Virion polypeptides and structure of SV 40

Summary Five virion polypeptides, VPI to VPV, have been identified in purified SV40 virus by polyacrylamide gel electrophoresis of which two, VPI and VPII, have been located in the virus capsid. The number of molecular subunits in VPI and VP II calculated from experimental data suggest that they represent the hexons and pentons, respectively, of a 72 unit icosahedral capsid structure.     

Immunochemical identification of viral and nonviral proteins of the respiratory syncytial virus virion.

We identified by immunochemical methods 13 polypeptides associated with the infectious respiratory syncytial virus virion. Eight of these polypeptides (VP200, VP84, VP66, VP43, VP40, VP37, VP28, and VP19) were identified as virus specific. Two other polypeptides, (VP) 22 and (VP) 12, are provisionally considered to be of viral origin. Three nonviral proteins are also intimately associated with the infectious virion. These nonviral proteins were identified as cellular actin and two proteins with bovine serum albumin immunospecificity. VP40 was identified as the major ribonucleoprotein. Based on biochemical and biophysical similarities with paramyxovirus proteins, other respiratory syncytial virus proteins are believed to have these specific viral functions: VP84, "hemagglutinin"; VP66, undissociated fusion protein, F1,2; VP43, F1; and VP19, F2, VP66 contains a major determinant involved in viral infectivity since all neutralizing antibodies tested, including a monoclone, precipitated this protein.     

Intracellular SV40 nucleoprotein complexes: synthesis to encapsidation

Five forms of SV40 nucleoprotein complexes have been characterized: (i) DNA-replicative intermediates, (ii) a 75 S complex which is the product of replicative forms, (iii) a 200 S previrion structure derived from the 75 S complex shortly after the completion of DNA replication, (iv) 250 S mature virion, and (v) a 75 S complex with accumulates in tsB11-infected cells at the nonpermissive temperature. These complexes were studied by pulse-labeling kinetics of viral DNA and proteins, CsCI gradient analysis, immunoprecipitation with anti-virion antibody, and two-dimensional electrophoretic analysis of the heterogeneity of the major capsid protein. Based upon the results of this study, a biosynthetic pathway for the production and maturation of the SV40 chromosome is proposed. The model involves the gradual addition of virion proteins onto the SV40 75 S chromosome: no capsid proteins are present on the tsB11 75 S complex; 2% of the mature virion VP1 complement and 1% of the VP3 complement are present in the transient wildtype 75 S complex; 84% of the virion VP1 complement, no VP2, and 32% of the VP3 complement are present in the 200 S pre-virion; while the virion contains VP1, VP2, and VP3. The phosphorylation and extent of heterogeneity of the major capsid protein VPI are identical in all of the wildtype SV40 nucleoprotein complexes.     

Immunological studies of the group B coxsackieviruses by the sandwich enzyme-linked immunosorbent assay (ELISA) and immunoprecipitation

A microplate double antibody sandwich ELISA was employed in an immunological study of the group B Coxsackieviruses. The assay, described in detail, detected high dilutions of virion antigen (less than 10 ng) in purified preparations and in crude infected cell extracts. Furthermore, by using a constant amount of antigen, group B virus antibodies in hamster antisera could be quantified with a sensitivity equivalent to the virus neutralization test. Titrations of virus antigens and antibodies were found to be type-specific when purified virions were employed in the assay. Urea disruption of virions exposed antigens common to all six group B viruses. The heterotypic reactivity of disrupted group B virions did not extend to the other viruses tested. Immunoprecipitation and SDS-PAGE analysis revealed that, of the four virion structural polypeptides (VPI to 4), VPI contained the major common antigenic determinants shared by members of the group B Coxsackieviruses.     

Nonhistone virion proteins of polyoma: characterisation of the particle proteins by tryptic peptide analysis by use of ion-exchange columns.

Highly purified polyoma virus particles contain four nonhistone polypeptides. Their approximate molecular weights are 86,000 (VP0), 48,000 (VP1), 35,000 (VP2), and 23,000 (VP3). These polypeptides have been isolated by SDS-polyacrylamide-gel electrophoresis. The primary structure of the various differentially labelled (¹⁴C]- or [³H]lysine plus arginine) polypeptides were compared by an analysis of their tryptic peptides by use of ion-exchange and paper chromatography. The results indicate that VP0 and VP1 have identical peptide maps and that VP0 is probably a dimer of VP1. All of the tryptic peptides derived from VP3 were also detected in the tryptic digest of VP2. Additional peptides that were found in the VP2 digest and not in the VP3 appears to be derived from VP2. Whether this is of biological significance is not known. VP1 shares very few, if any, tryptic peptides with VP2 and VP3. These results are consistent with there being two polyoma genes coding for the virion proteins. One gene codes for VP1 and the other for VP2.     

A structural model for picornaviruses as suggested from an analysis of urea-degraded virions and procapsids of coxsackievirus B3

Urea treatment (3 M, 15 min, 37 °C, pH 9) of coxsackievirus B3 inactivated virus infectivity and degraded the virus capsid into substructures recoverable on sucrose gradients. One substructure which sedimented around 20 S contained VP1 and VP3, the other substructures which sedimented at 5 S contained VP2 and VP4, respectively, as analyzed by SDS polyacrylamide electrophoresis. The VP2 and VP4 polypeptides in the 5 S peak were probably separate since their molar ratios differed over the peak, and VP4 could be removed by dialysis. Treatment of the virions with only 1 M urea for 5 min yielded four peaks of radioactivity on sucrose gradients which sedimented at about 150 S (undegraded virions), 75–80 S (capsids minus VP4), and the 20 S and 5 S structures referred to above, suggesting a stepwise degradation of B3 virions by urea. The procapsids also were degraded into substructures which were separated on sucrose gradients; one sedimenting at around 40 S containing mostly VPO, and the other sedimenting around 20 S containing only VP1 and VP3. When coxsackievirus B3-³⁵S cysteine-labeled virions were disrupted and analyzed on SDS gels, all polypeptides except VP4 were labeled, suggesting that VP2 and VP4 are distinct polypeptides. Analysis of urea-disrupted coxsackievirus B3 substructures provides the basis for a T = 3 structural model of the picornaviruses, with 12 pentamers (VP2 and VP4) and 20 hexamers (VP1 and VP3) per virion.     

Isolation of subviral components from transmissible gastroenteritis virus.

Exposure of purified transmissible gastroenteritis virus, a porcine coronavirus, to non-ionic detergents resulted in the removal of the surface projections and greater than 98% of the virus lipid. Virus RNA was associated with a subviral particle which had a sedimentation coefficient of 650S, compared with 495S for the intact virion, and which banded in Cs2SO4 gradients at 1-295 g/ml. Negatively stained preparations of subviral particles were shown by electron microscopy to contain spherical particles of 60 to 70 nm diam., similar in appearance to those derived from oncornaviruses. Polyacrylamide gel electrophoresis of the polypeptides from isolated subviral particles showed that these structures contained three of the four major virus structural proteins, the arginine-rich polypeptide VP2 and the two membrane glycopolypeptides VP2 and 4. The detergent-liberated surface projections, composed of a single species of sulphated glycopolypeptide, VPI, were isolated by rate-zonal centrifugation through sucrose gradients followed by precipitation with ammonium sulphate in the presence of bovine serum albumin.     

The polypeptide composition of avian infectious bronchitis virus particles

Summary Egg grown avian infectious bronchitis virus (IBV) centrifuged on sucrose density gradients was found to consist of a major virus peak of density 1.17 to 1.18 g/cm³ and occasionally two minor virus peaks of density 1.21 to 1.22 g/cm³ and 1.13 g/cm³. Three different IBV strains were examined and no morphological differences were detected between virus particles of different densities or from different strains. The polypeptides of the different density virus particles from the three IBV strains were analysed on polyacrylamide gels. In all cases 7 polypeptides were observed, although there were differences in the proportions of these polypeptides in particles of different densities and those from the different strains. The polypeptides have been called VP1 (molecular weight 130,000), VP2 (105,000), VP3 (97,000), VP4 (81,000), VP5 (74,000), VP6 (51,000) and VP7 (33,000). Additional polypeptides were produced if slightly harsher treatments were used.With 4 Figures     

Respiratory syncytial virus polypeptides: their location in the virion.

Purified respiratory syncytial (RS) virus contains, in addition to the six to seven polypeptides previously reported (S. Levine, 1977, J. Virol., 21, 427–431), a large polypeptide (VPO), MW > 160,000. Treatment of purified virus with trypsin removes the major glycoproteins, VP1 and 2. Treatment of purified virus with 2% Triton X-100 in HBSS (equivalent to 0.15 M NaCl) solubilizes the glycoproteins VP1 and 2 and a nonglycosylated protein, VP5, MW 28,000, which suggests that VP5 is an M protein. Treatment with 2% Triton X-100 in 0.4 M NaCl solubilizes all the virion proteins except VPO and VP3, which are also not solubilized in 0.8 M NaCl. The results suggest that VP3, MW 44,000, is the major nucleocapsid protein, and that VPO is not a superficial contaminant of the virus preparation, but instead is closely associated with the nucleocapsid. Only VP3 is present in nucleocapsids isolated from RS virus-infected cells by isopycnic centrifugation in CsCl.     

Resolution of the major poliovirus capsid polypeptides into doublets.

Using the pH gradient electrophoretic technique of Vrijsen and Boeyé, the coat protein of poliovirus types 1 and 2 was resolved into six components, Cl to C6, instead of the classical VP1-2-3 (the much smaller polypeptide VP4 was excluded from this study). The multiple components ran true upon reelectrophoresis, and there were no technical artifacts. Their resolution did not depend on a particular method for the preparation or disruption of the virion. The C1–C6 components of pH gradient electrophoresis and the classical VP1–VP3 polypeptides were examined with regard to (i) their migration in normal and pH gradient reelectrophoresis and (ii) their kinetics of leucine incorporation in emetine-stopped pulses. It is concluded that C1 and C2 were both derived from VPl, C3 and C4 from VP2, and C5 and C6 from VP3.     

Investigation of the structure of polio- and human rhinovirions through the use of selective chemical reactivity.

The configurations of poliovirus and human rhinovirus type 2 (HRV-2) virions and subviral particles were investigated by measuring the relative accessibility of the four virion polypeptides (VP1-4) to the labeling reagents [³H]acetic anhydride, ¹²⁵I, and [¹⁴C]iodoacetamide. The reaction of [³H]acetic anhydride with the intact virions of poliovirus or HRV-2 revealed that in both cases VP1 was labeled to a greater extent than the smaller polypeptides, VP2 and VP3. The smallest polypeptide, VP4, was not labeled at all, suggesting that it may be internal and may not contribute directly to the surface properties of native virions. Treatment of poliovirus at 47° or HRV-2 at pH 5 produces slower sedimenting A-particles which lack VP4 and resemble the particles produced during the early interaction of virus with host cells. The occurrence of a configurational change in the formation of A-particles was suggested by the observation that A-particles exhibited increased relative susceptibility to labeling of VP2 with [³H]acetic anhydride. Virions which had been disrupted by treatment with dodecyl sulfate at 100° were labeled with [³H]acetic anhydride approximately uniformly in all polypeptides including VP4. The labeling patterns of the polypeptides of poliovirions, A-particles, and disrupted virus with ¹²⁵I were qualitatively similar to those obtained with [³H]acetic anhydride. In contrast, VP1 in HRV-2 virions was relatively protected from labeling with ¹²⁵I and became accessible only in A-particles or disrupted virions. With both viruses, again, VP4 was not labeled with ¹²⁵I in intact virions but was labeled after virus disruption. Very little [¹⁴C]iodoacetamide was incorporated into native virions, although in a few experiments VP2 was labeled in HRV-2. Only the capsid polypeptides VP1, VP2, and VP3 were labeled with [¹⁴C]iodoacetamide following disruption with SDS, indicating that neither the poliovirus VP4 nor the HRV-2 VP4 contain free SH groups.     

Hemagglutination and structural polypeptides of a new coronavirus associated with diarrhea in infant mice

Summary The hemagglutination (HA) and receptor destroying enzyme (RDE) activities of a newly isolated mouse enteric coronavirus (designated as DVIM) are described. DVIM agglutinates mouse or rat red blood cells (RBC) at 4° C. At 37° C the agglutination was rapidly reversed. The optimal pH for HA and for RDE activities using mouse red cells were shown to be 6.5 and 7.3 respectively. Hemagglutination by DVIM was not inhibited by pretreatment of RBCs withVibrio cholerae filtrate or by pretreatment with Influenza-A neuraminidase. Therefore, the DVIM receptors on RBCs differ from the receptors of Influenza-A, and the RDE activity of DVIM acts specifically on this receptor.In addition, an analysis of the DVIM polypeptides showed that the virions contain five major, VP1 (M.W. 139,000), VP2 (68,000), VP3 (53,000), VP4 (38,000), VP5 (22,000) and two minor, VP1a (110,000), VP1b (100,000) polypeptides. VP1 and VP1b were digested by bromelain, suggesting that they constitute the surface glycoproteins.With 4 Figures     

Baculovirus structural polypeptides

The structural polypeptides of eight insect baculoviruses were studied using vertical slab polyacrylamide gel electrophoresis. All viruses revealed a complex but unique composition of 15 to 25 bands with molecular weights ranging from 15,000 to 160,000. Since certain baculoviruses have more than one nucleocapsid per viral envelope (multiples), comparisons were made of the multiples and singles (enveloped single nucleocapsids) for each virus. Quantitative and qualitative differences were documented to exist in polypeptide composition. Where possible, the envelopes of certain baculoviruses were selectively removed in order to identify the major capsid proteins. Autographa californica MNPV (NPV: nuclear polyhedrosis virus) capsids contained two major polypeptides, VP18.5 and VP37. Rachiplusia ou MNPV capsids contained several polypeptides of which VP16, VP17, VP18, VP30, and VP36 were considered major constituents. Anticarsa gemmatalis MNPV contained one major capsid protein, VP29, and several minor polypeptides. Major capsid proteins of Heliothis zea SNPV were VP16, VP28, and VP63; as were VP16, VP17, and VP31 of Trichoplusia ni granulosis virus (GV).     

Descriptive analysis of Ebola virus proteins

The virion proteins of two strains of Ebola virus were compared by SDS-polyacrylamide gel electrophoresis (PAGE) and radioimmunoprecipitation (RIP). Seven virion proteins were described; an L (180K), GP (125K), NP (104K), VP40 (40K), VP35 (35K), VP30 (30K), and VP24 (24K). The RNP complex of the virus contained the L, the NP, and VP30, with VP35 in loose association with them. The GP was the major spike protein, with VP40 and VP24 making up the remaining protein content of the multilayered envelope.     

Purification and characterization of vaccinia virus structural protein VP8

A major vaccinia virus core protein, designated VP8, has been purified from virions to homogeneity through DEAE-cellulose, CM-cellulose, and hydroxyapatite chromatography. VP8 migrates as a 25-kDa band in SDS-polyacrylamide gel electrophoresis and sediments as a monomeric species in neutral sucrose gradient centrifugation. This protein is a significant constitutent of the virion, comprising about 6.5% of the total viral polypeptides by mass. Analysis by filter binding and by sucrose gradient centrifugation shows that VP8 binds to double-stranded as well as to single-stranded DNA at low salt concentrations (25 mM NaCl). At higher salt concentrations (100 mM NaCl), the protein binds with a relatively greater affinity to single-stranded DNA. The results from sucrose gradient centrifugation indicate that VP8 probably binds noncooperatively to all structural forms of DNA. The protein is likely to be a component of the viral nucleoprotein complex.     

Fractionation of biologically active and inactive populations of human rhinovirus type 2.

Infectious virions of human rhinovirus type 2 (HRV-2) migrate during electrophoresis to pH 6.4 in a sucrose-stabilized pH gradient. As already reported, acidification of HRV-2 produces two kinds of noninfectious components both of which have lost the smallest virion polypeptide, VP4: One has lost RNA in addition to VP4 and sediments at about 80 S, while the other retains RNA and sediments at 135 S. The 80 S particles migrate to pH 4.5 upon electrophoresis while the 135 S particles migrate to pH 4.2. Natural top component of HRV-2 is also separable into two subpopulations that are isoelectric at pH 6.3 and pH 4.5. Only the particles isoelectric at pH 6.3 react with virion (D) specific serum and attach to HeLa cells. The polypeptide compositions of both the attaching and nonattaching fractions of natural top component are the same, and therefore the differences in activity and isoelectric point may reflect the arrangement of the polypeptides in the capsids. After a preparation of purified infectious virus is focused to its isoelectric point some particles are found at pH 4.5. These particles are relatively noninfectious, yet contain RNA and a full complement of polypeptides, and they sediment at 140 S. They are unable to attach to host cells. These results argue against a direct role of VP4 or RNA in the attachment process and support the view that protein conformation changes occur during the inactivation of HRV-2 virions.     

Biochemistry of Theiler''s murine encephalomyelitis virus isolated from acutely infected mouse brain: identification of a previously unreported polypeptide

The WW strain of Theiler's murine encephalomyelitis virus (WW-TMEV) was purified from homogenates of acutely infected mouse brain. Infectious WW-TMEV was found to have an estimated sedimentation coefficient of 156 (s20,w) and a density of 1.35 g/cm3 in CsCl. Electron microscopy revealed a homogeneous population of 26-nm nonenveloped particles. Iodination of sodium dodecyl sulfate (SDS)-disrupted virions revealed four major capsid proteins with molecular weights of 58,000, 37,000, 34,000, and 27,000. A 6,000-dalton polypeptide was observed after long exposures of autoradiograms. The 37,000-, 24,000-, 27,000-, and 6,000-dalton polypeptides corresponded to picornaviral VP1, VP2, VP3, and VP4 capsid polypeptides, respectively. Comparison of autoradiograms of virions radiolabeled before and after SDS disruption indicated that the 58,000-dalton protein, VP2, and VP3 preferentially bound 125I under the labeling conditions used. Direct evidence was obtained that VP2 and VP3 were derived from the 58,000-dalton polypeptide by isolation of the 58,000-dalton polypeptide from polyacrylamide gels run under nonreducing conditions and subjecting it to reelectrophoresis under reducing conditions. The effect of trypsin on purified virions and their polypeptides was also investigated. Trypsin-sensitive sites were found in the 58,000-dalton protein, VP1, and VP2. Our results indicate that, in addition to the four typical picornaviral capsid polypeptides, there is a 58,000-dalton polypeptide present in WW-TMEV, which is sensitive to trypsin and can be reduced into two of the capsid proteins, VP2 and VP3.     

Characterization of the minor polypeptides in the foot-and-mouth disease particle.

In addition to the four major polypeptides VP1 and VP4, foot-and-mouth disease virus particles contain two minor polypeptides, mol. wt. 40 X 10(3) (P40) and 52 X 10(3) (P52). Extensive purification procedures failed to remove these minor polypeptides from the virus particles. Polypeptide P40 co-electrophoresed in SDS-polyacrylamide gels with VP0, the probable precursor of VP2 and VP4 and was inaccessible to iodination in situ. The second minor polypeptide, P52, co-electrophoresed with the virus infection associated (VIA) antigen found in large amounts in harvests of the virus grown in BHK 21 cells. Polypeptide P52 was shown to be located near the surface of the virus particle by iodination experiments and by its removal on incubating the particles with trypsin or chymotrypsin. Pactamycin mapping showed that this polypeptide was not a precursor of the structural polypeptides. About one copy of P52 and 4 copies of P40 were found in the virus particles sedimenting at 146S. However a larger number of copies was found in those virus particles sedimenting faster than the 146S peak.     

Epitope mapping of Aleutian mink disease parvovirus virion protein VP1 and 2

Six overlapping fragments of the Aleutian Mink Disease parvoVirus (AMDV) virion protein VP1 and 2 (VP1/2) gene were inserted into the expression vector pMAL-c2. Four of the clones carried large overlapping fragments covering the entire VP1/2 gene. The remaining two clones covered specifically chosen regions within the VP1/2 gene. Using a Western blotting detection system, sera from AMDV-infected mink were tested against the recombinant polypeptides. These studies showed reactions primarily directed against the two AMDV polypeptides ranging from amino acids 297 to 518. Weaker reactions against other regions of the VP1/2 were also observed. The small fusion protein designed to cover the presumed AMDV VP1/2 loop 4 was purified by affinity chromatography and used to develop solid-phase immunoassays. Twelve small synthetic peptides were constructed and used as inhibitors. A peptide covering amino acids S428 to T448 was shown to block the reactivity of a pool of positive mink sera, indicating the presence of one dominant linear epitope.