Comparative analysis of the enveloped virions of two granulosis viruses of the armyworm, Pseudaletia unipuncta

The enveloped virions of two strains of a granulosis virus, a synergistic Hawaiian (GVH) and a nonsynergistic Oregonian (GVO) strain which infect the armyworm, Pseudaletia unipuncta, were liberated from their capsules with 0.02 N NaOH and purified by filtration through membrane filters. The envelopes were solubilized with 0.1% Triton X-100. The enveloped virions, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), had 9 to 12 polypeptides, 3 of which were associated with the naked virion. The two strains differed in their electrophoretic patterns mainly in the presence of two heavy polypeptides (89,000 and 97,000 daltons) and a 52,000-dalton polypeptide in GVH and in the absence of the heavy polypeptides and the presence of a 57,000-dalton polypeptide in GVO. The two heavy polypeptides were sensitive to proteinase. Two two-dimensional electrophoreses (first dimension with agarose gel electrophoresis and second dimension with SDS-PAGE and immunoelectrophoresis) of the envelope of GVH resolved two polypeptides, 37,000 and 48,000 daltons, which were serologically related to the synergistic factor present in the capsule of GVH; however their molecular weights differed from that of the GVH synergistic factor. In GVO, no polypeptide serologically related to the synergistic factor was detected in the envelope. The enveloped virions purified by differential filtration were infectious when fed to armyworm larvae, but the naked virions, free of envelopes, were not infectious.     

Structural components of mouse mammary tumor virus. I. Polypeptides of the virion.

Mouse mammary tumor virus (mMTV) obtained from MJY-alpha cell cultures and analyzed by polyacrylamide gel electrophoresis possesses four major polypeptides and six to eight minor components. Three of the major proteins, with estimated molecular weights of 60,000, 52,000 and 37,000, are glycoproteins, as demonstrated by labeling with [³H]glucosamine. Labeling with [³⁵S]sulfate revealed significant amounts of sulfate associated with the 60,000 (gp60) and 52,000 (gp52) glycoproteins, whereas sulfate was minimally incorporated into the 37,000 (gp37) dalton glycoprotein. At least three glycopeptide species containing both [³H]glucosamine and [³⁵S]sulfate labels were obtained after extensive Pronase digestion of mMTV, indicating that [³⁵S]sulfate is covalently linked to the carbohydrate component of mMTV glycoproteins. Variations in the polypeptide pattern of mMTV were observed when virions were grown and labeled under different culture conditions. The amount of gp60 decreased substantially, with an apparent increase in a minor polypeptide at 33,000 daltons, if virions were harvested from stationary cultures or if culture medium was not changed daily during viral harvests. When virions containing gp60 were incubated with medium from stationary cultures or with stationary cell layers, the level of gp60 decreased with a corresponding increase in the amount of radioactivity at 33,000 daltons. Cleavage of gp60 and gp52 was demonstrated by treatment of purified mMTV virions with trypsin or alpha-chymotrypsin (1–150 μg/ml) for 1 min to 3 hr. Virions were morphologically unaltered after incubation with either protease; however, both gp60 and gp52 were absent from the polypeptide patterns. The data suggest that gp52 is cleaved to form 33,000, 22,000, and possibly 37,000 dalton glycoproteins which remain associated with virions. Other mMTV virion polypeptides were resistant to treatment with protease.     

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.     

Immunological and physicochemical studies of influenza matrix (M) polypeptides.

Immunologically active preparations of M polypeptides were recovered from influenza A viruses and had a sedimentation coefficient of 3.3 s. The type-specific antigenic determinants of the M polypeptide were resistant to denaturation by heat at 100° for 2 min in the presence of detergents and to treatment with acidic-chloroform methanol. Mild proteolysis with trypsin or caseinase C produced fragments of 13,000 and 6,000 daltons. The 13,000-dalton fragments possessed some identical antigenic determinants to the whole 25,000-dalton M molecule and partially absorbed antibody to the 25,000-dalton polypeptide from a specific antiserum. Immunization of ferrets with M polypeptide or M polypeptide fragments had no preventative or enhancing effect on influenza virus infection, whereas immunization with homologous bromelain-released HA polypeptides conferred absolute protection to virus challenge.     

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.     

Effect of trypsin and chymotrypsin on the polypeptides of large and small plaque variants of foot-and-mouth disease virus: relationship to specific antigenicity and infectivity.

Large and small plaque variants of A12 foot-and-mouth disease virus were shown to have specific antigenic determinants. Large plaque virus antigenic specificity was destroyed by trypsin treatment, but the small plaque antigen was resistant despite cleavage of the trypsin-sensitive polypeptide. The cleavage of polypeptide VP3 by trypsin resulted in the formation of a new antigen not present on untreated virus. The effects of chymotrypsin and trypsin on the polypeptides of the plaque variants have been examined and related to changes in antigenicity, infectivity, and exposure of the polypeptides at the surface of the capsid. The results are discussed in relation to the orientation of the trypsin-sensitive polypeptide in the virus capsid.     

Evidence for common, intertypic antigenic determinants on poliovirus capsid polypeptides

Polyclonal antibodies prepared against individualized type 1 poliovirus structural polypeptides VP1, VP2, and VP3 were used to analyze the presence of common antigenic determinants among the three poliovirus serotypes. Each anti-VP antiserum immunoprecipitated specifically the polypeptide against which it had been prepared, as well as the corresponding polypeptide of the heterotypic viruses. Anti-VP1 antisera also reacted with type 1, type 2, and type 3 heat-denaturated poliovirions (C particles), whereas anti-VP2 and anti-VP3 sera formed immune complexes with type 1 and type 3, but not with type 2, C particles. It was concluded that capsid polypeptides VP1, VP2, and VP3 of the three serotypes share common antigenic determinants which are masked in mature virions (D particles), but can be unmasked, at least partially, upon heat inactivation of the virus.     

Structural polypeptides of mumps virus.

The structural polypeptides of egg grown mumps virus were analysed by SDS-polyacrylamide-slab-gel electrophoresis. Mumps virions contained eight major polypeptides with mol. wt. of 75, 73, 71, 61, 47, 44, 42 and 40 X 10(3). The 75 K and 61 K polypeptides were glycosylated. In virions treated with pronase and trypsin, the 75 K glycoprotein was removed more readily from the virus than the 61 K glycoprotein. The gradual removal of the 75 K glycoprotein was paralleled by a decrease of haemagglutinating activity. The large glycoprotein was cleaved into a 40 K glycoprotein by trypsin treatment. Pronase and trypsin treatment also removed the smallest 40 K non-glycosylated polypeptide. Thus this polypeptide appears to be located on the outside of the virion and probably represents a cleavage product of the large glycoprotein. Treatment of virions with 2% Triton-X 100 under alkaline conditions in the absence or presence of 2 M-KCl solubilized the two glycoproteins and a fraction of the 71 and 44 K polypeptides, but not the 73 and 47 K polypeptides. The two smallest polypeptides were solubilized by treatment with 2% Triton X-100 in the presence of 2 M-KCl. Since the 40 K polypeptide was interpreted to represent a cleavage product of the large surface glycoprotein the 42 K polypeptide was proposed to represent the membrane protein of mumps virus. The 44 K polypeptide co-migrated with Vero cell actin. The nature of the 47 K polypeptide could not be determined, but it is probably located in the central part of the virus. The 73 K polypeptide and in some experiments also the 71 K polypeptide were found in purified nucleocapsid preparations. It is concluded that mumps virus has a general polypeptide composition similar to other paramyxoviruses. However, the molecular weights of the different polypeptides of mumps virus differ markedly from the corresponding polypeptides in Newcastle disease virus and Sendai virus.     

Biosynthesis of Rauscher leukemia viral proteins: presence of p30 and envelope p15 sequences in precursor polypeptides.

Viral structural polypeptides p30 and a 17,000-dalton polypeptide, termed envelope p15, are formed in Rauscher leukemia virus (RLV)-infected N.I.H. Swiss mouse embryo fibroblasts by cleavage of high molecular weight precursor polypeptides. The evidence for this conclusion is based on the analysis of polypeptides precipitated from RLV-infected cells by antiserum directed against RLV structural proteins. High resolution sodium dodecyl sulfate (SDS)-polyacrylamide-gel electrophoresis (PAGE) of such immune precipitates from infected cells pulse-labeled with [³⁵S]methionine or pulse-labeled and then chased in unlabeled medium provides evidence that three size classes of unstable polypeptides are precursors to virion p30. They are: two polypeptides with an approximate molecular weight of 200,000 (termed Pr1a and b), an 80,000-dalton polypeptide (Pr3) and a 65,000-dalton polypeptide (Pr4). Ion-exchange chromatography of tryptic digests showed that methionine-containing tryptic peptides of p30 are present in these precursor polypeptides. Methionine-labeled tryptic peptide sequences of envelope p15 were present in a 90,000-dalton peptide fraction containing two components (Pr2a and b). The latter polypeptides comigrated with viral specific fucose-free glycoproteins not present in virions or uninfected cells.     

Detection by monoclonal antibodies of an antigenic determinant critical for poliovirus neutralization present on VP1 and on heat-inactivated virions

Hybridoma cell lines were established against poliovirus type 1 (Mahoney) heat-denatured virions (C particles). Each anti-C monoclonal antibody (McAb) immunoprecipitated specifically one of the individualized poliovirus capsid polypeptides VP1, VP2, or VP3. One of the anti-C McAb (C-3), reacting with VP1, neutralized homologous virus and immunoprecipitated infectious D particles. Its properties have been compared to those of a neutralizing anti-D McAb (D-Ic). In contrast with the C-3 antigenic site, the D-Ic epitope was not present on C particles nor on individualized structural polypeptide. This demonstrates that C-3 and D-Ic epitopes represent two independent antigenic determinants, both critical for poliovirus neutralization.     

Characterization of proteins of human papilloma viruses (HPV) and antibody response to HPV 1

The serological characterization of virus isolates from verrucae vulgares and plantar warts revealed that HPV 1 and HPV 4 are present in about 50% of these warts with HPV 1 being more prevalent, especially in plantar warts. Parallel to the high incidence of HPV 1 infections, about 50% of non-selected young adults contained antibodies against HPV 1. Only HPV 4 particles, however, reacted with serum from a patient with epidermodysplasia verruciformis when tested by immuno-electron microscopy.An examination of HPV 1 proteins indicated that the major structural proteins VP2 and VP3 are trypsin sensitive. Tryptic degradation leads to distinct polypeptides with molecular weights between 37,000 and 23,000 which may be correlated to minor protein components of HPV 1 preparations. HPV 1 histone-like proteins, which co-migrate with purified cellular histones in SDS gel electrophoresis were analyzed in an acetic acid urea system. It was shown that H3- and H4-like proteins differ from cellular histones. The reason for this difference and its meaning are discussed.     

Polypeptides of a viral DNA-protein complex form polyoma virus-infected cells.

A nascent viral DNA-protein complex was isolated from cells productively infected with polyoma virus (Py), by a conventional extraction procedure. This complex was purified by velocity sedimentation through neutral sucrose gradients, followed by ion-exchange chromatography. After in vitro labeling with ¹²⁵I, the protein moiety of the purified complex was analyzed by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis. Four polypeptides were thus detected: viral structural polypeptides VP1, VP2, and VP3, and a nonstructural polypeptide of a molecular weight of approximately 70,000. The latter represented as much as 48% of the ¹²⁵I radioactivity associated with all four polypeptides.     

Structure of the hepatitis A virion: identification of potential surface-exposed regions

Summary Iodination of highly purified hepatitis A (HAV) virus results in the selective labeling of two viral polypeptides, which are identified as the the VP 1 and VP 2 capsid polypeptides. Based upon the kinetics of labeling, the exposed region of VP 1 appears to be more accessible to iodination, although the ultimate proportion of label present within VP 1 and VP 2 is approximately equal. By utilizing iodinated whole virions, isolated VP 1, VP 2, and the tryptic digest derived from VP 1 and VP 2, binding by heterologous anti-160 S antibody indicated that a significant portion of the antibodies was directed against an epitope on VP 2 that was not affected by denaturation. Identification of the regions exposed for iodination on these two polypeptides was accomplished by tryptic digestion of the isolated polypeptides followed by characterization of the iodinated tryptic peptide by gel filtration and reverse-phase chromatography. The results indicate that tyrosine 100 on VP 2 and a large tryptic peptide composed of amino acids 222 through 260 on VP 1 which contains four tyrosine residues are two regions that are surface-exposed on these molecules.     

Characterization of novel populations of MVM virions containing covalent DNA-protein complexes

Virions of minute virus of mice were purified by sedimentation in sucrose gradients and chromatography on DEAE-cellulose columns and shown to consist of single-stranded viral DNA and the viral capsid polypeptides VP-1 (83 kDa) and VP-2 (64.5 kDa). A 63-kDa polypeptide distinct from the viral capsid polypeptide VP-3 (61.4 kDa) was found in some virion preparations. Virions sedimented at 135 and 110 S. The genomic single strands associated with purified 135 and 110 S virions were covalently bound to a protein as judged by the anomalous electrophoretic mobility of the DNA in agarose gels at pH 12.5. The protein was removed from the DNA by Pronase but remained bound after heating at 98 degrees in the presence of 0.1% sodium dodecyl sulfate. Nuclease digestion of the purified DNA-protein complex released several polypeptides ranging in size from 58 to 65 kDa. Restriction enzyme analysis of the purified DNA protein complex following its conversion to a duplex RF DNA in vitro showed that the protein was attached to the 5' termini of the DNA.     

Identification of the structural proteins of turkey enteric coronavirus

Summary Coronaviruses in the intestinal contents of turkey poults from Quebec flocks with outbreaks of enteritis were detected by electron microscopy. Five isolates were serially propagated in embryonic turkey eggs by inoculation of clarified intestinal contents into the amniotic cavity. Viral particles contained in the intestinal contents of infected embryos were purified by differential and isopycnic centrifugation in sucrose gradients. Intact virions present in fractions of densities 1.18 to 1.20 g/ml were precipitated with trichloroacetic acid and the protein content was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. At least seven polypeptides with molecular weights ranging from 27,000 to 180,000 were regularly detected after electrophoresis of SDS-solubilized virions. Homologous rabbit antisera to turkey coronaviruses and pooled sera from convalescent turkeys immunochemically stained five polypeptides of apparent molecular weights of 95,000, 72–75,000, 66,000, 52,000 and 27,000. A further polypeptide with a molecular weight of 140,000 appeared in the absence of 2-mercaptoethanol and probably represents a dimer of the 66,000 polypeptide. One Quebec isolate differed from the Minnesota strain by two of its polypeptides, but no antigenic variations could be demonstrated amongst the various isolates either by immuno-electron microscopy, hemagglutination inhibition, or enzyme immuno assays on nitrocellulose.This study was partly supported by the Medical Research Council of Canada and the Quebec Federation of Poultry Producers.     

Presence of a 43-kDa host-cell polypeptide in purified aphthovirions

A 43kDa cellular polypeptide (P43), which comigrates with host-cell actin in both SDS-PAGE and isoelectrofocusing slab gels, was found associated to 140 S aphthoviral particles purified from BHK 21 cells labeled with [35S]methionine prior to infection. Ultracentrifugation analysis of disrupted virions demonstrates that polypeptide P43 is not associated to VP1-3 containing 12 S subunits but remains, like viral polypeptide VP4, at the top of the sucrose gradients. In addition, in vitro iodination or trypsin treatment show that P43 is protected from the action of both procedures and therefore supports the hypothesis that host-cell polypeptide P43 is located within the viral particles.     

Detection of partially proteolysed cauliflower mosaic virus coat protein in infected leaf tissue by Western blotting

Cauliflower mosaic virus (CaMV) capsid polypeptides were detected by immunoelectroblotting ('Western blotting') 10-16 days after infection of Chinese cabbage leaves. The predominant polypeptides detected had molecular weights of 42,000 and 37,000 suggesting that in vivo proteolysis of the 55,000-58,000 molecular weight coat protein had taken place. The use of laboratory-made nitrocellulose membranes for Western blotting is reported. Phosphate-SDS buffer was more suitable than Tris-glycine buffer for the electrophoretic transfer of CaMV polypeptides. Specific antibodies prepared by absorption to intact CaMV were used as a probe for the viral coat proteins.     

Immunological properties of avian oncornavirus polypeptides.

The major polypeptides and glycoproteins of avian myeloblastosis virus and of the Prague strain of Rous sarcoma virus were isolated by gel filtration in guanidine hydrochloride (GuHCl). Hyperimmune sera prepared in rabbits against homogeneous preparations of each material were used to study the nature of the antigenic specificities on these molecules. The results indicated that (1) each component contained unique antigenic determinants; (2) each of four polypeptides (27,000, 19,000, 15,000, 12,000 daltons) contained group-specific (gs) reactivity; and (3) subgroup specific reactivity was found in the 19,000-dalton polypeptide.