Altered virion proteins of a temperature-sensitive mutant of polyoma virus, ts59.

The ts59 mutant of polyoma virus is blocked in a late step of infection at the restrictive temperature. Cellular and viral DNA synthesis proceed normally in ts59-infected cells at the restrictive temperature, but infectious progency virus particles are not assembled. The ts59 mutant complements early tsA mutants in mixed infection, and the temperature-sensitive mutation maps in the late region of the polyoma genome. The infectivity of ts59 virions is much heat labile than wild-type polyoma. All three nonhistone capsid proteins of ts59, VP1 (45,000 daltons) and the overlapping proteins VP2 (30,000 daltons) and VP3 (20,000 daltons), show altered mobilities when analyzed by SDS-polyacrylamide gel electrophoresis. The tryptic peptide patterns of all three ts59 virion proteins also differ from the tryptic peptide patterns of wild-type proteins. Analysis of the ts59 proteins synthesized in vitro and in infected cells suggests that the alterations in the ts59 virion proteins are caused by differences in primary structure rather than by post-translational modifications. The capsid proteins of convertant virions produced by marker rescue of the ts59 temperature-sensitive mutation, using various restriction endonuclease fragments of wild-type DNA, have been analyzed. Results of these studies suggest that (i) 26 map units is the furthest point, in a clockwise direction on the genetic map, that the information for the C-terminus of VP1 can be from the Eco·R1 cleavage site; (ii) the N-terminal end of VP2 extends beyond the N-terminal end of VP3; (iii) the temperature-sensitive phenotype of ts59 is correlated with a peptide alteration common to VP2 and VP3. The ts59 mutant contains two further peptide alterations not related to the temperature-sensitive phenotype: a C-terminal alteration in VP1 and an alteration unique to VP2. Cells infected by ts59 contain approximately fourfold lower amounts of viral capsid proteins and virus-specific messenger RNA at the restrictive temperature compared to the permissive temperature.     

A peptide comparison of proteins in the simian virus 40 virion.

Evidence is presented which suggests that (i) simian virus 40 (SV40) virion proteins VP-1 and VP-3 are independently coded; (ii) there is little sequence homology between the nonhistone virion proteins of SV40 and those of polyoma virions; and (iii) two of the histone-like protein species of SV40 are also present in polyoma virions.     

Relations between poliovirus polypeptides as shown by tryptic peptide analysis.

Poliovirus proteins were labelled in vivo with [35S]-methionine, and the major products of translation and cleavage were separated by electrophoresis and compared in terms of two-dimensional tryptic peptide maps visualized by autoradiography. The main intermediates p110 and p90 had few or no methionine-labelled sequences in common, but were both contained in, and therefore almost fully account for, the presumed primary translation product p210. The sequences of p79, a major stable product of cleavage and a non-structural protein, were almost completely contained in p90, which in turn is the major component of the larger intermediates p168 and p155. P110 is confirmed as the precursor of virus particle protein, and VP0 as the precursor of VP2. However, the sequences of p31, the other major product of translation that is not a stuctural protein, were not contained in any of the viral polypeptides mentioned above.     

Structures of polyoma virus: on the histone component and virion core.

We have observed that purified polyoma virus is able to take up an amount of calf thymus histone equivalent to 10 to 50% of its normal histone content under conditions allowing the binding of considerably lesser amounts of several other proteins. Some of the bound histone could not be released by procedures procedures routinely used for virus purification. We have also found that some of the histone present in purified polyoma virus could be selectively released without major breakdown of virus particles. Possible models for virus structure are discussed in the light of the present and other recent data.     

Polyoma virus proteins: a description of the structural proteins of the virion based on polyacrylamide gel electrophoresis and peptide analysis

Examination of the protein components of highly purified polyoma virions by electrophoretic separation in SDS-polyacrylamide gels has revealed the presence of three previously undetected protein bands. Two of these, designated P3a and P4a, were not found in lysates of infected cells and may represent modified products of their respective doublet partners, P3b and P4b. The third, P6a, along with P5, P6b, and P7 are electrophoretically indistinguishable from protein bands present in the nuclear fraction of both infected and uninfected cells, and represent the virion counterparts of the host cell histones. Comparisons of the major nonhistone proteins, P2, P3, and P4, by tryptic peptide analyses have shown that P3 and P4 are closely related, but that P2 appears to be independently coded.     

A mutation increasing the size of the polyoma virion proteins, VP2 and VP3

The ts48 mutant of polyoma virus carries a temperature-sensitive mutation (tsA) which affects the polyoma large T antigen, blocking viral DNA synthesis at the nonpermissive temperature. In addition, there are at least two other changes in the ts48 genome which affect virus-coded proteins. There is a single base change which abolishes an HpaII restriction enzyme cleavage site in the portion of the early region coding simultaneously for the polyoma medium and large T antigens. There is also a single base change in the late region of the genome which results in the synthesis of virion proteins, VP2 and VP3, larger than the corresponding wild type proteins. The change in the late region abolishes the normal termination codon for VP2 and VP3.     

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

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

Analysis of the 84K, 55K,and 48K proteins immunoprecipitable by SV40 T antibody from SV40-infected and -transformed cells by tryptic peptide mapping on cation-exchange columns

The 84K, 55K, and 48K proteins immunoprecipitable by SV40 T antibody from SV40-permissive and -transformed cells were analyzed by tryptic peptide mapping on cation-exchange columns. The 84K protein arises by proteolytic cleavage of large SV40 T antigen at its carboxy terminus. The 55K and 48K proteins are only slightly homologous, sharing at most only three methionine-containing tryptic peptides.     

Structure of the Mengo virion. I. Polypeptide and ribonucleate components of the virus particle

The genome of Mengo virus, “35 S” RNA, is a single polynucleotide chain having a molecular weight of 2.44 ± 0.08 × 10⁶ daltons. This value has been determined from studies using the methods of polyacrylamide gel electrophoresis, sedimentation in dimethyl sulfoxide, sedimentation after reaction with formaldehyde and electron microscope measurements of the viral replicative form. SDS-polyacrylamide gel electrophoresis of solubilized purified virus revealed the presence of four major polypeptides (α, β, γ, and δ) having estimated molecular weights of 32,500, 29,600, 23,700, and 10,600 daltons, respectively. In addition, two minor components were detected. The first (D2) has a molecular weight of 57,700 daltons and the second (?), a molecular weight of 39,000 daltons.     

Tryptic peptide analysis of the structural proteins of vesicular stomatitis virus.

The individual structural polypeptides of vesicular stomatitis virus have been examined by tryptic peptide analysis of 35S-methionine preparations labelled in vivo and 125I-preparations labelled in vitro. Isolates of the two classical serotypes of the virus (Indiana and New Jersey) and of a sub-type of the Indiana serotype, Brazil virus, were compared. The study showed that the major internal proteins of all three viruses gave similar maps, whereas the surface glycoproteins gave distinct maps that had very few spots in common. The map of the glycoprotein of Brazil virus, which has been shown previously to be more closely related serologically to Indiana virus than to New Jersey virus, did not show any greater similarity to the Indiana virus than to the New Jersey virus glycoprotein. On the other hand, peptide maps of the nucleoprotein and matrix protein showed Indiana and Brazil viruses to be more closely related to each other than to New Jersey virus.     

Temperature sensitive mutants of vesicular stomatitis virus: tryptic peptide maps of the proteins modified in complementation groups II and IV

The proteins specifically modified by mutations in complementations groups II and IV of vesicular stomatitis virus were identified using ion-exchange chromatography analysis. Labeled tryptic peptides from mutants and their revertants were compared and unambiguous results were obtained. For each mutant only one peptide in one protein was eluted at a place different from that of its revertant counterpart. The modified peptide was located in the M protein for ts 023 (III), as expected from previous data (F. Lafay (1974). J. Virol.14, 1220–1228). The modified peptide was found in the NS protein for ts 052 (II) and in the N protein for ts 0111 (IV). From these results we conclude that NS protein is encoded by VSV gene II and N protein, by gene IV.     

Synthetic peptide antisera: their production and use in the cloning of matrix proteins

Small amounts (0.1-1.0 nmole) of purified (greater than 90%) proteins isolated from small quantities of connective tissues can readily be microsequenced and 20 to 40 N-terminal amino acids determined. A synthetic peptide (10 or more amino acids long) can be produced and either injected directly into rabbits for antiserum production (generally peptides greater than 20 amino acids in length) or conjugated to a carrier protein prior to injection. The antisera have proven useful in the isolation of cDNA clones, for both locating clones producing the protein in expression libraries and in the subsequent confirmation of the sequence (preferably using a part of the sequence not directly used in the production of the original antiserum).     

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.     

Electrophoretic analysis of the structural polypeptides of polyoma virus mutants.

We have analysed the electrophoretic mobility of the structural polypeptides of wild-type polyoma virus (Py) and some of its temperature-sensitive (ts) mutants. Mutant ts-10 was found to contain two polypeptides migrating slightly slower than their wild-type counterparts. We suggest that these two structural polypeptides of Py are either coded for or dependent on a single viral gene and possibly are cleavage products from a precursor molecule.