A putative receptor for Venezuelan equine encephalitis virus from mosquito cells.

We have identified a cellular protein from a continuous mosquito cell line (C6/36) that appears to play a significant role in the attachment of Venezuelan equine encephalitis (VEE) virus to these cells. VEE virus bound to a 32-kDa polypeptide present in the C6/36 plasma membrane fraction, and binding to this polypeptide was dose dependent and saturable and competed with homologous and heterologous alphaviruses. These observations suggest that this polypeptide binds virus via a receptor-ligand interaction. The 32-kDa polypeptide was expressed on the surfaces of C6/36 cells, and monoclonal antibodies directed against either this cell polypeptide or the VEE virus E2 glycoprotein, which is thought to be the viral attachment protein, interfered with virus attachment. Collectively, these data provide evidence suggesting that the 32-kDa polypeptide serves as a receptor for VEE virus infection of cells. We have characterized this cell polypeptide as a laminin-binding protein on the basis of its ability to interact directly with laminin as well as its immunologic cross-reactivity with the high-affinity human laminin receptor.     

Transformation by Rous sarcoma virus: a cellular substrate for transformation-specific protein phosphorylation contains phosphotyrosine

Transformation of chicken embryo fibroblasts by Rous sarcoma virus (RSV) is caused by a single viral gene, src, which encodes a phosphoprotein, pp60src, with the enzymatic activity of a protein kinase. The relative abundance of a 36,000 molecular weight (36K) phosphorylated polypeptide which can be detected by two-dimensional electrophoresis of 32P-labeled phosphoproteins is greatly increased in RSV-transformed fibroblasts. We have reported previously that phosphorylation of the 36K polypeptide is an early event in the process of transformation and that protein synthesis is not required for its appearance. Here we identify a nonphosphorylated 36K polypeptide, present in both uninfected and transformed cells, which is homologous to the 36K phosphoprotein as judged by limited proteolysis and by tryptic peptide mapping. We conclude that the 36K phosphoprotein is generated by phosphorylation of this 36K polypeptide. It has recently been shown that pp60src phosphorylates tyrosine residues in vitro: phosphotyrosine and also phosphoserine are present in the 36K phosphoprotein isolated from RSV-transformed cells. On the basis of these results we propose that the 36K polypeptide present in chicken fibroblasts is a substrate for the protein kinase activity of pp60src. Phosphorylation of this polypeptide may be important in cellular transformation by Rous sarcoma virus.     

Identification of Sendai virus mRNA species.

Cell-free translation of separated Sendai virus mRNA species identified the message for polypeptide M and suggested the identity of the message for polypeptide NP.     

Measles virus polypeptides in infected cells studied by immune precipitation and one-dimensional peptide mapping.

Measles virus does not turn off host cell polypeptide synthesis, making it difficult to precisely identify the polypeptides specified by the virus during the infectious cycle. By using the technique of immune precipitation with measles-specific antisera, the host cell background has been eliminated, and new observations have been made concerning measles virus polypeptides H, P, NP, F, and M. The H polypeptide is first synthesized as a monomer which is processed by further glycosylation and by the formation of disulfide-bonded dimers. Polypeptide P (70,000 daltons) has been found to occur also as a 65,000-dalton molecule, P2, and both forms of the molecule are equally phosphorylated. Polypeptide NP is processed from a cleavage-sensitive form (which undergoes cleavage during the process of isolation to form polypeptide 6 [41,000 daltons]) to a form which is resistant to this cleavage. The fusion and hemolysin polypeptide is first found in the cells as a 55,000-dalton precursor, F0, which is clearly resolved from the NP polypeptide on gel electrophoresis. The measles virus F0 protein identified in previous reports had not been resolved from the 60,000-dalton NP polypeptide. The M protein occurs in the infected cells as two distinct bands, and, as in the case of Sendai virus, one of these two M protein bands represents a phosphorylated form of the other.     

Two small virus-specific polypeptides are produced during infection with Sindbis virus.

We have identified and characterized two small virus-specific polypeptides which are produced during infection of cells with Sindbis virus, but which are not incorporated into the mature virion. The larger of these is a glycoprotein with an approximate molecular weight of 9,800 and is found predominantly in the medium of infected cells. Three independent lines of evidence demonstrate conclusively that this 9,800-dalton glycoprotein is produced during the proteolytic conversion of the precursor polypeptide, PE2, to the virion glycoprotein E2. This small glycoprotein is therefore analogous to the virion glycoprotein E3 of the very closely related alphavirus, Semliki Forest virus. The 9,800-dalton glycoprotein of Sindbis virus, unlike the E3 glycoprotein of Semliki Forest virus, is not, however, present in the viral particle. The other virus-specific polypeptide is 4,200 daltons in size, does not appear to be a glycoprotein, and is neither incorporated into the mature virus nor released into the culture medium. The gene for this small polypeptide is present in the viral 26S mRNA (the mRNA which encodes all the viral structural polypeptides) and appears to be located in the portion of the mRNA which encodes the two viral glycoproteins. The possibility that this 4,200-dalton polypeptide functions as a signal peptide during the synthesis of the viral membrane glycoproteins is discussed.     

Cell-free translation of avian erythroblastosis virus RNA

Avian erythroblastosis virus (AEV) RNA rescued from nonproducer cells by superinfection with a helper virus is translated into three polypeptides in the messenger-dependent rabbit reticulocyte lysate. A 75,000 molecular weight polypeptide (P75AEV) is synthesized from 28S RNA and is encoded by the 5' section of the AEV RNA, including gag-related and AEV-specific sequences. The P75AEV synthesized in infected cells and the P75AEV synthesized in the cell-free system are electrophoretically identical. A 44,000 molecular weight polypeptide (P44AEV) is synthesized from 20-24S RNA, apparently from the 3' section of the AEV-specific RNA sequence. A minor 37,000 molecular weight polypeptide (P37AEV) is synthesized from 20S AEV RNA. A comparison is drawn between the cell-free products of MC29 and AEV RNAs.     

Core particles of hepatitis B virus and ground squirrel hepatitis virus. II. Characterization of the protein kinase reaction associated with ground squirrel hepatitis virus and hepatitis B virus.

The recently described protein kinase activity in hepatitis B virus core antigen particles (Albin and Robinson, J. Virol. 34:297-302, 1980) has been demonstrated here in the liver-derived core particles of ground squirrel hepatitis virus. Both protein kinase activities were initially associated with DNA polymerase-positive heavy core particles in CsCl density equilibrium gradients and shifted to polymerase-negative cores during the course of purification. The major core-associated polypeptide of each virus was the dominant species labeled. A variable number of other polypeptide species were also labeled by this reaction. Tryptic peptide mapping of both major and minor phosphorylated polypeptides of each virus resulted in similar patterns, suggesting that many of the sites of phosphorylation were the same in the components of each core particle. Hydrolysis of these phosphorylated core particles revealed a major phosphoamino acid as serine and a minor phosphoamino acid as threonine. The products of the protein kinase reaction in both human hepatitis B and ground squirrel hepatitis virus core particles, then, share many characteristics. The possible function(s) of this protein kinase activity is discussed in the light of similarly characterized activities in other animal viruses.     

Identification of a common antigen of herpes simplex virus bovine herpes mammillitis virus, and B virus.

In immunoelectrophoretic analyses one common antigen was demonstrated in antigen preparations from herpes simplex virus types 1- and 2- (HSV-1 and HSV-2), bovine herpes mammillitis (BHM) virus-, and B virus-infected cells solubilized by Triton X-100. The antigen was also demonstrated in solubilized purified HSV-1 and BHM virus. The common antigen was identified as antigen 11 of HSV-1 or HSV-2. Differences were found in the polypeptide composition of the related antigens when isolated from the four different herpesviruses, but a glycopolypeptide with a molecular weight of 125,000 was present in each of the four different antigen preparations, indicating that this polypeptide carried the common antigenic determinants.     

Comparison of the expression of the src gene of Rous sarcoma virus in vitro and in vivo.

We have compared the polypeptide products of the src gene of several strains of Rous sarcoma virus produced by in vitro translation of heat-denatured 70S virion RNA in the nuclease-treated reticulocyte lysate with those present in chick cells transformed by these viruses. We have done this by immunoprecipitation, using sera from rabbits injected at birth with Schmidt-Ruppin Rous sarcoma virus. In vitro translation results in the synthesis of at least nine polypeptides which appear to be encoded by the src gene. These range in size from 17,000 to 60,000 daltons. The sera from tumor-bearing rabbits precipitated these polypeptides arising from the in vitro translation of RNA from Schmidt-Ruppin Rous sarcoma virus of both subgroup A and subgroup D and from one stock of Prague Rous sarcoma virus of subgroup C. In each case, all of this family of related polypeptides could be precipitated except the smallest, the 17,000-dalton polypeptide. No precipitation of analogous polypeptides resulting from the translation of RNA from other strains of Rous sarcoma virus was observed. Cells transformed by these three strains of Rous sarcoma virus contain easily detectable amounts of a polypeptide, p60src, essentially identical to the 60,000-dalton in vitro product. With one exception, they do not contain significant amounts of polypeptides analogous to the smaller in vitro products which can be precipitated by these sera. Cells transformed by one stock of Schmidt-Ruppin Rous sarcoma virus of subgroup A did contain a 39,000-dalton polypeptide, which was related, by peptide mapping, to the 60,000-dalton polypeptide and was similar in size to a precipitable in vitro product. The 60,000-dalton polypeptide present in transformed cells appeared to be phosphorylated 10 to 25 min after its synthesis, metabolically very stable, and not derived from a precursor polypeptide. All immunoprecipitates from transformed cells which contained p60src also contained an 80,000-dalton phosphoprotein. This polypeptide is unrelated to p60src, as determined by peptide mapping, and may well be a host cell polypeptide which is specifically associated with p60src.     

Protective antigens of the vaccinia virus

The comparison of the polypeptide composition of 3 vaccinia virus strains, L-IVP, B-51 and CM-63, has revealed that strains L-IVP and B-51 are similar in their polypeptide composition, while in strain CM-63 capsid polypeptide with a molecular weight of 34000 daltons is absent or has altered electrophoretic mobility. As the result of the isolation of vaccinia envelopes (from strain L-IVP) and the electrophoretic separation of their polypeptides in plates with polyacrylamide gel 10 polypeptides have been obtained in 7 fractions, each containing 1 or 2 polypeptides. The immunization of rabbits with individual fractions has demonstrated that the formation of virus-neutralizing antibodies is induced mainly by 4-5 polypeptides in 3 fractions, having the highest molecular weight (54000-31000 daltons) and constituting about 19% of all proteins in the whole virion. The low-molecular envelopes polypeptides have been found to play no essential role in inducing the formation of virus-neutralizing antibodies. The highest antibody titers (1: 15625) have been detected in antisera to the preparations of whole vaccinia virus envelopes.     

Biophysical studies of infectious pancreatic necrosis virus.

The molecular weight of infectious pancreatic necrosis virus (IPNV) has been determined by analytical ultracentrifugation and dynamic light scattering. The sedimentation coefficient of the virus was found to be 435S. The average value for molecular weight is (55 +/- 7) x 106. The virus genome consists of two segments of double-stranded RNA (molecular weights, 2.5 x 106 and 2.3 x 106), which represents 8.7% of the virion mass. The capsid protein moiety of IPNV consists of four species of polypeptides, as determined by polyacrylamide gel electrophoresis. The number of molecules of each polypeptide in the virion has been determined. There are 22 molecules of the internal polypeptide alpha (molecular weight, 90,000), 544 molecules of the outer capsid polypeptide beta (molecular weight, 57,000), and 550 and 122 molecules, respectively, of the internal polypeptides gamma1 (molecular weight, 29,000) and gamma2 (molecular weight, 27,000). IPNV top component contains only the beta polypeptide species, and its molecular weight is estimated to be 31 x 106. The hydrodynamic diameter and electron microscopic diameter (calculated by catalase crystal-calibrated electron microscopy) of IPNV was compared with those of reovirus and encephalomyocarditis virus. Due to the swelling of the outer capsid, reovirus particles were found to be much larger when hydrated (96-nm diameter) than when dehydrated (76-nm diameter), having a large water content content and low average density. In contrast, IPNV particles are more rigid, having nearly the same average diameter under hydrous (64 nm) as under anhydrous conditions (59.3 nm). Encephalomyocarditis virus has a very low water content and does not shrink at all when prepared for electron microscopy.     

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.     

Identification of a viral protein involved in post-translational maturation of the encephalomyocarditis virus capsid precursor.

Translation of encephalomyocarditis virus RNA in a cell-free system from uninfected Krebs ascites cells results in the synthesis of a major polypeptide product with a molecular weight of approximately 112,000. In contrast, when the viral RNA is translated in a cell-free system from virus-infected cells, this polypeptide is absent and the largest polypeptide produced has a molecular weight of about 100,000. This latter polypeptide comigrates on sodium dodecyl sulfate-gels with in vivo virus capsid precursor A, and the two have identical patterns of CNBr-generated peptides. A polypeptide having a molecular weight of 12,500 is also a major translation product in the system from infected cells (but not from uninfected cells). This polypeptide appears to be generated by cleavage of the NH-2-terminal portion of the viral RNA-dependent polypeptides by a proteolytic activity present in the infected cell-free system. This proteolytic activity copurifies with the 23,000-molecular weight viral capsid protein gamma, found in infected cells, through chromatography on DEAE-cellulose and cellulose phosphate. This suggests that gamma is itself a proteolytic enzyme involved in maturation of the viral capsid precursor.     

Functional analysis of the herpes simplex virus UL42 protein.

The herpes simplex virus UL42 gene encodes a multifunctional polypeptide (UL42) that is essential for virus DNA replication. To further understand the relationship between the structure of UL42 and the role that it plays during virus replication, we analyzed an extensive set of mutant UL42 proteins for the ability to perform the three major biochemical functions ascribed to the protein:binding to DNA, stably associating with the virus DNA polymerase (Pol), and acting to increase the length of DNA chains synthesized by Pol. Selected mutants were also assayed for their ability to complement the replication of a UL42 null virus. The results indicated that the N-terminal 340 amino acids of UL42 were sufficient for all three biochemical activities and could also support virus replication. Progressive C-terminal truncation resulted in the loss of detectable DNA-binding activity before Pol binding, while several mutations near the N terminus of the polypeptide resulted in an altered interaction with DNA but had no apparent affect on Pol binding. More dramatically, an insertion mutation at residue 160 destroyed the ability to bind Pol but had no effect on DNA binding. This altered polypeptide also failed to increase the length of DNA product synthesized by Pol, and the mutant gene could not complement the growth of a UL42 null virus, indicating that the specific interaction between Pol and UL42 is necessary for full Pol function and for virus replication. This study confirms the validity of the Pol-UL42 interaction as a target for the design of novel therapeutic agents.     

Proteolytic activity of the nonstructural polypeptide p22 of encephalomyocarditis virus

A nonstructural polypeptide, p22, of encephalomyocarditis virus has been partially purified from extracts of virus-infected cells. Evidence is presented suggesting that this polypeptide, rather than some contaminants, possesses a proteolytic activity. Purified preparations of p22 containing no other detectable virus-specific proteins, cleave a high-molecular-weight virus-specific precursor-polypeptide into capsid polypeptides, ε, α, and γ, nonstructural polypeptide G as well as several low-molecular-weight products.     

Use of a mammalian internal ribosomal entry site element for expression of a foreign protein by a transfectant influenza virus.

The ribonucleoprotein transfection system for influenza virus allowed us to construct two influenza A viruses, GP2/BIP-NA and HGP2/BIP-NA, which contained bicistronic neuraminidase (NA) genes. The mRNAs derived from the bicistronic NA genes have two different open reading frames (ORFs). The first ORF encodes a foreign polypeptide (GP2 or HGP2) containing amino acid sequences derived from the gp41 protein of human immunodeficiency virus type 1. The second ORF encodes the NA protein; its translation is achieved via an internal ribosomal entry site which is derived from the 5' noncoding region of the human immunoglobulin heavy-chain-binding protein mRNA. The GP2 (79 amino acids) and HGP2 (91 amino acids) polypeptides are expressed in cells infected with the corresponding transfectant virus. The HGP2 polypeptide, which contains transmembrane and cytoplasmic domains identical to those of the hemagglutinin (HA) protein of influenza A/WSN/33 virus, is packaged into virus particles. This novel influenza virus system involving an internal ribosomal entry site element may afford a way to express a variety of foreign genes in mammalian cells.     

Purification of Epstein-Barr virus DNA polymerase from P3HR-1 cells.

The Epstein-Barr virus DNA polymerase was purified from extracts of P3HR-1 cells treated with n-butyrate for induction of the viral cycle. Sequential chromatography on DNA cellulose, phosphocellulose, and blue Sepharose yielded an enzyme preparation purified more than 1,300-fold. The purified enzyme was distinct from cellular enzymes but resembled the viral DNA polymerase in cells infected with herpes simplex virus type 1 or 2. The active enzyme had an apparent molecular weight of 185,000 as estimated by gel filtration on Sephacryl S-300. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a major polypeptide corresponding to a molecular weight of ca. 110,000. This polypeptide correlated with the catalytic function of the purified enzyme, whereas the other, less abundant polypeptides did not. By immunoblotting, the 110,000-molecular-weight polypeptide could be identified as a viral polypeptide. It could not be determined whether the native enzyme was composed of more than one polypeptide.     

Immediate glycosylation of Sindbis virus membrane proteins

The mechanism by which the membrane proteins of Sindbis virus are initially glycosylated during growth of the virus in chick cells was studied. The experiments suggest strongly that the two viral glycoproteins are glycosylated before release from the polysome, and that this glycosylation involves transfer of a large 1800 dalton oligosaccharide to the polypeptide chains. The donor of the oligosaccharide is most probably a lipid.