Internal Viral Proteins Research Articles

A host range restriction in infectious pancreatic necrosis virus maps to the large RNA segment and involves virus attachment to the cell surface

Infectious pancreatic necrosis virus (IPNV) strain JV can replicate in both chinook salmon embryo (CHSE-214) cells and fathead minnow (FHM) cells; but another strain (OV) cannot replicate in FHM cells. In addition, a spontaneous host range mutant of JV has been isolated which has the same host range restriction as OV. By genetic reassortment of the two RNA segments in IPNV, we have determined that the gene responsible for the host range phenotype in both OV and the mutant maps to the large RNA segment which encodes the major capsid protein, the internal virion protein, and a nonstructural protein. Attachment of virions to the surface of FHM cells was impaired in OV and in the mutant. We have succeeded in transcapsidating OV RNA into the capsid of JV, thereby facilitating the entry of OV RNA into FHM cells. FHM cells infected with this transcapsidated virus showed abundant OV-specific antigen synthesis, suggesting that the host range block was solely at the level of virus attachment to the cell surface.

Precursor polypeptides to structural proteins of visna virus.

Visna virus is a retrovirus which replicates in fibroblast-like cells of the sheep choroid plexus through a lytic cycle. Visna virions contain three major low-molecular-weight proteins (p30, p16, and p14) which, together with the genomic RNA and several molecules of reverse transcriptase, constitute the core structure of the virions. The core is surrounded by an envelope containing a major glycoprotein (gp135). By analogy with the oncoviruses, these three groups of structural proteins (i.e., the internal proteins, the envelope glycoprotein, and the reverse transcriptase) are probably encoded by the gag, env, and pol genes, respectively. To elucidate the genetic organization of the visna virus genome and its expression, we studied the synthesis of viral proteins in infected sheep choroid plexus cells. Intracellular viral proteins were detected by immunoprecipitation of pulse-labeled cell extracts with monospecific sera raised against p30, p16, and gp135 and resolution of the proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoprecipitation with anti-p30 and anti-p16 sera allowed the characterization of the 55,000-dalton polypeptide precursor to internal virion proteins p30, p16, and p14 (Pr55(gag)). Tryptic peptide mapping confirmed the precursor-product relationship between Pr55(gag) and the three internal proteins. In addition, a gag-related polypeptide of 150,000 daltons was also detected. This polypeptide, which was less abundant than Pr55(gag), is a likely precursor to the viral reverse transcriptase (Pr150(gag-pol)). Pr55(gag) and Pr150(gag-pol) are not glycosylated. The precursor related to major envelope protein gp135 is a glycosylated polypeptide with an average molecular weight of 150,000 (gPr150(env)). Pulse-chase experiments indicated that gPr150(env) matures into glycoprotein gp135 intracellularly; however, gp135 was never preponderant in cell extracts. The non-glycosylated from of gPr150(env), which accumulated in the presence of 2-deoxy-d-glucose, appeared as a polypeptide of about 100,000 daltons. These results indicated that visna virus codes for the largest non-glycosylated env-related precursor among all of the retroviruses and therefore probably contains the largest env gene.

Identification of the proteins encoded by each genome segment of infectious pancreatic necrosis virus.

Abstract Infectious pancreatic necrosis virus (IPNV) contains two segments of double-stranded RNA. Temperature-sensitive (ts) mutants were isolated from two serotypes (1 and 2) of IPNV after treatment with nitrosoguanidine. Crosses between is mutants showed that two recombination groups (named I and II) were present within each serotype. Crosses between serotypes yielded viable ts + recombinant viruses that were shown by gel electrophoresis to contain one RNA segment from each parent. Analysis of the proteins of each hybrid showed that the large RNA coded for the major capsid protein, the internal virion protein, and a nonstructural protein, and the small RNA coded for the 100,000-dalton protein.

A fluorescence photobleaching study of vesicular stomatitis virus infected BHK cells. Modulation of G protein mobility by M protein.

The mobility of vesicular stomatitis virus (VSV) G protein on the surface of infected BHK cells was studied by using the technique of fluorescence photobleaching recovery. The fraction of surface G protein that was mobile in that time scale of the measurement (minutes) was at least 75%, a relatively high value among cell surface proteins so far observed. For studies of the effect of an internal viral protein (M protein) on G protein mobility, cells infected with wild-type VSV were compared with those infected with temperature-sensitive VSV mutants of complementation group III, which contains lesions in the M protein. At the permissive temperature, a pronounced decrease in the mobile fraction of surface G was observed for each of three mutants studied, while mobility of surface G at the nonpermissive temperature was indistinguishable in mutant and wild-type infected cells. A significantly lower mobile fraction of G protein was also observed in SV40 transformed 3T3 cells infected with wild-type VSV, but not in 3T3 or chick embryo fibroblast cells similarly infected. None of the variables tested had a measurable effect on the lateral diffusion coefficient of the mobile G protein. These results are interpreted as modulation of the mobility of a specific cell surface protein by a specific intracellular protein.

Mechanisms in T cell leukemogenesis. II. T cell responses of preleukemic BALB/c mice to Moloney leukemia virus antigens.

The T cell responses of Moloney leukemia virus (MoLV)-infected preleukemic BALB/c mice were examined. The major in vitro response detectable was T cell blastogenesis in response to the major viral envelope protein MoLV gp71 and an internal viral protein p12. The majority of the preleukemic mice had readily detectable responses to gp71, whereas the presence of a response to p12 was less consistent. With both antigens, T cell blastogenesis showed typical antigen response characteristics similar to those detected in other immune responses to C-type viruses. Proliferation was dependent on a Thy-1+, Lyt-1+, 2- population and was macrophage-independent. In contrast to most immune responses to C-type viruses, which are temporally restricted, T cell blastogenesis was detectable throughout the preleukemic period of 4 to 16 wk of age. During this period neither gp71-specific T cells nor PHA-responsive T cells were found to express viral antigens. The correlations between T cells responding to gp71 and leukemia were examined. Under conditions in which MoLV inoculation of BALB/c mice does not induce leukemia, no T cell responses were deectable. These results suggest a causal relationship between the presence of antigen-specific T cells and the ability of MoLV to induce leukemia. The results are discussed with reference to the possible role of chronic immune stimulation in virus-induced leukemogenesis.

Isolation of native internal structural proteins of avian oncovirions by SP-Sephadex column chromatography

Upon treatment of avian oncovirions with 1% Triton X-100 in a buffer pH 5.0 of low ionic strength containing 1% 2-mercaptoethanol, p27, p19 and p15 internal virion proteins have been quantitatively and selectively solubilized. These proteins have been separated by ion-exchange chromatography on a SP-Sephadex column, yielding well-resolved peaks of p27, non-phosphorylated p19, phosphorylated p19 and p15 proteins. The identity and purity of the isolated proteins has been documented by gel electrophoresis and amino acid composition. High proteolytic activity of the p15 protein indicates that the isolated proteins had not been denatured. This method may be useful for the preparation of pure oncoviral proteins suitable for studies of their chemical, immunological and especially functional properties.

Evolution of pandemic influenza virus strains.

It was found by the technique of molecular hybridization that the pandemic influenza virus strains of 1957 (H2N2) and 1968 (H3N2) evolved by reassortment of RNA segments from the foregoing pandemic strains, replacing four genes including those coding for haemagglutinin (HA) and neuraminidase (NA) (1957) or only that coding for the haemagglutinin (1968), respectively. The earlier pandemic strains from 1918-9 (as deduced from the swine influenza strain (Hsw1N1), which is assumed to be a survivor of the Spanish influenza), from 1933-4 (H0N1), and from 1947 (H1N1) were derived from each other through a number of point mutations only. The Russian strain from 1977 (H1N1) is genetically almost identical with the FW strain from 1950 (H1N1). In contrast to the conserved genes coding for the internal viral proteins, the genes coding for the viral surface glycoproteins consist of a relatively small highly conserved part which presumably is responsible for the functional integrity of the gene products, and the relatively large variable region which probably determines the antigenic property of their gene products. This structural feature of the surface glycoprotein genes explains the fast antigenic drift by mutations in the variable region and selection by the immune response of the host.

An enzyme-linked immunoassay for the detection of antibodies to the mouse mammary tumor virus: Application to human breast cancer

An enzyme-linked immunoassay (ELISA) was developed, using the mouse mammary tumor virus (MMTV) fixed to wells of a microtiter plate, for the determination of antibodies to MMTV. The intensity of the final color change was dependent upon virus or viral antibody concentration. MMTV antibody was readily detectable in sera diluted as much as 1 : 2800. Fixed MMTV bound antibodies to an internal viral protein (p 28) as well as to viral envelope components (gp 52, gp 34), demonstrating that the virus was rendered permeable by our procedure. Applying this assay to human sera, significant differences ( P < 0.005) in IgG binding to MMTV were detected between sera of breast cancer patients, benign breast disease patients and healthy individuals. 26% of breast cancer-derived sera contained MMTV binding antibody; 10% of benign sera or 8% of normal sera were also positive. The reactivity of human IgG with MMTV was blocked by prior incubation of the virus with antisera to gp 34 or, to a lesser extent, with gp 52. The results demonstrate that MMTV antibodies can be quantitated by this simple, rapid and inexpensive procedure.

Vicinal relationships between the major structural proteins of murine mammary tumor virus

Spatial relationships between the major structural proteins of murine mammary tumor virus (MuMTV), a type-13 RNA tumor virus, were investigated. Untreated virions, when analyzed by diagonal gel electrophoresis, contained two major species of disulfide-linked protein oligomers, i.e., a high-molecular-weight aggregate of p18 and a dimer composed of gp34 and p28. These complexes are termed “native crosslinked oligomers.” In addition, induced crosslinking by dithiobis succinimidyl propionate (DTSP) of vicinal proteins of whole virions or rosettes (viral membranes with attached spikes or projections) was also investigated. Diagonal gel electrophoretic analyses of DTSP-crosslinked virions revealed dimers of gp55 · gp55 and gp55 · gp34 as well as the native crosslinked oligomers. No DTSP-induced crosslinking of internal viral proteins was observed. Similar studies with rosettes indicated the formation of DTSP-induced crosslinked dimers consisting of gp55·gp55 and gp34 · gp34 in addition to gp34 · p28. From these studies, we conclude that the MuMTV projection is a homooligomer of gp55 which is directly apposed to the transmembrane protein, gp34, on the external surface. In addition, a portion of the gp34 transmembrane protein interacts internally with the major core shell protein (p28) as a native crosslinked oligomer. The latter observation most probably accounts for the eccentric location of the MuMTV nucleoid. A model which derives from these studies is discussed vis-à-vis virus assembly and previous studies of MuMTV structure.

Conserved peptides in the proteins of vesicular stomatitis virus

The structural proteins of several isolates of the Indiana serotype (Orsay, Glasgow, and San Juan) of vesicular stomatitis virus (VSV) were compared by sizing peptides generated by digestion with Staphylococcus aureus V8 protease or α-chymotrypsin. These digestions yielded nearly identical peptide patterns for all the VSV proteins except for the glycoprotein, indicating that the major surface antigen, even of these closely related isolates, is subject to change. When two serologically distinct VSV isolates (Indiana and New Jersey) were similarly examined, each protein had a peptide pattern characteristic of that serotype. The glycoprotein of the two serotypes had no peptides in common, while the internal viral proteins L, N, NS, and M showed various degrees of similarity. Digests of the L (polymerase) and M (membrane) protein, in particular, generated groups of small peptides with identical mobilities, suggesting that there may be core regions in the Indiana and New Jersey strain which are conserved. These conserved regions may in turn represent essential functions of rhabdovirus proteins and are promising targets for broad-spectrum antivirals.

Purification and characterization of proteins excreted by cells infected with herpes simplex virus and their use in diagnosis

A virus-specific glycoprotein, excreted by rabbit kidney cells infected with herpes simplex virus, type 1, has been purified and radioiodinated. This problem has an electrophoretic mobility in polyacrylamide gels similar to that of one of the largest glycoproteins of the virions of herpes simplex virus (Molecular Weight ∼132,000) and shares antigenic determinants with an internal virion protein. After radioiodination, this glycoprotein can be used for the detection of virus-specific antigens in patients with herpes simplex virus encephalitis.

A single gene determines the host range of influenza virus.

THE genome of influenza virus consists of eight distinct segments of single stranded RNA as determined by electrophoresis in polyacrylamide gels1–3. Each of these segments codes for one of the eight virus specified polypeptides4–9 and is therefore believed to be a single gene which functions by a monocistronic messenger RNA10. Because different strains of influenza may vary in base sequence homology9 or in the electrophoretic mobilities of their RNA segments and infected cell polypeptides11,12, it is often possible to determine the parental origin of every gene in an interstrain recombinant. Such procedures have been used to investigate the physiological properties associated with some of the influenza virus specified proteins9,13,14. I describe here the application of this approach to an investigation into the genetic control of host range. The results show that the difference in host range between two avian influenza viruses depends solely on the properties of one of the internal viral proteins associated with RNA polymerase activity.

Matrix protein from influenza A virus and its role in cross-protection in mice

The matrix (M) protein of influenza A virus, one of the two group-specific internal proteins of the virion, was isolated in a pure form, and its immunogenicity was stable to heating at 100 degrees C for 2 min. Mice immunized with isolated M protein in complete Freund adjuvant and subsequently infected with influenza virus cleared virus more rapidly from their lungs than did unimmunized mice. Despite the rapid clearance of virus, the mice developed pneumonia that was at least as severe as in unimmunized mice. Preliminary studies suggest that the rapid clearance of influenza virus from the lungs of mice immunized with M protein may be initiated by a cell-mediated rather than a humoral response. The mechanism by which a cross-reactive internal virion protein can initiate clearance of the different subtypes of influenza is not clear. Perhaps the M protein is exposed on the surface of the virus-infected cell and is responsible for the cross-reactivity at the cytotoxic T-cell level recently detected between influenza A virus subtypes.

Genetic analysis of adenovirus type 2: VII. Cleavage-modified affinity for DNA of internal virion proteins

The core structures derived from wild-type (wt) Ad2 and ts1 virions, by disintegration with N-laury sarcosine and pyridine, were analyzed biochemically and by electron microscopy. Disintegration of wt virus with Sarkosyl yielded cores of density 1.58 g/ml (in CsCl) whereas the particles synthesized by ts1 at 39° (ts1-39°) generated subviral structures ranging in density from 1.58 to 1.72, indicating the increased lability of the mutant virions. The wt core (1.58 g cm −3) contained predominantly polypeptide VII, while the ts1-39° cores only contained traces of this polypeptide. Significantly, Pre-VII, which is the principal form of this polypeptide in the ts1-39° virions, was completely absent from the cores. Electron microscopy of wt and ts1-Sarkosyl cores revealed identical types of structures on negative staining, although the ts1-39° cores were found to unfold more rapidly in the presence of high salt. The polypeptide analysis of the pyridine cores showed significant differences between wt and ts1. Wild-type core contained polypeptides V, VII, and 14K; ts1-33° core contained V, Pre-VII, VII, traces of IVa2, Pre-VI, VI, and possibly IX and X; while ts1-39° core contained V, Pre-VI, and Pre-VII. These findings clearly establish structural differences between wt and ts1 cores, most important the differential affinity of precursor polypeptides for DNA in ts1 virions under different conditions of disintegration. It is suggested that the loss of infectivity by the mutant is due to the altered composition and structure of the viral core.

Comparative primary structure analysis of the p30 protein of woolly monkey and gibbon type C viruses

The primary structures of the major internal virion protein (p30, approximate molecular weight, 30,000) of gibbon (GaLV) and woolly monkey [SSV(SSAV)] type C viruses were compared by tryptic peptide mapping and NH 2-terminal amino acid sequence analysis covering the initial 28 residues. No differences were detected by either technique. The results are consistent with the high degree of relationship indicated by serological techniques. While the p30 of mouse type C viruses is immunochemically distinct from GaLV and SSV (SSAV) p30s, the amino acid sequence comparisons indicate that the major differences in the NH 2-terminal sequence are based on a large number of inserted residues in the latter two proteins.

Defective cleavage of a precursor polypeptide in a temperature-sensitive mutant of avian sarcoma virus

Noninfectious virus particles released at the nonpermissive temperature from cells infected with the temperature-sensitive mutant LA334 of avian sarcoma virus B77 have been investigated. These particles bud atypically, have improperly formed core structures, are heterogeneous in size and on a density gradient, and are also slightly more dense than wild-type virus. One major new viral-coded protein of 23,000 molecular weight, p23, and several minor abnormally cleaved and/or partially degraded viral-specific peptides are incorporated into the noninfectious particles. As a result of tryptic peptide analyses of some of these new proteins, it could be proposed that the defect of this mutant is in the cleavage of pr76, the 76,000 molecular weight precursor for the internal viral proteins of avian tumor viruses (ATV). In addition, we have detected one methionine-labeled viral tryptic peptide with an altered amino acid composition, which may represent the site of the temperature sensitive lesion.

Autogenous Humoral Immunity to Baboon Xenotropic Endogenous Type C Virus 2

A radioimmune precipitation assay was used to evaluate the humoral immune response of baboons (Papio cynocephalus) to their endogenous, xenotropic, type C (M7) virus. Using 125I or [3H]leucine-labeled intact virus preparation, we demonstrated that baboons possess naturally occurring virus-binding antibodies to this virus. Antibody appeared directed against envelope and not internal viral proteins. Immunoglobulins responsible for virus-binding were predominant in the IgG and IgA serum fractions, whereas IgM participated to a minor degree. The biologic activities of these immunoglobulins were investigated by means of a focus reduction assay for virus neutralization with a murine sarcoma virus (M7) pseudotype. We determined that, despite the presence of considerable virus binding, none of the normal baboon sera tested possessed neutralizing activity for M7 virus.

Expression of murine oncornavirus proteins in synchronized cells

Normal rat kidney (NRK) cells and NRK cells transformed by Kirsten murine sarcoma-leukemia virus (NRK-K) were synchronized by double thymidine block. Cells were examined by indirect cytoplasmic immunofluorescence during synchronous growth for the presence of the 10,000, 12,000, and 30,000 dalton internal virion proteins (p10, p12, and p30) and for the 71,000 dalton major viral glycoprotein (gp 71) using monospecific antisera to p10, p12, p30, and gp 71 proteins of Friend murine leukemia virus. Expression of p30 and gp 71 was increased during late S + G 2 phase and was decreased during M and G 1 phases of the cell cycle in NRK-K cells. Expression of p30 was similarly coordinated to the cell cycle in NRK cells. Patterned expression of p10 and p12 was not detected.

Translation of avian myeloblastosis virus RNA in a reticulocyte cell-free system

AMV-RNA was translated into a precursor polypeptide of 76,000–80,000 daltons in a reticulocyte cell-free system. Besides this high molecular weight precursor, several smaller precursor polypeptides and the four major internal structural viral proteins were also synthesized. These virus-specific translation products were detectable after immunoprecipitation with antiserum against the gs-antigens of AMV.

Relationship of infectious murine leukemia virus and virus-related antigens in genetic crosses between AKR and the Fv-1 compatible strain C57L.

In a further genetic study of murine leukemia virus (MuLV) and its components we examined the backcross C57L X (C57L X AKR). This population was selected because strains AKR and C57L are both Fv-1n, and the restriction which the Fu-1b allele imposes on the output of virus was thereby obviated. The segregants were scored for three characters: (a) infectious Gross-AKR-type MuLV (V), in the tail; (b) group-specific antigen indicative of p30 internal viral protein, in spleen; and (c) GIX antigen, now thought to be indicative of gp69/71 viral envelope glycoprotein, on thymocytes. Our conclusions are: (a) It is confirmed that the AKR mouse has two unlinked chromosomal genes, Akv-1 and Akv-2, each of which can independently give rise to the life-long high output of MuLV that is characteristic of AKR mice. (b) Of the eight phenotypes that could possibly be derived from segregation of the three pairs of independent alternative traits, seven were observed, but on progeny testing only three were shown to reflect stably heritable genotypes; these were V+p30+GIX+ and V-p30-GIX- (the parental types) and V-p30+GIX+. A third, newly identified AKR gene, designated Akvp, segregating independently of Akv-1 and Akv-2, also determines expression of p30 and GIX but in this case independently of XC-detectable MuLV. (c) The four remaining observed phenotypes, which did not breed true on progeny testing, involved mostly antigen-negative parents yielding antigen-positive progeny; it is likely that these discrepancies represented suppression of phenotype by a maternal resistance factor.