Polypeptide composition of spleen necrosis virus, a reticuloendotheliosis virus.

The polypeptide composition of virions of spleen necrosis virus, a reticuloendotheliosis virus, was determined using electrophoresis on sodium dodecyl sulfate-containing, 10 percent polyacrylamide gels. Ten polypeptides were resolved. Four of these were present in minor and somewhat variable amounts. Two proteins, gp71 and gp22, contained D-glucosamine and were located on the outer surface of the lipid envelope, as demonstrated by lactoperoxidase-catalyzed iodination and by bromelain digestion. The results suggest that two of the minor proteins, p36 and p26, were also located on the outer surface, although they lacked D-glucosamine. Treatment of the virus with 0.25 percent Nonidet P-40 and 1 percent dithiothreitol produced a subparticle with a buoyant density of approximately 1.31 g/cm-3. This particle was relatively enriched with polypeptides p77, p62, and p50 and contained small amounts of three other polypeptides.     

Sialylatin of glycoproteins of murine mammary tumor virus, murine leukemia virus, and Mason-Pfizer monkey virus.

Neuraminidase treatment of mouse mammary tumor virus, Rauscher murine leukemia virus, and Mason-Pfizer monkey virus resulted in loss of their capacity to inhibit hemagglutination of influenza virus. Hemagglutination-inhibition activity of these RNA tumor viruses could be restored by in vitro resialylation catalyzed by sialyl transferase. The major glycoprotein in the intact envelope of desialylated and, to some extent, native virions could be specificallly labeled in vitro with CMP-(14C) sialic acid. These studies further characterize the individual glycoproteins of mouse mammary tumor virus, Rauscher murine leukemia virus, and Mason-Pfizer monkey virus.     

Identification of new protein kinase-related genes in three herpesviruses, herpes simplex virus, varicella-zoster virus, and Epstein-Barr virus.

By using amino acid sequence patterns (motifs) diagnostic of conserved regions within the catalytic domains of protein kinases, homologous open reading frames of three herpesviruses were identified as protein kinase-related genes. The three sequences, herpes simplex virus gene UL13, varicella-zoster virus gene 47, and Epstein-Barr virus gene BGLF4, resemble serine/threonine kinases rather than tyrosine kinases.     

The entry into host cells of Sindbis virus, vesicular stomatitis virus and Sendai virus.

We have compared the mechanisms of entry into host cells of three enveloped viruses: Sendai virus, vesicular stomatitis virus (VSV) and Sindbis virus. Virus entry by membrane fusion should antigenically modify the surface of a newly infected cell in such a way that it will be killed by anti-viral antibody and complement. On the other hand, virus entry by a mechanism involving uptake by the cell of the whole virion should not make cells sensitive to antibody and complement. As expected, cells newly infected with Sendai virus were readily and completely lysed by anti-Sendai antibody and complement. In marked contrast, however, cells newly infected with either Sindbis virus or VSV were killed by anti-viral antibody and complement only when infected at an extremely high multiplicity of infection, in excess of 1000 plaque-forming units per cell. We favor the following explanation for these results with Sindbis virus and VSV: a very large majority of the Sindbis and VSV virions entered the infected cells by some means other than membrane fusion, presumably engulfment of the whole particle. Efficient entry by way of membrane fusion may therefore not be a general characteristic of enveloped viruses.     

Persistent cyclic herpes simplex virus infection in vitro. II. Localization of virus, degree of cell destruction, and mechanisms of virus transmission

Hampar, Berge (National Institute of Dental Research, Bethesda, Md.). Persistent cyclic herpes simplex virus infection in vitro. II. Localization of virus, degree of cell destruction, and mechanisms of virus transmission. J. Bacteriol. 91:1959-1964. 1966. The localization of virus, degree of cell destruction, and mechanisms of virus transmission in persistent herpes simplex virus-infected cultures were studied. The major fraction of infectious virus was associated with the medium and a minor fraction was associated with the attached cells. Virus in the medium was further separable into a sedimentable (cellular) fraction and a nonsedimentable (extracellular) fraction. The sedimentable fraction was comprised of cellular debris, most of which appeared to contain viral antigen, and intact cells of which less than 10% contained infectious virus. Cell destruction during the cycle involved more than 99.9% of the maximal number of cells present. Infection could be transmitted by extracellular virus, cell-to-cell transfer, and reattachment of infectious cellular material. The results indicated that transmission by reattachment was probably mediated through the cellular debris rather than the intact cells.     

Incomplete protection, but suppression of virus burden, elicited by subunit simian immunodeficiency virus vaccines.

We compared the efficacy of immunization with either simian immunodeficiency virus (SIV) Env glycoprotein (Env), Env plus Gag proteins (Gag-Env), or whole inactivated virus (WIV), with or without recombinant live vaccinia vector (VV) priming, in protecting 23 rhesus macaques (six vaccine and two control groups) from challenge with SIVmac251 clone BK28. Vaccination elicited high titers of syncytium-inhibiting and anti-Env (gp120/gp160) antibodies in all vaccinated macaques and anti-Gag (p27) antibodies in groups immunized with WIV or Gag-Env. Only WIV-immunized macaques developed anticell (HuT78) antibodies. After homologous low-dose intravenous virus challenge, we used frequency of virus isolation, provirus burden, and change in antibody titers to define four levels of resistance to SIV infection as follows. (i) No infection ("sterilizing" immunity) was induced only in WIV-immunized animals. (ii) Abortive infection (strong immunity) was defined when virus or provirus were detected early in the postchallenge period but not thereafter and no evidence of virus or provirus was detected in terminal tissues. This response was observed in two animals (one VV-Env and one Gag-Env). (iii) Suppression of infection (incomplete or partial immunity) described a gradient of virus suppression manifested by termination of viremia, declining postchallenge antibody titers, and low levels (composite mean = 9.1 copies per 10(6) cells) of provirus detectable in peripheral blood mononuclear cells or lymphoid tissues at termination (40 weeks postchallenge). This response occurred in the majority (8 of 12) of subunit-vaccinated animals. (iv) Active infection (no immunity) was characterized by persistent virus isolation from blood mononuclear cells, increasing viral antibody titers postchallenge, and high levels (composite mean = 198 copies per 10(6) cells) of provirus in terminal tissues and blood. Active infection developed in all controls and two of three VV-Gag-Env-immunized animals. The results of this study restate the protective effect of inactivated whole virus vaccines produced in heterologous cells but more importantly demonstrate that a gradient of suppression of challenge virus growth, reflecting partial resistance to SIV infection, is induced by subunit vaccination. The latter finding may be pertinent to studies with human immunodeficiency virus vaccines, in which it is plausible that vaccination may elicit significant suppression of virus infection and pathogenicity rather than sterilizing immunity.     

Antigenic cross-reactions among herpes simplex virus types 1 and 2, Epstein-Barr virus, and cytomegalovirus.

Polyvalent rabbit antisera against herpes simplex virus type 1 and 2 (HSV-1 and HSV-2), cytomegalovirus (CMV), and Epstein-Barr virus (EBV), monospecific antisera against affinity-purified HSV-2 glycoproteins gB and gG, and a panel of monoclonal antibodies against HSV and EBV proteins were used to analyze cross-reactive molecules in cells infected with the four herpesviruses. A combination of immunoprecipitation and Western blotting with these reagents was used to determine that all four viruses coded for a glycoprotein that cross-reacted with HSV-1 gB. CMV coded for proteins that cross-reacted with HSV-2 gC, gD, and gE. Both CMV and EBV coded for proteins that cross-reacted with HSV-2 gG. Antigenic counterparts to the p45 nucleocapsid protein of HSV-2 were present in HSV-1 and CMV, and counterparts of the major DNA-binding protein and the ribonucleotide reductase of HSV-1 were present in all the viruses. The EBV virion glycoprotein gp85 was immunoprecipitated by antisera to HSV-1, HSV-2, and CMV. Antisera to CMV and EBV neutralized the infectivity of both HSV-1 and HSV-2 at high concentrations. This suggests that cross-reactivity between these four human herpesviruses may have pathogenic as well as evolutionary significance.     

Influenza virus neuraminidase: structure, antibodies, and inhibitors.

The determination of the 3-dimensional structure of the influenza virus neuraminidase in 1983 has served as a platform for understanding interactions between antibodies and protein antigens, for investigating antigenic variation in influenza viruses, and for devising new inhibitors of the enzyme. That work is reviewed here, together with more recent developments that have resulted in one of the inhibitors entering clinical trials as an anti-influenza virus drug.     

RNA recombination in brome mosaic virus, a model plus strand RNA virus.

Studies on the molecular mechanism of genetic recombination in RNA viruses have progressed at the time when experimental systems of efficient recombination crossovers were established. The system of brome mosaic virus (BMV) represents one of the most useful and most advanced tools for investigation of the molecular aspects of the mechanism of RNA-RNA recombination events. By using engineered BMV RNA components, the occurrence of both homologous and nonhomologous crosses were demonstrated among the segments of the BMV RNA genome. Studies show that the two types of crossovers require different RNA signal sequences and that both types depend upon the participation of BMV replicase proteins. Mutations in the two BMV-encoded replicase polypeptides (proteins 1a and 2a) reveal that their different regions participate in homologous and in nonhomologous crossovers. Based on all these data, it is most likely that homologous and nonhomologous recombinant crosses do occur via two different types of template switching events (copy-choice mechanism) where viral replicase complex changes RNA templates during viral RNA replication at distinct signal sequences. In this review we discuss various aspects of the mechanism of RNA recombination in BMV and we emphasize future projections of this research.     

Identification of a common antigen of herpes simplex virus bovine herpes mammillitis virus, and B virus.

In immunoelectrophoretic analyses one common antigen was demonstrated in antigen preparations from herpes simplex virus types 1- and 2- (HSV-1 and HSV-2), bovine herpes mammillitis (BHM) virus-, and B virus-infected cells solubilized by Triton X-100. The antigen was also demonstrated in solubilized purified HSV-1 and BHM virus. The common antigen was identified as antigen 11 of HSV-1 or HSV-2. Differences were found in the polypeptide composition of the related antigens when isolated from the four different herpesviruses, but a glycopolypeptide with a molecular weight of 125,000 was present in each of the four different antigen preparations, indicating that this polypeptide carried the common antigenic determinants.     

Antigenic and structural relationships of the surface antigens of hepatitis B virus, ground squirrel hepatitis virus, and woodchuck hepatitis virus.

The surface antigens of human hepatitis B (HBsAg), ground squirrel hepatitis (GSHsAg), and woodchuck hepatitis (WHsAg) viruses were compared serologically, and their major polypeptides were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and tryptic peptide mapping. Results showed that both GSHsAg and WHsAg are antigenically cross-reactive, that their major pairs of polypeptides have identical mobilities on sodium dodecyl sulfate gels, and that the major polypeptides of GSHsAg and WHsAg migrate faster in sodium dodecyl sulfate-polyacrylamide gel electrophoresis than do the corresponding bands of HBsAg. The peptide maps of the major (P-22) surface antigen polypeptides of GSHsAg and WHsAg showed that they shared over half of their spots. Peptide mapping of HBsAg subtypes indicated a close relationship between the major polypeptides (P-24) of adw and adr and a more distal relationship to ayw. Only about 25% of the spots shared by the combined HBsAg subtypes were also found in the peptide maps of GSHsAg and WHsAg, indicating at least some structural homology among the major polypeptides of the human and animal virus surface antigen particles. This is also reflected in the serological cross-reactivity among HBsAg, GSHsAg, and WHsAg. Further, the detection of ground squirrel and woodchuck antigens by Ausria II radioimmunoassay, combined with peptide mapping data indicating the common origin of these viruses, suggests that the common a determinant is shared by each and is restricted to approximately 25% of the sequences in their major polypeptides.     

Biochemical, biophysical, and biological properties of densonucleosis virus. I. Structural proteins.

The polypeptide composition of highly purified densonucleosis virus was studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The viral proteins showed a different behavior in sodium dodecyl sulfate-gels in comparison with the marker proteins. Therefore, the molecular weights were estimated by analyzing the retardation of the electrophoretic mobility of these proteins in gels with increasing polyacrylamide concentrations. Four structural proteins with molecular weights of 49,000, 58,500, 69,000, and 98,000 were found, ant they were designated p49, p59, p69, and p98, respectively. There are several indications that p98 is a dimer of p49. The relative quantity of the structural proteins in a virion suggests that at least p49 (accounting for +/-70% of total protein mass) is a capsid protein and that there will be 12 capsomers per virion.     

Glycoprotein 110, the Epstein-Barr virus homolog of herpes simplex virus glycoprotein B, is essential for Epstein-Barr virus replication in vivo.

The Epstein-Barr virus (EBV) glycoprotein gp110 has substantial amino acid homology to gB of herpes simplex virus but localizes differently within infected cells and is essentially undetectable in virions. To investigate whether gp110, like gB, is essential for EBV infection, a selectable marker was inserted within the gp110 reading frame, BALF4, and the resulting null mutant EBV stain, B95-110HYG, was recovered in lymphoblastoid cell lines (LCLs). While LCLs infected with the parental virus B95-8 expressed the gp110 protein product following productive cycle induction, neither full-length gp110 nor the predicted gp110 truncation product was detectable in B95-110HYG LCLs. Infectious virus could not be recovered from B95-110HYG LCLs unless gp110 was provided in trans. Rescued B95-110HYG virus latently infected and growth transformed primary B lymphocytes. Thus, gp110 is required for the production of transforming virus but not for the maintenance of transformation of primary B lymphocytes by EBV.     

Infectious, linear, unintegrated DNA of Moloney murine leukemia virus.

A closed circular, double-stranded infectious DNA of Moloney leukemia virus has been described previously. The present report characterizes a second type of infectious, unintegrated viral DNA which is linear, largely double stranded, and of mass comparable to that of the closed circular viral DNA. The linear form is of nonpermuted sequence, and SalI endonuclease cleaves at one site 45% from one end.     

Host-specificities of papillomavirus, Moloney murine sarcoma virus and simian virus 40 enhancer sequences.

The bovine papillomavirus (BPV-1), Moloney murine sarcoma virus (MoMuSV) and simian virus 40(SV40) genomes have been shown to contain sequences termed 'enhancers' which activate the expression of linked genes. DNA fragments containing these three enhancers have been inserted into recombinant plasmids upstream from the herpes simplex virus thymidine kinase (tk) gene, and their effect on tk expression monitored. Two types of assay have been used. Firstly, the ability of recombinant plasmids to transform TK- recipient cells to a TK+ phenotype was measured. Secondly, the amount of tk-specific RNA and TK enzyme activity transiently expressed after DNA transfection was determined. Both types of assay gave similar results. The enhancers increased tk gene expression by regulating the amount of full length tk mRNA present shortly after transfection independent of gene copy number. Furthermore, marked species specificity in the relative efficiencies of different enhancers was observed, including that of the BPV-1 enhancer for the first time. The MoMuSV enhancer showed preference for murine fibroblasts, while the papillomavirus enhancer showed a marked preference for bovine cells. In contrast, the SV40 enhancer gave the same relative increase in tk gene expression in the murine, rat, bovine and human cells tested.