- Volume 68, Issue 4, 1987

We investigated the properties of amber mutants of coliphage T5 defective in a late stage of phage assembly. The non-functional heads synthesized by mutants am25 or aw 158 were unable to combine with functional tails to produce viable phage particles. These mutants were shown to lack a single protein, designated the head-tail linker protein (HTLp), which was identified by polyacrylamide gel electrophoresis and Western blot analysis and had an M r of 18000. An extract containing the HTLp, when added to the HTLp-deficient heads, restored their ability to combine with functional tails.

Cross-neutralization assays were done using 85 strains of poliovirus type 1 with five groups of monoclonal antibodies. These strains were classified into 10 subgroups which had marked differences in antigenicity. Subgroups P1-2 (28%) and P1-5 (43%) were dominant and have been epidemic in China in recent years. These two subgroups were antigenically different from the Sabin-1 strain, but according to their responses to one group of monoclonal antibodies they had antigenic epitopes in common with the Mahoney and Brunhilde strains. Similarly, 91 strains of type 3 poliovirus were classified into six subgroups with another five groups of monoclonal antibodies. The results showed that strain P3/Yunnan/2/84, which was isolated from cases of poliomyelitis in a local epidemic in the Yunnan province of China in 1984, and strain P3/Finland/23127/84, which was isolated in Finland in 1984, were both antigenically different from the Sabin-3 strain and the reference virulent strain.

In Felis catus whole foetus D cells infected with feline infectious peritonitis virus (FIPV), strain 79–1146, six virus-specific, poly(A)-containing RNA species of about 20, 9·6, 5·2, 3·8, 2·8 and 1·6 kb were found. By translation in vitro the 3·8 and 2·8 kb RNAs were shown to encode the 25K envelope protein and the 45K nucleocapsid protein, respectively. The partial map of the FIPV genome was compared with genomic maps of porcine, murine and avian coronaviruses. Differences in these maps suggest that transcription units have been lost or gained during coronavirus divergence.

The complete nucleotide sequence of the human parainfluenza virus type 3 (HPIV3) fusion (F) protein gene has been determined. The HPIV3 F gene is 1851 nucleotides long including six U residues in the genomic RNA, which probably direct synthesis of the first few nucleotides in the F mRNA polyadenylate tail. The HPIV3 F gene contains a single long open reading frame coding for 539 amino acids. The predicted molecular weight of the unglycosylated precursor F0 protein was 60031. Four potential carbohydrate acceptor sites were identified. Comparison of the HPIV3 F protein sequence with the F gene sequences of two other paramyxoviruses, Sendai virus and simian virus 5, indicated a very close evolutionary relationship between HPIV3 and Sendai virus. Sequence analysis of HPIV3 F gene flanking regions identified signals which appear to be responsible for polymerase recognition and polyadenylation.

The ultrastructure and distribution of some viral and cellular antigens was examined in lymphoid C91/PL and fibroblastic HOS/PL cells infected with human T-cell leukaemia virus type I. Tubuloreticular structures in the endoplasmic reticulum, characteristic of virus-infected or interferon-treated cells, were found in both cell types. Virions were observed particularly in the HOS/PL cells in budding and mature forms and in coated pits, coated vesicles and lysosomes, indicative of receptor-mediated endocytosis. Using immunogold indirect labelling, viral antigens, recognized by sera of infected patients and by monoclonal antibody, were detected in patches on the cell surface and in material loosely attached to the envelope of some virions. Beta2- microglobulin was associated with virion envelopes from C91/PL and HOS/PL cells, but major histocompatibility complex class 1 (HLA) antigens were not associated with virions even when produced by HLA-positive HOS/PL cells.

Vaccinia virus cores prepared in vitro can be uncoated by incubation with extracts of cells infected with vaccinia virus, as determined by the conversion of the genome to DNase susceptibility. This uncoating activity had all the characteristics of the corresponding in vivo activity and of the agent responsible for non-genetic reactivation. Thus, it was not induced by heat-inactivated virus, nor was it produced in the presence of inhibitors of RNA or protein synthesis. The uncoating protein induced by cowpox virus will uncoat vaccinia virus cores. The uncoating protein was concentrated from infected cell extracts by ultrafiltration and purified by gel filtration and ion-exchange and affinity chromatography. It was characterized as a trypsin-like protease with a mol. wt. of 23050. Cores treated with the purified uncoating protein had an altered sedimentation rate but no differences between treated and untreated cores were detected by electron microscopy or polyacrylamide gel electrophoresis.

Primary cultures of non-proliferating hepatocytes isolated by the two-step collagenase perfusion method from woodchuck naturally infected with hepatitis virus (WHV) were used to study WHV propagation in vitro. Hepatocytes carrying WHV DNA exhibited a very high level of survival and retained their morphological characteristics for 2 to 3 months. Over this time, they were found to produce virus-specific proteins and release viral particles with DNA polymerase activity into the medium. Using Southern blot analysis and a recombinant hepatitis B virus DNA plasmid probe, intracellular and extracellular viral DNA was consistently detected. Only extrachromosomal forms of WHV DNA were observed and no integration could be demonstrated in the DNA of the cells. The WHV DNA patterns were repeatedly identical with a characteristic smear starting from 3·3 kb associated with other smaller DNA fragments which presumably represented intermediate replicative forms of viral DNA. Furthermore, dot blot hybridization of the total RNA revealed the presence of WHV-specific transcripts in cells after 3 weeks of culture. All these results are compatible with the maintenance of active WHV replication in vitro although it was somewhat reduced after the first day of culture. This provides a mammalian model for hepadnavirus replication studies in stable primary hepatocyte cultures.

Replicative form (RF) DNA of the lymphotropic strain SL3 of Aleutian disease virus was isolated from infected cell cultures. A novel intermediate of about 7·6 kilobases was demonstrated in Hirt lysates in addition to single-stranded viral, double-stranded monomer and dimer RF DNA. The monomer RF DNA exhibited a length heterogeneity of 70 bp and 160 bp at its 3′ and 5′ termini. The two major monomer RF DNA species each contained hairpins in the extended or the foldback configurations.A central fragment between map units 0′15 and 0′88 was cloned into plasmid pUC18. The recombinant clone expressed virus-specific proteins ranging from 32000 to 74000 mol. wt.

The population of herpesvirus saimiri (HVS) genomes extracted from extracellular virions are double-stranded, linear DNA molecules of about 160 kilobase pairs (kbp) each composed of a central segment of 110 to 112 kbp and 36% (G + C) (i.e. ‘light’ or L-DNA) linked to direct reiterations of a 1·44 kbp repeat unit of 71 % (G + C) (i.e. ‘heavy’ or H-DNA) at each terminus. In this paper, we show that the population of HVS DNA molecules contains approximately equal concentrations of genomes with all possible integral numbers of complete repeat units (i.e. from > 30 to 1) at either ‘left’ or ‘right’ ends but that all molecular ends are derived by a unique cleavage at a site close to the single Apal restriction endonuclease site of the H-DNA repeat unit. Junctions of proximal H-DNA repeat units with L-DNA occur at, or very close to, the sequence present at the molecular ends. The transition from L- to H-DNA occurs abruptly at this site at the ‘right’ end of the L-DNA component but some rearranged restriction enzyme cleavage sites typical of H-DNA are found within the first 0·8 kbp of the L-DNA sequences at the ‘left’ H-L DNA junction. HVS appears to provide an extreme example of the general process whereby herpesvirus DNAs are matured from concatemeric intermediates by a site-specific cleavage/recombination process involving random choice between equivalent sites for the initiation of the process and with choices between alternative termination sites being limited by a headful packaging mechanism.

Varicella-zoster virus (VZV) and Epstein-Barr virus (EBV) are important human pathogens which belong to different subfamilies of the herpesviruses: the Alpha- and Gammaherpesvirinae, respectively. Computer comparisons of the amino acid sequences of proteins predicted from the published complete VZV and EBV DNA sequences resulted in the detection of EBV counterparts to 29 of the 67 unique VZV genes. Conserved genes were detected only in the UL component of each genome, and are located in three major regions, within which conserved genes are generally colinear. However, the three regions are arranged differently in the two genomes. These results make it possible in principle to propose the functions of EBV genes on the basis of the functions of their VZV counterparts. The data also allow identification of the types of events which may have occurred during divergence of VZV and EBV, as representatives of the Alpha- and Gammaherpesvirinae, from a common ancestor.

Cytoplasmic RNA was prepared from biopsy material obtained from the nasopharynx of normal individuals and nasopharyngeal carcinoma (NPC) patients. Similar RNA preparations were prepared from lymphocytes of healthy donors and infectious mononucleosis patients. RNA was radioactively labelled in vitro and hybridized to cloned fragments of B95-8 Epstein-Barr virus (EBV). In all seropositive cases the minimum pattern of EBV-specific RNA expression was like that observed previously in latently infected, EBV-positive lymphoblastoid cell lines or Burkitt’s lymphoma biopsies. A novel observation was that all of the control biopsies from normal nasopharynxes expressed EBV-specific RNA, some of which appeared to be associated with a more active state of EBV infection. The pattern of expression in NPC patients was similar to that of the normal donors but showed some variations in complexity. In general the virus-specified small RNAs were not present in normal nasopharyngeal tissue but were expressed in the NPC biopsies.

Intraperitoneal (i.p.) immunization with herpes simplex virus type 1 (HSV-1) or an anti-idiotypic antibody (anti-id C) prepared against a monoclonal antibody specific for glycoprotein C of HSV-1, tolerizes mice for an HSV-1 delayed type hypersensitivity (DTH) response. This tolerization could be adoptively transferred to naive X- irradiated mice by splenic T cells, and was specific for HSV-1 DTH. Thus, DTH- tolerized mice responded to vaccinia virus or HSV-2 challenge, while remaining tolerized for HSV-1 DTH. In addition, these animals demonstrated a form of split tolerance such that HSV antibody, cytotoxic T cell and lymphoproliferative responses were detected in vitro. Thus, anti-id C induced a T cell population capable of specifically suppressing the HSV-1 DTH response, mimicking the effect of i.p. and intravenous immunization with HSV-1 found previously.

Mouse L cell lines have been developed which constitutively express glycoproteins B (gB) and D (gD) of herpes simplex virus type 1. When used to study the immune response of mice to the viral glycoproteins, it was found that both gB and gD induce a delayed type hypersensitivity response and both also induce an antibody response, but only the cell line expressing gD could stimulate the production of neutralizing antibody. Virus-specific cytotoxic T lymphocytes (CTLs) recognized gB expressed by the cell line and this line could also induce CTLs in mice. Recognition of gD by major histocompatibility complex class I restricted CTLs was never seen. Vaccination of mice with the cell lines provided protection from viral challenge and inhibited the establishment of a latent infection, although gD proved to be the better protective immunogen.

Seventeen Thy-1+ cell clones were induced in A/J mice immunized with the HEP- Flury strain of rabies virus after repeated stimulations with antigens in vitro. Ten clones with cell surface phenotypes Thy-1+, Lyt-1−,2+ were cytotoxic T lymphocytes (CTL) which lysed the virus-infected target cells under H-2 restriction. Target cells expressed the G and M2 structural proteins of rabies virus on their surface; however, target lysis by CTL clones was not blocked by anti-rabies antibody or by monoclonal antibodies to these proteins. All of the CTL clones efficiently and equally lysed target cells infected with three different strains of rabies virus and were cross-reactive for target cells infected with one (Duvenhage virus) of three different rabies serogroup viruses. Another five clones having phenotype Thy-1+, Lyt-1+,2− did not show any cytotoxic activity. The proliferation response of these clones to antigen stimulation was virus- specific and H-2-restricted. These clones were able to grow in culture medium without any or with the addition of low concentrations of T cell growth factor, in contrast to CTL clones, and were considered to be helper T lymphocytes (HTL). Both CTL and HTL clones produced y-interferon in response to antigen stimulation. The remaining two clones were Thy-1+, Lyt-1−,2−, asialo-GM1+, and were not cytotoxic to target cells even in the presence of anti-rabies antibody but were cytotoxic to YAC-1 cells. Further studies with these clones should allow us to investigate more closely the role of T cells in the pathogenesis of rabies.

Poly(rI). poly(rC) induces a lytic reaction in interferon-treated mouse LM cells. We have attempted to determine whether the intracellular penetration of poly(rI). poly(rC) is a prerequisite for cell lysis and to gain some insight into the pathway followed. We found that poly(rI). poly(rC) coupled to Sepharose beads was unable to generate lysis of interferon-treated cells whereas the cells underwent lysis after microinjection of poly(rI).poly(rC). Some inhibitors of endocytosis were found to inhibit the development of the lytic reaction. Lysosomotropic amines or a low temperature (19 °C) blocked endocytosis of poly(rI). poly(rC) but did not prevent its uptake. The internalization of poly(rI). poly(rC) was energy-dependent and was blocked when sodium azide and 2-deoxyglucose were added simultaneously. We conclude that poly(rI). poly(rC) is internalized and reaches an acidic compartment before triggering the lytic reaction in the cell.

Normal guinea-pig serum (GPS) lacking detectable antiviral antibody efficiently neutralized the infectivity of influenza B virus grown in chick embryos or MDCK cells. The inhibitor was heat-labile and sensitive to trypsin digestion. This β-like inhibitor required Ca2+ and the complement components C1 and C4 for its activity. In contrast, GPS did not inactivate influenza A virus. Influenza B virus from which the neuraminidase activity of the spikes on the viral envelope had been eliminated by trypsin digestion was also inactivated to a level comparable to untreated virus. Complement component C1 alone bound directly to influenza B virus and inhibited its haemagglutinin activity. We suggest that the β like inhibitor in GPS is a component of the classical complement pathway which is triggered by the protein moiety of influenza B virus haemagglutinin, leading to virus neutralization.

The replication of HVJ (Sendai virus) in C6 rat glial cells was found to be inhibited by treatment of the cells with papaverine, an inhibitor of cAMP phosphodiesterase, but not with cAMP or dibutyryl cAMP. In addition, cyclic GMP which often manifests a reciprocal relationship to cAMP did not counteract the inhibition of HVJ yield by papaverine. Both viral genome replication and transcription were suppressed slightly by treatment of the cells with papaverine. In the cells cultured in the presence of papaverine, the synthesis of viral proteins and their phosphorylation occurred at normal rates. Membrane immunofluorescence and cell surface immunoprecipitation showed that the viral glycoproteins HN and F0 were expressed on the cell surface of the papaverine-treated cells. Moreover, all the viral structural proteins were associated with plasma membrane isolated from the treated cells. These results indicate that papaverine treatment suppresses some part of the process of virus budding at the plasma membrane.

Prostaglandin J2 (PGJ2) was found to suppress dramatically Sendai virus replication in African green monkey kidney cells in culture. PGJ2 was not toxic at the active dose to uninfected cells and did not significantly inhibit macromolecular synthesis, but it specifically stimulated the synthesis of a polypeptide of 74000 mol. wt. In Sendai virus-infected cells, PGJ2 partially inhibited virus protein synthesis and caused an alteration in the mobility of the virus glycoprotein HN in SDS-PAGE, corresponding to a decrease of about 4000 in its mol. wt. We propose that the PGJ2-induced alteration in the molecular structure of the HN protein prevents the insertion of this protein into the cell membrane thereby blocking virus maturation. The α, β-unsaturated carbonyl group in the cyclopentane ring of the PGJ2 molecule may be necessary for antiviral activity.