and the previously characterized protein kinase (casein kinase II) from BHK-21 cells. The activity of the kinase that phosphorylates the P protein did not vary in CV-1 cells on treatment with α- or γ-interferon, both of which inhibited VSV replication. Similarly, casein kinase II activities in Raji and SIRC cells, which do not normally support VSV growth, were the same as in BHK-21 cells. Thus restriction of VSV replication in these cells, in contrast to T lymphocytes, was not associated with a deficiency in the host casein kinase II activity.

We report here that activation of protein kinase C (PKC) results in the inhibition of adenovirus virus-associated (VA) gene transcription in vitro. The involvement of PKC in this inhibition is supported by the fact that the addition of PKC inhibitors to transcription reactions in which the PKC cofactor phosphatidyl serine (PS) was present resulted in increased levels of transcription compared to those in reactions in which PKC activity was stimulated in the absence of PKC inhibitors. Furthermore, based on these in vitro studies we propose that the inhibition of VA gene transcription is possibly due to the inability of the VA gene to form an active transcription complex following the activation of PKC. This conclusion was drawn from in vitro data which demonstrated that PS stimulation of endogenous PKC present in cell extracts resulted in failure to isolate a fully initiated, stable transcription complex.

Amino acid residues 101 to 180 of hepatitis B surface antigen (HBsAg) were predicted by sequencing the corresponding part of the S gene of hepatitis B virus (HBV) DNA in 46 HBsAg-positive sera, which had been subtyped by immunodiffusion with respect to d/y, w/r, w1 to w4 and q. The sequences of the nine different HBV serotypes defined by these specificities were found to be homogeneous proving that they represent consistent variations of HBV at the genomic level. Residue 127 was found to be important as were Pro, Thr and Leu for w1/w2, w3 and w4, respectively. Five residues were found to differ between ayw1 and ayw2. These were at positions 134 (Phe instead of Tyr), 143 (Thr instead of Ser), 159 (Ala instead of Gly), 161 (Tyr instead of Phe) and 168 (Val instead of Ala). However, all these residues were shared by ayw1 and adw2, implying that Arg122 was also important for w1 expression. All genomes expressing r, apart from one ayr strain, had an Ile126, which might explain the pseudo-allelism of w1 to w4 in relation to r, since this substitution might influence the w epitope. There were two regions where adw4q − and adrq − differed from all the q + subtypes. These were located at residues 158 and 159, and at residues 177 and 178, where both the q − subtypes had amino acid substitutions in adjacent positions. The mapping of the epitopes defining these antigenic specificities will help to link information on the world-wide distribution of HBsAg subtypes to future molecular epidemiology with regard to HBV.

Clinically malignant Buschke-Löwenstein tumours and benign condylomata acuminata are caused by human papillomaviruses (HPVs), predominantly HPV-6 and -11. In some cases, the HPV-6 genomes found in Buschke-Löwenstein tumours and in verrucous carcinomas differ from HPV-6b isolated from a benign genital wart, by rearrangements of the upstream regulatory region (URR). To evaluate the frequency and role of mutations of the URR of HPV-6 we analysed 42 condylomata acuminata and four Buschke-Löwenstein tumours by the polymerase chain reaction and restriction enzyme cleavage. Using only four different restriction enzymes we could demonstrate four distinct restriction patterns, indicating that naturally occurring HPV-6 isolates display a high degree of DNA polymorphism within the URR. One Buschke-Löwenstein tumour and two condylomata acuminata yielded rearranged URRs with DNA duplications. All three lesions harboured multiple HPV-6 variants, suggesting that cellular or environmental factors facilitate the development of rearrangements. Therefore, rearrangements of the URR may represent only secondary events in condylomata acuminata and Buschke-Löwenstein tumours which do not necessarily confer a higher malignant potential to the infected cell.

Vaccinia virus gene D6R encodes the small subunit of the virion early gene transcription initiation factor. Three temperature-sensitive mutations have been mapped to this gene. The biochemical phenotype exhibited by each mutation was examined. All mutants displayed altered viral protein synthesis in pulse-labelling analyses at both the permissive and non-permissive temperatures. The onset of early protein synthesis was delayed, and the rate of early protein synthesis was reduced in each case. Furthermore the shut-off of both host and early protein synthesis was delayed. In pulse-chase experiments, the stability of the D6R protein in E93- or C46-infected cells was shown to be reduced at 40°C relative to that at 31°C. Early mRNA was quantified in cells at 2 h post-infection and shown to be reduced substantially. The ability of each mutant virus to support transcription in vitro was examined at both temperatures and, of the three mutants, only S4 transcription was shown to exhibit reversible temperature sensitivity.

The relative levels, rates of synthesis and stabilities of the abundant Epstein—Barr virus (EBV)-encoded small RNAs, EBER-1 and EBER-2, were examined in Daudi Burkitt's lymphoma cells. Although both RNAs are transcribed at approximately equal rates, the steady-state level of EBER-1 is at least 10-fold greater than that of EBER-2. This is shown to be due to a much faster rate of turnover of EBER-2. In the presence of actinomycin D, the half-lives of EBER-1 and EBER-2 are 8 to 9 h and 0.75 h, respectively. Following treatment of the cells with human interferon (IFN) α the transcription of both RNAs is strongly inhibited. However, the level of EBER-1 increases up to twofold, indicating a further stabilization of this RNA. In IFN-treated cells, EBER-2 accumulates in the form of truncated products. Nuclease protection experiments indicated that this is due to a post-transcriptional modification of the 3′ end of the molecule. These data show that the effects of IFN treatment on the expression of these two viral gene products are very complex in cells latently infected with EBV.

The DNA polymerase gene of the Lymantria dispar multinucleocapsid nuclear polyhedrosis virus (LdMNPV) was cloned and sequenced. The predicted DNA polymerase protein (1113 amino acids, 115.9K) was found to have an amino acid identity of 48% with the corresponding gene of the Autographa californica MNPV (AcMNPV). It contains five domains associated with substrate binding, primase interaction, and pyrophosphate hydrolysis and three domains associated with 3′»5′ exonuclease activity common to other DNA polymerases. A region with a conserved TATA promoter and a CAGT mRNA start site sequence motif was identified and shown to be transcribed by RNA polymerase II, indicating that the LdMNPV DNA polymerase gene is expressed as an early gene. An open reading frame possibly expressed as a late gene, oriented in the opposite direction and overlapping the N-terminal coding region of the DNA polymerase gene was found in the LdMNPV sequence and was shown to be conserved in the same position in AcMNPV.

The kinetic and equilibrium attachment of Autographa californica nuclear polyhedrosis virus (AcMNPV) to seven insect cell lines was evaluated. Kinetic experiments revealed differences of up to 10-fold in the infection rates among cell lines. Equilibrium binding also varied between cell lines and was saturable. The Tn 5B1-4 and Tn F cell lines had the highest virus binding affinities and infection rates and exhibited diffusion-limited attachment. The rate of infection appears to be limited by the rate of attachment. For the Tn 5B1-4 cells the physical to infective particle ratio for AcMNPV was 5.3. From the Scatchard analyses, the cell lines Tn 5B1-4 and Tn F displayed affinities of 2.35 × 1010 m −1 and 1.60 × 1010 m −1, respectively, with 6000 and 13700 binding sites per cell. The insect cell line Hz 1075, which is not susceptible to AcMNPV infection, displayed a much lower, but saturable, binding of AcMNPV with 900 sites/cell and an affinity of 1.1 × 1010 m −1. Unlabelled AcMNPV, but not Lymantria dispar MNPV could compete with labelled AcMNPV for binding sites. There were 93 to 96% reductions in virus cell binding following pretreatments of cells with three proteases, suggesting the involvement of a cellular protein component in virus binding. Tunicamycin, an inhibitor of N-linked glycosylation and expression of some membrane proteins on the cell surface, reduced virus binding in a dose-dependent manner suggesting a role for glycoprotein(s) in binding. However there was no evidence for the direct involvement of oligosaccharides in attachment. Metabolic inhibitors of oligosaccharide trimming and competition binding assays using simple sugars caused no measurable reductions in virus binding. These findings suggest that AcMNPV attachment to insect cells is receptor-mediated via a glycoprotein component(s); the direct involvement of oligosaccharide moieties in binding is unlikely.

Protein synthesis has been studied in pupae of the silkworm Bombyx mori (Bm) infected with nuclear polyhedrosis virus (BmNPV) at various stages of the pupal period. Nascent proteins were labelled by injection of [35S]methionine into pupae and then analysed by SDS-PAGE. Temporal regulation of synthesis of infected cell-specific proteins (ICSPs) in pupae was demonstrated by electrophoretic analysis of the proteins labelled at different times post-infection (p.i.). The rate of ICSP synthesis reached a maximum at 4 to 5 days p.i., exceeding the rate of synthesis of cellular proteins in uninfected pupae by about twofold. The viral proteins p10 and polyhedrin were the most abundant products synthesized late in the infection. Both proteins were found to be associated with the nuclear matrix after fractionation of nuclei from infected pupae. Two virus-induced phosphoproteins, pp35 and ppB, were found to be the major acceptors of labelled phosphate from [γ-32P]ATP during in vitro phosphorylation of proteins in pupal homogenates, nuclei and nuclear extracts. These proteins had electrophoretic mobilities comparable to those of structural phosphoproteins of BmNPV virions with M rs of 35K and 11K to 16K, respectively. The latter polypeptide was identified as the major DNA-binding protein of the virus. The susceptibility of silkworms to BmNPV decreased markedly during the pupal period. Following injection of BmNPV all young pupae acquired polyhedrosis and finally died whereas most of the older pupae did not exhibit disease and completed metamorphosis normally. Moreover, the later in the pupal period the silkworms were infected, the lower the production of polyhedrin in diseased pupae.

Protein kinases induced by Bombyx mori nuclear polyhedrosis virus in pupae of the silkworm B. mori were examined by activity gel analysis using phosvitin as a protein substrate. The method involved PAGE of the soluble fraction from pupae under native conditions and in the presence of SDS, followed by in situ renaturation of proteins and recovery of protein kinase activity in the intact gel. A novel protein kinase able to phosphorylate phosvitin was detected in the infected pupae from 2 days post-infection. This enzyme was not present in uninfected silkworms at any stage of the pupal period. The novel kinase activity was found by SDS-PAGE to be associated with a single polypeptide with an apparent M r of 50K. However, on electrophoresis under native conditions its activity was associated with a set of polypeptides with similar but not identical electrophoretic mobilities. Microheterogeneity of the catalytically active polypeptides suggests that the virus-induced protein kinase undergoes post-translational modification during the course of infection.

The entomopoxviruses (insect poxviruses) of eastern spruce budworm (Choristoneura fumiferana), two year cycle spruce budworm (C. biennis) and the Indian red army worm (Amsacta moorei) are being studied in our laboratory for their potential as biological insecticides and expression vectors. These viruses characteristically replicate in the cytoplasm of insect cells and produce occlusion bodies that serve to protect the virion from the environment. By analogy to mammalian pox-viruses, they should also contain a viral thymidine kinase (TK) that functions in viral DNA synthesis. The replication of the A. moorei entomopoxvirus was inhibited by bromodeoxyuridine whereas the baculovirus of Autographa californica was insensitive to this drug. This result was a biochemical indication that entomopoxviruses contained a kinase that phosphorylated this nucleoside analogue and thus viral DNA synthesis was inhibited. TK genes from the three different insect poxviruses were identified, cloned and sequenced. The sequences of the TK genes of the entomopoxviruses were closely related and exhibited 63.2% identity and 9.9% similarity at the protein level. However, there was only 36.7% identity and 13.6% similarity when these enzymes were compared to their mammalian poxvirus counterpart in vaccinia virus. Finally, one entomopoxvirus TK gene was expressed in Escherichia coli mutants lacking the enzyme. These bacteria were converted to a phenotype that could incorporate radioactive thymidine into their chromosomal DNA. The results presented in this paper provide impetus for the design of a recombinant entomopoxvirus expression system in which foreign genes could be introduced into the viral TK locus under selective pressure from bromodeoxyuridine.

The purpose of this study was to construct an intergenic transfer vector which can be used for the generation of recombinant myxoma viruses (MVs) expressing a foreign gene insert. Recombinant MVs expressing the Escherichia coli lacZ gene were constructed in vitro by transfection of MV-infected rabbit cells with a transfer expression vector, and isolated under growth conditions selecting for transient expression of the E. coli gpt gene. The effect of inserting foreign DNA sequences between the viral thymidine kinase gene and open reading frame MF8a upon the transcription of these genes was investigated.

We have previously shown that most Rous sarcoma proviruses in Rat-1 cells are simple insertions, at apparently random sites, and are not transcribed. A small minority of simple insertions are transcribed and these show a bias to insertion at sites closely 3′ to presumptive cellular CG-rich islands. However, most transcribed proviruses are complex, typified by contiguous duplications of proviral DNA 5′ to a complete proviral unit. The cellular sites of these complex insertions are unknown and their structure and significance incompletely documented. We report here more extensive analyses of proviral duplications, with the following findings. The 5′ duplications predominantly involve proviral regions known to contain enhancer functions, substantiating earlier data. The cellular insertion sites are not biased to CG-rich loci at the level displayed by simple transcribed proviruses. The detailed structure of two duplications, and partial analysis of several others, strongly favours their genesis by illegitimate template transfer at reverse transcription, followed by self-recombination. These findings show that aberrant reverse transcription can generate duplications that dispense with the need for an expressed provirus to be integrated at a favourable cellular site.