Viral Structural Polypeptides Research Articles

Studies on the role of M protein in virus assembly using a is mutant of HVJ (Sendai virus)

A temperature-sensitive mutant of HVJ, HVJ cl.151, was isolated from BHK cells persistently infected with HVJ and characterized. HVJ c1.151 virion had an M polypeptide different in apparent molecular weight from that of HVJ wild-type, that is 36,000 and 34,000 daltons, respectively. HVJ c1.151 was blocked in a late function required for virus maturation. M protein antigen of HVJ c1.151 was detected in infected cells by immunofluorescent microscopy only at permissive temperature but not at nonpermissive temperature, although GP and NP antigens were detected at both temperatures. Further, analysis of the infected cells by SDS-polyacrylamide gel electrophoresis showed that viral structural polypeptides P, F 0, NP, and F and nonstructural polypeptide C were synthesized in infected cells at nonpermissive temperature and these structural polypeptides were incorporated into virions upon temperature shiftdown, whereas polypeptides HN and M, which may be synthesized at nonpermissive temperature, were not able to be incorporated into virions upon temperature shift down. Thus, the temperature-sensitive lesion of HVJ c1.151 is considered to be in HN and M proteins. Membrane fluorescense, immunoferritin electron microscopy, and SDS-polyacrylamide gel electrophoresis of plasma membranes showed that migration of F 0, and F to the cell surface occurred normally even at nonpermissive temperature. Immunoferritin electron microscopy also demonstrated that the viral glycoproteins which arrived at the plasma membrane were dispersed on the entire surface of the membrane at the nonpermissive temperature and that viral components synthesized at this temperature could not assemble at the plasma membrane. In addition, it was found that antibody-induced redistribution of viral glycoproteins on the surface of cells infected with HVJ c1.151 and incubated at nonpermissive temperature occurred very rapidly; in contrast, such redistribution of viral glycoproteins occurred more slowly and less completely in cells incubated at permissive temperature, suggesting that viral glycoproteins on the plasma membrane of cells at nonpermissive temperature have a high degree of mobility in the plane of the membrane as compared with those on the plasma membrane of cells at permissive temperature. These results suggest strongly that a function which fixes the viral glycoproteins at restricted areas of plasma membrane to form a viral envelope is blocked in HVJ c1.151-infected cells at nonpermissive temperature. Analysis of plasma membrane by SDS-polyacrylamide gel electrophoresis showed that viral glycopolypeptides but no NP were present on membranes isolated from HVJ cl.151-infected cells at nonpermissive temperature in spite of the presence of a large amount of NP in the whole cells, whereas plasma membranes isolated from cells at permissive temperature contained all viral structural polypeptides. The possible roles of M protein of HVJ in formation of the viral envelope and association of nucleocapsid with the envelope during assembly are discussed based on these results.

Structural studies on Rauscher murine leukemia virus: isolation and characterization of viral envelopes.

A preparative method for isolating pure viral envelopes from a type-C RNA tumor virus, Rauscher murine leukemia virus, is described. Fractionation of virions of Rauscher murine leukemia virus was studied after disruption of the virions with the detergents sodium dodecyl sulfate of Nonidet P-40 in combination with ether. Fractionation was performed through flotation in a discontinuous sucrose gradient and, as appeared from electron microscopic examination, a pure viral envelope fraction was obtained in this way. By use of sensitive competition radioimmunoassays or sodium dodecyl sulfate-polyacrylamide gel electrophoresis after immunoprecipitation with polyvalent and monospecific antisera directed against Rauscher murine leukemia virus proteins, the amount of the gag and env gene-encoded structural polypeptides in the virions and the isolated envelope fraction was compared. The predominant viral structural polypeptides in the purified envelope fraction were the env gene-encoded polypeptides gp70, p15(E), and p12(E), whereas, except for p15, there was only a relatively small amount of the gag gene-encoded structural polypeptides in this fraction.

Analysis of baculovirus genomes with restriction endonucleases

The viral DNAs from nine wild-type insect baculoviruses have been isolated and the EcoR-1 restriction endonuclease fragment patterns compared. Genomic heterogeneity could be detected in the DNA restriction patterns of four of these wild-type baculoviruses. Three infectious virus forms (two that are occluded in the nucleus and an extracellular virus that has budded from the plasma membrane of infected cells) of a nuclear polyhedrosis virus with multiple nucleocapsids per envelope (MNPV) from the lepidopteran insect, Autographa californica are shown to be phenotypically distinct by comparison of viral structural polypeptides by polyacrylamide gel electrophoresis and autoradiography of L[ 35S]methio-nine-labeled virus proteins. The three phenotypic forms were cloned by successive plaque purification and eight distinct variants were identified from 11 plaque-purified viruses by genotypec analysis with EcoR-1 and HindIII restriction endonuclease. Isolation of variants from the three phenotypic forms has shown that each of the infectious forms is heterogeneous and that no segregation of genotypes among the three forms was evident. The characteristic restriction fragment patterns of several variants were maintained upon multiple passage in cell culture.

Surface expression of murine leukemia virus structural polypeptides on host cells and the virion.

The expression of murine leukemia virus structural polypeptides on the surface of cells producing exogenous Friend leukemia virus, endogenous ecotropic AKR and xenotropic BALB/c virus was investigated. Antisera to Friend virus gp71, p30, p15E, p12 and p10 were employed in a complement-dependent chromium release assay and to immunoprecipitate lactoperoxidase iodinated surface polypeptides prior to analysis in polyacrylamide gel electrophoresis. With the latter technique gag-gene encoded proteins and their precursors were not discovered on the viral and cellular surface membranes. Only env-gene encoded polypeptides gp85, gp71, and p15E were detectable. p15E is embedded into the lipid membrane. gp85 is formed by disulfide linkage of p15E to surface-exposed gp71. The ratio of gp71 to gp85 is variable and apparently determined by the host cell. Antibodies of strong cytotoxicity are those against type- and group-specific epitopes of gp71 as well as type-specific epitopes of p12.

Protein structure of lymphocytic choriomeningitis virus: Identification of the virus structural and cell associated polypeptides

Lymphocytic choriomeningitis virus (LCMV) has been found to contain 3 major structural polypeptides. The largest of these was shown to be a nonglycosylated nucleoprotein (NP) of approximately 63,000 daltons which was found internally in the virion and was associated with a dense ribonucleoprotein complex. Two glycopeptides, termed GP-1 and GP-2, of approximately 54,000 and 35,000 daltons, respectively, were shown to be localized on the virion surface by proteolytic digestion of intact virus. Peptide maps of GP-1 and GP-2 indicated that they were independent polypeptides and that GP-2 was not a cleavage product of GP-1. Furthermore, the tryptic peptide map of NP was distinct from the two glycopeptides. Pulse-labeling studies of the intracellular synthesis of LCMV specific polypeptides in infected BHK-21 cells demonstrated synthesis of the nucleoprotein by 6 hr after infection at a multiplicity of infection of 10, corresponding to the beginning of log phase of viral replication. In cytosol extracts of LCMV-infected cells enhanced by immune precipitation an additional apparently nonstructural glycopeptide with a molecular weight of 74–75,000 was observed. This cell associated glycopeptide, designated GP-C, was readily detectable after 24–48 hr of infection, and appeared to accumulate with increasing time of incubation. While the relationship of GP-C to the virion glycopeptides GP-1 and GP-2 has not been determined, immune precipitation studies have shown that GP-C does not share antigenic determinants with NP. The probable relationships between the polypeptides of lymphocytic choriomeningitis virus both to the pathogenesis of virus induced immune diseases and to the structure of other arenaviruses have been discussed.

Intracellular synthesis of measles virus-specified polypeptides.

The intracellular synthesis of measles-specified polypeptides was examined by means of polyacrylamide gel electrophoresis of cell extracts. Since measles virus does not efficiently shut off host-cell protein synthesis, high multiplicities of infection were used to enable viral polypeptides to be detected against the high background of cellular protein synthesis. The cytoplasm of infected cells contained viral structural polypeptides with estimated molecular weights of 200,000, 80,000, 70,000, 60,000, 41,000, and 37,000. All of these structural polypeptides, with the exception of P1, the only virion glycoprotein (molecular weight congruent to 80,000), were also found in the nuclei. In addition, two nonstructural polypeptides with estimated molecular weights of 74,000 and 72,000 were also present in the cytoplasm of infected cells. The initial synthesis of the smaller, nonstructural polypeptide began later in infection than the structural polypeptides. Pulse-chase experiments failed to detect any precursor-product relationships. The intracellular glycosylation and phosphorylation of the viral polypeptides were found to be similar to those found in purified virions.

Studies on viral DNA · protein complexes isolated at different times after infection of monkey kidney cells with Simian virus 40

The major protein components of the DNA complex, isolated from SV40-infected monkey cells, are the major viral structural polypeptide VP1 and cellular histones. At early times (24 h) after infection, VP1 is present in large amounts relative to histones, whereas at late times (48 h), the complex contains mostly histones. The amount of VP1 in the complex can be correlated to the amount of “free” VP1 present in the cells, i.e. VP1 not yet incorporated into virus particles. At early times about 40% of VP1 is “free” VP1; at late times, most of the VP1 is incorporated into virus particles. In contrast, viral DNA is produced in huge excess and only about 13% is incorporated into virions. In agreement with the above result, we find that only about 16% of the DNA in the DNA complex can be chased into virions. There is, apparently, no turnover of newly synthesized VP1 that is associated with the DNA complex at late times after infection.

Polypeptide Synthesis in Simian Virus 5-Infected Cells

Polypeptide synthesis in three different cell types infected with simian virus 5 has been examined using high-resolution polyacrylamide slab gel electrophoresis, and all of the known viral polypeptides have been identified above the host cell background. The polypeptides were synthesized in infected cells in unequal proportions, which are approximately the same as they are found in virions, suggesting that their relative rates of synthesis are controlled. The nucleocapsid polypeptide (NP) was the first to be detected in infected cells, and by 12 to 14 h the other virion structural polypeptides were identified, except for the polypeptides comprising the smaller glycoprotein (F). However, a glycosylated precursor (F(0)) with a molecular weight of 66,000 was found in each cell type, and pulse-chase experiments suggested that this precursor was cleaved to yield polypeptides F(1) and F(2). No other proteolytic processing was found. In addition to the structural polypeptides, the synthesis of five other polypeptides, designated I through V, has been observed in simian virus 5-infected cells. One of these (V), with a molecular weight of 24,000, was found in all cells examined and may be a nonstructural viral polypeptide. In contrast, there are polypeptides present in uninfected cells that correspond in size to polypeptides I through IV, and similar polypeptides have also been detected in increased amounts in cells infected with Sendai virus. These findings, and the fact that the synthesis of all four of these polypeptides is not increased in every cell type, suggest that they represent host polypeptides whose synthesis may be enhanced upon infection. When a high salt concentration was used to decrease host cell protein synthesis in infected cells, polypeptides IV and (to a lesser extent) I were synthesized in relatively greater amounts than other cellular polypeptides, as were the viral polypeptides. The possibility that these polypeptides may play some role in virus replication is discussed.

Morphogenesis of bovine leukemia virus

The morphogenesis of bovine leukemia virus (BLV) was studied in short-term cultures of leukocytes from cows with persistent lymphocytosis and in BLV-producing cell lines. Few budding particles were found. They consisted of one shell underneath the cell membrane with granules attached to the inner side. When the shell is completed the budding particle could follow two pathways: It could (a) bud from the cell membrane to give rise to a free immature particle or (b) mature while still in contact with the cell, by condensation of the shell and the granules into a nucleoid, and subsequently bud from the cell membrane as a mature virion. A different pathway of morphogenesis, probably followed by the majority of the virions, is proposed based on the following observations: (1) Low numbers of budding particles on the cell membrane, (2) condensation of electrondense material within the cytoplasm resembling virus particles in the first stage of budding, and (3) immature and mature particles lying free in the cytoplasm. This pathway of morphogenesis suppose the formation of immature and mature particles within the cytoplasm without a budding process. Immunoferritin studies on these BLV-producing cells using bovine and goat anti-BLV sera have shown labeling of the BLV particles. The cell surface, however, was labeled only rarely and then in small areas. This means that on the cell surface of BLV-producing cells very few viral structural polypeptides are present. A comparison of the morphology of BLV and several other oncornaviruses leads to the conclusion that the morphogenesis of BLV is different from that of the type B and C and other similar particles such as Mason-Pfizer monkey virus and bromodeoxyuridine-induced guinea pig leukemia virus.

In vitro translation of Harvey murine sarcoma virus RNA

The viral RNA of the Harvey strain of murine sarcoma virus (Ha-SV), which does not encode for any known viral structural polypeptides, has been translated in a nuclease-digested, cell-free system. The major protein product of the in vitro translation reaction has a molecular weight of 21,000 and is initiated faithfully with [35S]formylmethionine from formyl-[35S]methionyl-tRNAFMET. This polypeptide is clearly distinct from the RNA of the Moloney strain of type C helper virus used to pseudotype the Ha-SV. The intensity of the 21,000-dalton polypeptide on gels correlates well to the concentration of Ha-SV RNA in different viral RNA preparations. These experiments indicate that a polypeptide marker for Ha-SV is now available for the first time. The possibility that this protein is the product of the rat portion of the Ha-SV genome is discussed.

The properties of three baculoviruses from closely related hosts

The baculoviruses of three important and closely related pest insects, Spodoptera littoralis, S. exempta and S. frugiperda, were purified and several of their properties were compared. These include data on virus morphology, physical properties of the virus particle, serological properties, structural polypeptides, and viral DNA. The effect of the endogenous polyhedron alkaline protease on the polyhedron and virus particle proteins was also investigated. Biochemical and biophysical differences between the viruses were found which were reflected in their serological properties. The feasibility of using a simple serological technique for specific identification of each virus has been demonstrated.

Synthesis of Sindbis virus nonstructural polypeptides in chicken embryo fibroblasts.

The identification of eight previously undescribed polypeptides in chicken embryo cells infected with Sindbis virus is reported. Seven of these polypeptides were distinguishable from the virus structural polypeptides and their precursors by their molecular weights and tryptic peptide maps. The eighth was closely related to pE2 (Schlesinger and Schlesinger, 1973), a precursor to one of the virus particle glycoproteins. Pulse-chase experiments and the use of an inhibitor of proteolytic cleavage allowed a division of the seven nonstructural (NS) polypeptides into three stable end products (NS p89, NS p82, and NS p60) and four precursors (p230, p215, p150, and p76). The labeling kinetics after synchronous initiation of translation indicated that synthesis of the NS polypeptides started at a single site and showed that the order of the genes coding for the NS polypeptides was (5' leads to 3') NS p60, NS p89, and NS p82. Short-pulse experiments under conditions of both synchronized and nonsynchronized translation suggested that cleavage of the primary translation product of the NS genes occurred only after its synthesis was completed and that the first cleavage removed the C-terminal polypeptide. From these and other experiments, we propose a detailed scheme for the synthesis and processing of Sindbis virus NS polypeptides.

Analysis of structural polypeptides of purified human cytomegalovirus

Human cytomegalovirus strain C87 was purified by the following procedures. (i) Extracellular virus was concentrated by centrifugation at 100,000 X g for 90 min and passed through a Bio-Rad Bio-Gel A-15m column. Most of the virus was recovered in the void volume. (ii) After two consecutive isopycnic potassium tartrate gradient centrifugations (20 to 50%), coinciding peaks of plaque titer, protein, and radioactivity were found at a density of from 1.20 to 1.21 g/cm3. To characterize the structural polypeptides of human cytomegalovirus and to establish relative purification criteria, virus was purified from two mixtures: (i) [35S]methionine-labeled extracellular virus mixed with an equal volume of unlabeled normal culture fluid; (ii) unlabeled extracellular virus mixed with an equal volume of [357a1methionine-labeled normal culture fluid. The extent of purification, as judged by the ratio of cellular to viral radioactivity, was 39-fold; i.e. about 2.5% of the protein in the purified virus preparation could be accounted for by host protein contamination. Electrophoresis of purified [35S]methionine-labeled virus on a polyacrylamide gel slab showed that there were at least 33 viral structural polypeptides (VPs), and their molecular weights ranged from 11,000 to 290,000. Autoradiograms obtained from electropherograms of purified [14C]glucosamine labeled virus showed six bands. Four of these were so broad that several VPs corresponded to each of the glycosylated bands. When heavy (two fractions close to 1.21 g/cm3) and light (two fractions close to 1.20 g/cm3) fractions of the PFU peak from the second potassium tartrate gradient were analyzed separately, the number of polypeptides observed was the same, but the relative amounts of some polypeptides differed. The major polypeptide, VP17, was found in greater amounts in the heavy fraction (35%) than in the light fraction (22%). The amount of DNA as a percentage of the weight of protein was 2% for the light fraction and 1% for the heavy fraction.

Multiplication of parvovirus LuIII in a synchronized culture system. IV. Association of viral structural polypeptides with the host cell chromatin

Newly synthesized structural polypeptides of parvovirus LuIII, VP1 (62,000 daltons) and VP2 (74,000 daltons), were detected in nuclei of synchronized, infected HeLa cells at 11 to 12 h postinfection, i.e., after cells had passed through the S phase of the cell cycle. At this time, most of intranuclear viral polypeptides were associated with the chromatin acidic proteins. However, 13 to 14 h postinfection, about one-third of intranuclear VP1 and VP2 also could be extracted in the fraction containing nuclear sap proteins. According to pulse-chase experiments, VP1 and VP2 accumulated in the chromatin with a time lag of 20 to 30 min. About 90% of these chromatin-associated viral polypeptides represented empty viral capsids. In addition, chromatin prepared at 14 h postinfection contained 90 to 95% of the total intranuclear viral 16S replicative-form DNA. Since viral replicative-form DNA and empty viral capsids seem to be associated specifically with cellular chromatin, we assume that this subnuclear structure is the site of the synthesis of progeny viral DNA and the formation of complete virions.

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

Abstract 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 125 I, 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 125 I radioactivity associated with all four polypeptides.

Structural polypeptides of mammalian type C RNA viruses. Isolation and immunologic characterization of a low molecular weight polypeptide, p10.

Low molecular weight polypeptides of several mammalian type C RNA tumor viruses were purified by sequential ion exchange chromatography and molecular sizing techniques. These included a polypeptide with a molecular weight of 10,000 to 11,000, p 10, from two type C viruses of mouse origin. Rauscher- and Moloney-murine leukemia virus (MuL virus), and from an infectious type C virus isolate of the woolly monkey. The p12 structural polypeptides of these viruses as well as Rauscher-MuL virus p15 were also purified. By using radioimmunoassays developed for each polypeptide, it was possible to demonstrate that all three low molecular weight polypeptides, p15, p12, and p10, were immunologically unique. Among type C viral structural polypeptides, p10 has been least well characterized immunologically. The results of the present study indicate that p10 is virus-coded and possesses strong group-specific antigenic determinants. By use of appropriate immunoassays, broadly reactive interspecies determinants shared by mammalian type C virus isolates of murine, feline, and primate origin, were also demonstrated. The interspecies antigenic determinants of p10 were shown to be as broadly cross-reactive as those exhibited by the major type C virus structural polypeptide, p30.

The post-translational processing of semliki forest virus structural polypeptides in puromycin treated cells

The post-translational processing of semliki forest virus structural polypeptides in puromycin treated cells

Structural and enzymatic characterization of viper C-type virus.

The structural polypeptides of purified viper range in molecular weight from 11,000 to 97,000 daltons and consist of 3 major and about 13 minor polypeptides. The virus contains both protein kinase and reverse transcriptase activities. Several of the structural polypeptides are phosphorylated in vitro by the virus-associated protein kinase. However, most (possibly all) of the viral structural polypeptides are not phosphorylated in vivo. DeoxyATP is as efficient as ATP in donating phosphate for in vitro phosphorylation of viral proteins. In vitro protein phosphorylation always precedes transcription and the virus-associated protein kinase and reverse transcriptase activities can be partially separated by sedimentation in a sucrose gradient.

Adenovirus protein maturation at 42 degrees C.

Incubation of adenovirus type 2 infected cells at 42 degrees C resulted in an inhibition of assembly of virus particles although all the major viral structural polypeptides and virus-induced cellular polypeptides so far identified were detected by electrophoretic analysis. Selective high salt-acid-urea extraction of low mol. wt. polypeptides revealed the absence of protein VII at 42 degrees C whereas precursor polypeptide P-VII and core protein V were found. Pulse-chase and temperature shift experiments indicated that cleavage of P-VII into VII was a reversible thermosensitive process, requiring de novo protein synthesis after shift-down to 37 degrees C. Virus particles assembled at 37 degrees C after transfer from 42 to 37 degrees C contained both viral DNA and polypeptides pre-labelled during the eclipse phase at 42 degrees C, including core protein VII.

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 Pr1 a 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 (Pr2 a and b). The latter polypeptides comigrated with viral specific fucose-free glycoproteins not present in virions or uninfected cells.