Hog cholera virus: molecular composition of virions from a pestivirus.

Virions from hog cholera virus (HCV), a member of the genus Pestivirus, were analyzed by using specific antibodies. The nucleocapsid protein was found to be a 14-kDa molecule (HCV p14). An equivalent protein could also be demonstrated for virions from another pestivirus, bovine viral diarrhea virus. The HCV envelope is composed of three glycoproteins, HCV gp44/48, gp33, and gp55. All three exist in the form of disulfide-linked dimers in virus-infected cells and in virions; HCV gp44/48 and gp55 each form homodimers, whereas gp55 is also found dimerized with gp33. Such complex covalent interactions between structural glycoproteins have not been described so far for any RNA virus.

Oligomer formation of the gB glycoprotein of herpes simplex virus type 1.

Oligomer formation of the gB glycoprotein of herpes simplex virus type 1 was studied by sedimentation analysis of radioactively labeled infected cell and virion lysates. Fractions from sucrose gradients were precipitated with a pool of gB-specific monoclonal antibodies and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Pulse-labeled gB from infected cell was synthesized as monomers and converted to oligomers posttranslationally. The oligomers from infected cells and from virions sedimented as dimers, and there was no evidence of higher-molecular-weight forms. To identify amino acid sequences of gB that contribute to oligomer formation, pairs of mutant plasmids were transfected into Vero cells and superinfected with a gB-null mutant virus to stimulate plasmid-specified gene expression. Radioactively labeled lysates were precipitated with antibodies and examined by SDS-PAGE. Polypeptides from cotransfections were precipitated with an antibody that recognized amino acid sequences present in only one of the two polypeptides. A coprecipitated polypeptide lacking the antibody target epitope was presumed to contain the sequences necessary for oligomer formation. Using this technique, two noncontiguous sites for oligomer formation were detected. An upstream site was localized between residues 93 and 282, and a downstream site was localized between residues 596 and 711. Oligomer formation resulted from molecular interactions between two upstream sites, between two downstream sites, and between an upstream and a downstream site. A schematic diagram of a gB oligomer is presented that is consistent with these data.

Rotavirus spike structure and polypeptide composition.

Negatively stained preparations of rotavirus imaged with a low dose of electrons provide sufficient contrast to reveal surface projections or spikes. The number of spikes found projecting from different particles indicates that not all 60 peripentonal sites are occupied. Treatment at pH 11.2 with 250 mM ammonium hydroxide specifically removes the spikes, yielding smooth double-shelled particles of the same diameter as that of the native virus. Protein analysis confirms that the released spikes are composed of polypeptide VP4 (or its two cleavage products VP5* and VP8*) and that the smooth particle retains the other major outer shell protein VP7. Spikeless particles can be decorated by a monoclonal antibody specific for the major immunodominant neutralizing domain of VP7, implying that removal of the spikes does not denature the VP7 that is retained on the surface of the smooth particle.

Localization of the influenza virus nucleoprotein: cell-associated and extracellular non-virion forms.

Both the supernatant of influenza virus-infected chick embryo cells and allantoic fluid containing influenza virus were shown to contain non-virion nucleoprotein (NP), which reacted readily with anti-NP monoclonal antibodies. Adsorption onto erythrocytes and centrifugation at 70,000 g for 2 h resulted in the removal of about 20% of the extracellular NP, whereas centrifugation at 100,000 g for 4 h eliminated about 50%, and practically all [3H]uridine-labelled virions. These results suggest that of the extracellular NP about 30% exists in the form of ribonucleoprotein, about 20% is precipitated with virions and about 50% occurs as free molecules. Comparative analysis of the kinetics of the accumulation of NP in the supernatant of infected cells, on the cell surface and inside the cells in relation to virus production, showed that there is a significant correlation between them.

Biophysical and biochemical properties of an unusual birnavirus pathogenic for rotifers.

A cytoplasmic dsRNA virus, rotifer birnavirus (RBV), has recently been isolated from the rotifer Brachionus plicatilis and is associated with a high mortality rate. Histologically, the viral lesions consist of characteristic inclusions, particularly amorphous dense bodies containing occluded particles. Purified virions are about 59 nm in diameter, single-shelled and display four capsomers per edge. The purified virions have a buoyant density of 1.290 (full particles) and 1.250 (empty particles) in CsCl gradients. Four major structural polypeptides of MrS 60K, 52K, 33K and 27K were detected by SDS-PAGE. The genome is composed of two linear segments of dsRNA with MrS of 2.45 x 10(6) and 2.31 x 10(6); additionally, small circular ssRNA molecules were detected by electrophoresis in overloaded agarose gels, but their significance is currently unknown. Except for this last feature and the structural instability of purified virions under freeze storage, all the other biochemical and biophysical characters indicate that RBV is a member of the Birnaviridae family with, for the moment, a unique position in this group.

Genetic and biochemical characterization of vaccinia virus genes D2L and D3R which encode virion structural proteins.

Polyclonal antisera raised against fusion proteins containing portions of the vaccinia virus D2L and D3R proteins were prepared. Immunoprecipitation of pulse-labeled infected cell extracts and Western blot analysis demonstrated that genes D2L and D3R encode 16.9- and 27-kDa proteins, respectively. Both are synthesized late during infection and there is no evidence for proteolytic processing of either protein. Western blots of purified virus and subvirion fractions showed that D2L and D3R are virion components, residing in a detergent-insoluble fraction, containing viral core structural proteins. Trypsin sensitivity experiments suggest that each is found in an equivalent position within the virus core. Pulse-chase analysis showed that both proteins exhibit biphasic stability in which an unstable nascent component is replaced by a stable form. This observation suggests that the stable component results from the insertion of D2L and D3R into an immature core structure. The DNA sequence of four ts mutants previously mapped to genes D2L and D3R is reported. Analysis of the ability of each mutant to synthesize and process viral proteins showed that protein synthetic patterns were indistinguishable from wild type, however, three of the four mutants were defective in the processing of the major virion structural precursor, p4a. Unlike the biphasic stability observed in wild-type infected cells, D2L and D3R were totally degraded in cells infected at 40 degrees with any of the four ts mutants. Stability of the D2L and D3R proteins, in cells treated with rifampicin, is unaffected which demonstrates that a block in morphogenesis is not directly responsible for the observed instability of the mutant proteins.

Expression of the major inner capsid protein of the group B rotavirus ADRV: primary characterization of genome segment 5.

A complete cDNA copy of the fifth RNA segment of the human group B rotavirus, ADRV, has been cloned into plasmid AD512. Gene segment 5 contains 1269 bases and encodes one long open reading frame of 391 amino acids beginning at base 31 and terminating at base 1203. The gene 5 polypeptide, expressed in vitro in a rabbit reticulocyte lysate, comigrates with the 44-kDa major inner capsid protein present on EDTA treated ADRV virions. The gene 5 protein is immunoprecipitable by hyperimmune serum to ADRV, human ADRV convalescent serum and by a group B-specific monoclonal antibody. In addition, this protein shares amino acid identity and similarity with the VP6 proteins from group C and group A rotavirus strains. The ADRV VP6 equivalent protein appears to be more closely related to the group C VP6 than the Group A VP6 polypeptide and a common ancestral rotavirus VP6 precursor protein is suggested. As a result, the fifth RNA segment of ADRV defines the major inner capsid protein, or VP6 equivalent, in the group B rotavirus. Expression of the ADRV VP6 equivalent protein is potentially useful for evaluating the prevalence of serum antibodies to group B rotavirus in human and animal populations as well as for generating antibodies for the direct detection of group B rotavirus antigen.

[The anomalous isoelectric properties of influenza virus matrix protein M1]. / Anomal'nye izoélektricheskie svoistva matriksnogo belka M1 virusov grippa.

The isoelectric point (pI) values of matrix protein M1 of influenza A, B, and C viruses, calculated theoretically on the basis of its primary structure, were found to be about 10.0. However, experimental pI determination by electrofocusing in ampholyte-containing polyacrylamide gel showed it to be 5.0 for M1 protein isolated from A/WSN/33 (H1N1) and A/Aichi/2/68 (H3N2) viruses by mild deproteinization with nonionic detergents. This marked discrepancy between experimental and theoretical pI values indicated that influenza virus matrix protein M1 possessed an unusual tertiary structure and/or intensive posttranslational addition of charged residues.

A Gly1 to Ala substitution in poliovirus capsid protein VP0 blocks its myristoylation and prevents viral assembly.

Capsid protein VP4 of poliovirus is acylated with myristic acid via an amide linkage to its N-terminal glycine residue. Our previous studies suggested that myristic acid plays a role in poliovirus assembly and in the early events of infection. In order to understand better its role in the assembly process, we introduced a Gly1 to Ala amino acid substitution in the myristoylation signal sequence of VP4. This substitution prevented VP0 myristoylation in vivo and abolished the infectivity of genomic transcripts harbouring the mutation. These mutated RNAs were still able to replicate in the transfected cells but the assembly processes were inefficient and no mature virions could be detected.

Two new formulas for triangulation number T.

The present paper reports two new formulas for calculating triangulation number T. T = L2/l2 and T = 1.45 r2/l2, where L is the distance between pentons, l the distance between any two adjacent capsomeres, r the radius of viral nucleocapsid. The formulas have been verified and applied. It is worth noticing that the triangulation number, viral size and distance between capsomeres are fully connected by the formula r/the square root of Tl = 0.83, and the capsid parameters of all icosahedral viruses are unified in one constant, 0.83.

cDNA cloning and nucleotide sequence of the wheat streak mosaic virus capsid protein gene.

The 3'-terminal region of wheat streak mosaic virus (WSMV) genomic RNA was cloned and a cDNA sequence of 1809 nucleotides upstream of the poly(A) tract was determined. The sequence contains a single open reading frame of 1662 nucleotides and a 3' untranslated region of 147 nucleotides. Translation products from WSMV RNA and WSMV cDNA transcripts were immunoprecipitated by WSMV capsid protein antiserum, indicating that the 3'-terminal region of WSMV RNA encodes the capsid protein. Five potential N-terminal capsid protein protease cleavage sites were identified, which would yield proteins ranging from 31.7K to 46.8K. Alignment of the deduced amino acid sequence of the WSMV capsid protein with those of other potyviruses showed significant, but limited, identity as compared to the alignment of two or more aphid-transmitted potyviruses. Although WSMV has characteristics distinct from potyviruses, because of its particle morphology, translation strategy apparently based on polyprotein processing, the ability to form cytoplasmic cylindrical inclusions and the degree of capsid protein homology with aphid-transmitted potyviruses, it should be considered a member of the potyvirus group.

Identification and characterization of a novel non-infectious herpes simplex virus-related particle.

During gradient purification of herpes simplex virus type 1 (HSV-1) two bands of particles were observed: a sharp lower band and a more diffuse upper band. The lower band contained almost exclusively HSV-1 virions (H particles) whereas the upper band consisted of membrane-enclosed particles (L particles). These L particles resembled the virions in appearance, but lacked the viral nucleocapsid and were not infectious. Many polypeptides of the viral envelope and the tegument were common to both types of particles. The H particles had polypeptide profiles typical of HSV virions. The L particles contained at least three phosphoproteins (175K, 92K and 55K) and a further two phosphorylated polypeptides not normally observed in virion profiles which comigrated with the 134K and 60K glycoproteins. This clearly indicates that the novel L particles were not merely virions which had formed without the inclusion of a nucleocapsid or virions which had subsequently lost their nucleocapsid during preparative handling. Thus these novel L particles are genuine products of the infectious processes occurring when HSV-1 replicates.

[Multiple forms of the NS1 protein as the main component of the nonvirion ("soluble") antigen of the tick-borne encephalitis virus]. / Mnozhestvennye formy belka NS1 kak osnovnogo komponenta nevirionnogo ("rastvorimogo") antigena virusa kleshchevogo éntsefalita.

The nonstructural virus-specific NS1 protein of tick-borne encephalitis (TBE) virus was found to be the main antigenic component of the nonvirion ("soluble") antigen which had been shown previously to present a complex protein structure closely associated with the infected cell membranes. In the presence of sodium dodecylsulphate, beta-mercaptoethanol and 8M urea NS1 protein could be detected in the form of oligomeric molecules which disintegrated to monomers after heating at 100 degrees C for 5 min. The infected cell membrane-associated NS1 protein was shown to differ in electrophoretic mobility from the NS1 protein released into the culture fluid in the course of virus infection. The conditions for detection of NS1 protein and its oligomeric forms by PAG electrophoresis and immune blotting were determined. Dimeric forms of NS1 protein were found to contain products of its proteolysis. NS5 protein was found together with NS1 protein in TBE nonvirion antigen and also possessed antigenic activity.

Characterization of the subunit particles of feline calicivirus.

In the culture fluid from cells infected with feline calicivirus (FCV) F4 strain, the infectious and smaller non-infectious subunit particles were detected by complement fixation (CF) test after sucrose gradient centrifugation. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analyses confirmed the existence of the subunit particles of FCV, and showed that the infectious and subunit particles were mainly composed of 65K capsid protein. The subunit particles were further purified by ion-exchange chromatography and sucrose gradient centrifugation. The purified subunit and infectious particles had the same neutralizing epitope on 65K protein detected by immunoblot analysis with a neutralizing monoclonal antibody. Antigenic comparison between the infectious and subunit particles by the CF tests using an antiserum against heterologous strain of FCV F14 indicated that the subunit particles might have more highly conserved antigens of FCV than the infectious particles.

Rotational mobility of Sendai virus glycoproteins in membranes of fused human erythrocytes and in the envelopes of cell-bound virions.

The rotational mobility of Sendai virus envelope glycoproteins (F, the fusion protein, and HN, the hemagglutinin/neuraminidase) was determined by using erythrosin (ER)-labeled monovalent Fab' antibody fragments directed specifically against either F or HN. By use of time-resolved phosphorescence anisotropy, the rotational mobility of Er-Fab'-viral glycoprotein complexes was studied both in the envelopes of unfused virions bound to erythrocyte ghosts and in the target cell membrane after fusion had occurred. The rotational correlation times (phi) of Er-Fab'-labeled F and HN were rather similar in the envelopes of bound unfused virions, but highly different in membranes of fused cells. The different phi values indicate that F and HN diffuse separately in the target cell membrane and for the major part are not complexed together. The temperature dependence of the phi values of the Er-Fab'-viral glycoprotein complexes revealed a breakpoint at 22 degrees C for the F protein both in bound virions and in the membranes of fused cells, and for the HN proteins in the envelopes of bound virions. In all these cases, the phi values increased between 4 and 22 degrees C, demonstrating a reduction in the rate of rotational diffusion. Further elevation of the temperature reversed the direction of the change in phi. This phenomenon may reflect a temperature-dependent microaggregation of F and HN saturating at ca. 22 degrees C and presumably related to the fusion mechanism since the breakpoint temperature correlates closely with the threshold temperature for virus-cell and cell-cell fusion.(ABSTRACT TRUNCATED AT 250 WORDS)

The nucleocapsid protein isolated from HIV-1 particles binds zinc and forms retroviral-type zinc fingers.

The role of zinc in retroviral gag protein function has been addressed through the application of high-resolution nuclear magnetic resonance spectroscopy to samples of the nucleocapsid protein (NCP, p7) isolated directly from infectious HIV-1 particles. Unlike reports for the NCP from avian myeloblastosis virus (AMV) particles [Jentoft et al. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7094], we find that the HIV-1 NCP binds 2 equiv of zinc tightly and stoichiometrically. Two-dimensional NMR spectroscopic studies reveal that zinc binding induces formation of folded domains that are conformationally similar to (if not identical with) structures observed previously for relevant retroviral-type (RT) zinc finger peptides [formerly called zinc fingerlike peptides; Summers et al. (1990) Biochemistry 29, 329]. This finding is consistent with the hypothesis that the inability of mutant proteins (with substituted Cys and His residues) to package viral RNA results from deficient zinc-binding capability, which may have significant consequences in the development of vaccines for the prevention of AIDS.

Ubiquitinated conjugates are found in preparations of several plant viruses.

Recently D.D. Dunigan, R.G. Dietzgen, J.E. Schoelz, and M. Zaitlin (Virology 165, 310-312, 1988) demonstrated that a small proportion of the subunits of tobacco mosaic virus particles were conjugated with the small protein ubiquitin. We have now detected ubiquitinated conjugates in immunoblots of virion preparations of several other plant viruses, using anti-human ubiquitin antiserum. Based on their polyacrylamide gel migrations, plant virus-associated ubiquitin-immunoreactive proteins were considered to be possible virus structural protein-ubiquitin conjugates of the following viruses: barley stripe mosaic, brome mosaic, cowpea mosaic (two proteins), cowpea severe mosaic (two proteins), and satellite panicum mosaic. Ubiquitinated conjugates were not detected in immunoblots of preparations of cucumber mosaic virus and Cymbidium mosaic virus. The significance of ubiquitinated viral proteins remains to be determined.

Monoclonal antibodies to the peplomer glycoprotein of coronavirus mouse hepatitis virus identify two subunits and detect a conformational change in the subunit released under mild alkaline conditions.

Monoclonal antibodies (MAbs) directed against the E2 glycoprotein of mouse hepatitis virus (MHV) have been classified according to their ability to bind to either of the two purified 90,000-molecular-weight subunits (90K subunits) of the 180K peplomeric glycoprotein E2. Correlation with previously reported information about these MAbs suggest that both of the subunits of E2 are important for viral infectivity and cell fusion. Incubation of trypsin-treated virions at pH 8.0 and 37 degrees C released only the E2N subunit from virions. The pattern of MAb reactions suggested that a conformational change occurred in the E2N subunit in association with its release from virions under mildly alkaline conditions at 37 degrees C, the same conditions which are optimal for coronavirus-induced cell fusion.

Ubiquitin in avian leukosis virus particles.

We have identified unconjugated ubiquitin as a component of avian leukosis virus (ALV). Quantitation both by immunoblotting and by protein staining showed that ubiquitin makes up about 0.5% of total viral protein, corresponding to 100 molecules per virion. This level is about fivefold higher than the level of unconjugated ubiquitin in the cytosol, when expressed as a fraction of total protein. Other abundant low molecular weight proteins in the cytosol were not detected in virions, indicating that packaging occurs in a specific manner. A naturally occurring ALV mutant that lacks the env gene was found to package normal levels of ubiquitin, ruling out involvement of the viral glycoproteins as carriers of ubiquitin. We examined disrupted virus particles as well as lysates of infected cells for the presence of gag protein-ubiquitin conjugates. No conjugates could be detected, suggesting that ubiquitin does not enter virions linked to gag protein.

Variable expression of preS1 antigen in serum during chronic hepatitis B virus infection: an accurate marker for the level of hepatitis B virus replication.

The expression of the preS1 antigen of hepatitis B virus in sera from chronic HBsAg carriers was studied using a specific monoclonal antibody F35.25 in an original, double-immunoradiometric assay. The antibody F35.25 recognized an epitope located between amino-acid residues 32 and 53 on the preS1 sequence of the large HBsAg protein. This domain could be involved in the recognition of hepatitis B virus by hepatocyte receptors. PreS1 antigen detection by monoclonal antibody F35.25 closely correlated with the presence of complete virions in the serum of HBsAg carriers, as demonstrated by ultracentrifugation-gradient experiments and electron-microscopical examination. Of the 19 HBsAg carriers with chronic liver disease, preS1 antigen was detected in 17 (90%): all of the 11 HBeAg- and hepatitis B virus-DNA--positive cases (group 1) and six of eight anti-HBe--positive cases with low levels of hepatitis B virus replication (group 2). PreS1 antigen/HBsAg ratios parallel to preS1 antigen titers were significantly higher in the HBeAg-positive group (34% and 1:10(6] than in the anti-HBe--positive group (18% and 1:10(2]. In contrast, preS1 antigen was not detected in 18 (90%) of the 20 HBsAg healthy carriers positive for anti-HBe and negative for serum hepatitis B virus-DNA (group 3). Our results show that in chronic HBsAg carriers the serum expression of preS1 antigen correlates well with the level of hepatitis B virus replication (serum hepatitis B virus-DNA and/or liver HBcAg) and that it may be useful in assessing the clinical importance of the chronic viral infection.