Virion-Bound Protein Kinase in Semliki Forest and Sindbis Viruses

Semliki forest virus and Sindbis virus (Alphaviruses belonging to the togavirus group) grown in BHK-21 cells possessed very low levels of virion-associated protein kinase activity. For comparison, vesicular stomatitis virus, also grown in BHK-21 cells, contained a virion-bound protein kinase which had a specific activity 80 times greater than that of the Alphaviruses. The Alphavirus protein kinase was unmasked by the nonionic detergent Nonidet P-40 but was not activated by cyclic nucleotides. Phosvitin was the best exogenous phosphate acceptor for assaying the viral enzyme in vitro. Phosphoprotein phosphatase activity was also detected in the Alphaviruses. Both in vivo and in vitro, all of the viral structural polypeptides were phosphorylated, and the phosphorylated amino acids were found to be serine and threonine. The viral nucleocapsid protein was about four times more efficient as a phosphate acceptor than were the envelope proteins. From 33 to 50% of the total protein kinase was bound to the viral nucleocapsid, and the specific activity of this enzyme was 4 to 10 times greater than that associated with the viral envelope.     

Phosphorylation of Animal Virus Proteins by a Virion Protein Kinase

Compared with several other enveloped viruses, purified virions of frog virus 3 contained a relatively high activity of a protein kinase which catalyzed the phosphorylation of endogenous polypeptides or added substrate proteins. Virions also contained a phosphoprotein phosphatase activity which released phosphate covalently linked to proteins. It was possible to select reaction conditions where turnover of protein phosphoesters was minimal, as the phosphatase required Mn(2+) ions for activity whereas the protein kinase was active in the presence of Mg(2+) ions. Electrophoretic studies in polyacrylamide gels containing sodium dodecyl sulfate indicated that at least 10 of the virion polypeptides were phosphorylated in the in vitro protein kinase reaction. Characterization of these phosphoproteins demonstrated that the phosphate was incorporated predominantly in a phosphoester linkage with serine residues. The protein kinase was solubilized by disrupting purified virions with a nonionic detergent in a high-ionic-strength buffer and was separated from many of the virion substrate proteins by zonal centrifugation in glycerol gradients. The partially purified protein kinase would phosphorylate polypeptides of many different animal viruses, and maximal activity was not dependent on added cyclic nucleotides. These properties distinguished the virion protein kinase from a well characterized cyclic AMP-dependent protein kinase which phosphorylated viral proteins only to a small extent.     

Characterization of phosphoproteins and protein kinase activity of virions, noninfectious enveloped particles, and dense bodies of human cytomegalovirus.

Phosphorylation of the proteins of human cytomegalovirus (CMV) virions, noninfectious enveloped particles (NIEPs), and dense bodies was investigated. Analyses of particles phosphorylated in vivo showed the following. Virions contain three predominant phosphoproteins (i.e., basic phosphoprotein and upper and lower matrix proteins) and at least nine minor phosphorylated species. NIEPs contain all of these and one additional major species, the assembly protein. Dense bodies contain only one (i.e., lower matrix) of the predominant and four of the minor virion phosphoproteins. Two-dimensional (charge-size) separations in denaturing polyacrylamide gels showed that the relative net charges of the predominant phosphorylated species ranged from the basic phosphoprotein to the more neutral upper matrix protein. In vitro assays showed that purified virions of human CMV have an associated protein kinase activity. The activity was detected only after disrupting the envelope; it had a pH optimum of approximately 9 to 9.5 and required a divalent cation, preferring magnesium to manganese. In vitro, this activity catalyzed phosphorylation of the virion proteins observed to be phosphorylated in vivo. Peptide comparisons indicated that the sites phosphorylated in vitro are a subset of those phosphorylated in vivo, underscoring the probable biological relevance of the kinase activity. Casein, phosvitin, and to a minor extent lysine-rich histones served as exogenous phosphate acceptors. Arginine-rich and lysine-rich histones and protamine sulfate, as well as the polyamines spermine and spermidine, stimulated incorporation of phosphate into the endogenous viral proteins. Virions of all human and simian CMV strains tested showed this activity. Analyses of other virus particles, including three intracellular capsid forms (i.e., A, B, and C capsids), NIEPs, and dense bodies, indicated that the active enzyme was not present in the capsid. Rate-velocity sedimentation of disrupted virions separated the protein kinase activity into two fractions: one that phosphorylated exogenous casein and another that phosphorylated primarily the endogenous virion proteins.     

Structural components of the arenavirus Pichinde.

Purified Pichinde virions grown in monolayers of BHK-21 cells were found to contain three major species of virion proteins as described previously (Ramos et al., J. Virol. 10:661-667, 1972). Two of the proteins were glycosylated (G1, molecular weight = 64,000; G2, molecular weight = 38,000) and were present in similar proportions on the outer surface of the virions. A third protein (N, molecular weight = 66,000) was not glycosylated and, in association with the viral RNA species, was the major protein component of the viral nucleocapsids. An estimate of the approximate number of molecules of these three major proteins per virion was made. Minor amounts of other proteins were also routinely observed in Pichinde virus preparations. None of the three major protein species were phosphorylated to any significant exten, nor did they contain sulfated components. Two virion RNA species (L and S), but no 18S rRNA species, were detected in Pichinde virus preparations obtained from infected BHK-21 cells.     

Glycolipid Content of Vesicular Stomatitis Virus Grown in Baby Hamster Kidney Cells

Vesicular stomatitis virions grown in baby hamster kidney (BHK-21-F) cells were found to contain hematoside (neuraminosyl-galactosyl-glucosyl-ceramide). This ganglioside, which was the only detectable glycolipid in the virion, is also the only glycolipid found in significant amount in BHK-21-F cells. Approximately 87% of the total neuraminic acid in the virion was found to be linked to protein and 13% to lipid.     

Sialoglycoprotein of Vesicular Stomatitis Virus: Role of the Neuraminic Acid in Infection

Neuraminidase free of proteolytic activity substantially reduced the infectivity of vesicular stomatitis (VS) virus but less effectively than trypsin. The only sugar residue hydrolyzed by neuraminidase was N-acetyl neuraminic acid, ~89% of which was liberated from virion glycoprotein and the rest from virion glycolipid. Desialylation of virion glycoprotein but not of glycolipid resulted in progressive loss of infectivity. Sialyl transferase prepared and partially purified from BHK-21 cells catalyzed resialylation by CMP-[¹⁴C]sialic acid of the glycoprotein of neuraminidase-treated VS virions and supersialylation of unhydrolyzed VS viral glycoprotein. Resialylation of desialylated VS virions resulted in substantial (26-fold) restoration of their infectivity. We conclude that terminal neuraminic acids of VS viral sialoglycoprotein play an important role in initiation of infection with this virus.     

Phosphorylation of Vesicular Stomatitis Virus In Vivo and In Vitro

The structural protein, NS, of purified vesicular stomatitis virus (VSV) is a phosphoprotein. In infected cells phosphorylated NS is found both free in the cytoplasm and as part of the viral ribonucleoprotein (RNP) complex containing both the 42S RNA and the structural proteins L, N, and NS, indicating that phosphorylation occurs as an early event in viral maturation. VSV contains an endogenous protein kinase activity, probably of host region, which catalyzes the in vitro phosphorylation of the viral proteins NS, M, and L, but not of N or G. The phosphorylated sites on NS appear to be different in the in vivo and in vitro reactions, and are differentially sensitive to alkaline phosphatase. After removal of the membrane components of purified VSV with a dextran-polyethylene glycol two-phase separation, the kinase activity remains tightly associated with the viral RNP. However, viral RNP isolated from infected cells shows only a small amount of kinase activity. The protein kinase enzyme appears to be a cellular contaminant of purified VSV because an activity from the uninfected cell extract can phosphorylate in vitro the dissociated viral proteins NS and M. The virion-associated activity may be derived either from the cytoplasm or the plasma membrane of the host cell since both of these cellular components contain protein kinase activity similar to that found in purified VSV.     

Protein Composition of the Structural Components of Vesicular Stomatitis Virus

Digitonin, a sterol glycoside which complexes with cholesterol, stripped off the envelope of vesicular stomatitis (VS) virions and liberated two viral structural proteins, 83% of P6 and 53% of P4. Deoxycholate also disrupted VS virions but released nucleocapsid cores which could be identified by higher buoyant density, ratio of incorporated (3)H-uridine to (14)C-protein, and electron microscopy. The major nucleocapsid protein was P5 but varying amounts of the minor protein aggregate P2 were present, depending on the concentration of urea used for extraction. P2 appeared to be a polymer of P5. Two other minor structural proteins, P1 and P3, could not be located in the virion. From these data, we conclude that the three microscopically identifiable structures of VS virions are each composed primarily of a single major protein, as follows: P6 = envelope protein, P4 = protein of underlying "shell," and P5 = nucleocapsid protein.     

Phosphorylation in vivo of a vaccinia-virus structural protein found associated with the ribosomes from infected cells.

When vaccinia-virus-infected cells were labeled with radioactive phosphate in the absence of viral gene expression an additional phosphoprotein, containing phosphoserine, was found specifically associated with the ribosomes. The phosphoprotein was removed from the ribosomes following a 0.5 M KCl washing or after EDTA treatment. This additional phosphoprotein was found in infected cells after either a long (3-4 h) or a short (30 min) labeling period; it was detected when the infected cells were incubated in the presence or absence of an inhibitor of RNA or protein synthesis. This phosphoprotein originated from the phosphorylation of vaccinia virion structural protein VP11b (Mr 11,000) at a specific site since only a single major phosphopeptide was obtained after trypsin digestion. This phosphoprotein was also present in purified vaccinia virions labeled with radioactive phosphate. VP11b protein was phosphorylated in vitro by the protein kinase associated with the cores. When the reaction was carried out at an alkaline pH the phosphorylation in vitro occurred at different sites in the protein; at neutral pH the phosphorylation of VP11b was more specific and, as judged by tryptic peptide analysis, occurred mainly at the same site as in the phosphorylation in vivo. A role for the involvement of phosphoprotein VP11b in the establishment of the shut off of host protein synthesis by vaccinia virus is suggested.     

Preferential phosphorylation of NP-protein of influenza A2 virus by virion-associated protein kinase.

Influenza A2 virions were found to contain protein kinase activity which was stimulated, like in other virion-associated kinases, with Mg++ and Nonidet-P 40 but not with cyclic AMP. The kinase phosphorylated only the NP-protein fraction of the influenza virions in the in vitro reaction. In contrast, none of the influenza virion proteins were phosphorylated significantly during the process of virus production in infected chorioallantoic membranes. The in vitro and in vivo phosphorylations of influenza viral proteins were compared with those of Sendai virus (HVJ).     

Mosquito cells infected with vesicular stomatitis virus yield unsialylated virions of low infectivity.

Vesicular stomatitis virus propagated in and released from Aedes albopictus cells had the normal complement of viral proteins; the glycoprotein contained carbohydrate but no sialic acid. These virions had markedly reduced hemagglutinating activity and exhibited a very high ratio of physical particles to infectious virus. In vitro sialylation of vesicular stomatitis virions grown in mosquito cells resulted in a 100-fold increase in both infectivity and hemagglutination titers to levels approaching those of virus grown in BHK-21 cells. These experiments provide an example of host-controlled modification of viral infectivity.     

The varicella-zoster virus immediate-early protein IE62 is a major component of virus particles.

Varicella-zoster virus (VZV) open reading frame (ORF) 62 potentially encodes a protein with considerable amino acid homology to the herpes simplex virus (HSV) immediate-early regulatory polypeptide ICP4 (or IE3). To identify and characterize its protein product(s) (IE62), we used a rabbit antiserum prepared against a synthetic peptide corresponding to the C-terminal 13 amino acids of the predicted protein. This antiserum reacted with phosphorylated polypeptides of 175 to 180 kDa that were made in VZV-infected cells and in cells infected with a vaccinia virus recombinant expressing IE62, but not in control-infected cells, confirming its specificity and reactivity to the IE62 protein. The antiserum recognized a 175-kDa polypeptide in purified virions that comigrated with a major structural protein. Comparison of this reactivity with that of an antipeptide antiserum directed against the VZV ORF 10 product (homologous to the HSV major structural protein VP16) indicates similar levels of ORF 62 and ORF 10 polypeptides in VZV virions. In contrast, antipeptide antiserum directed against the VZV ORF 29 product, the homolog of the HSV major DNA-binding protein, failed to recognize any protein in our virion preparations. Treatment of virions with detergents that disrupt the virion envelope did not dissociate IE62 from the nucleocapsid-tegument structure of the virion. Differential sensitivity of VZV virion IE62 to trypsin digestion in the presence or absence of Triton X-100 indicates that IE62 is protected from trypsin degradation by the virus envelope; since it is not a nucleocapsid protein, we conclude that it is part of the tegument. Finally, we show that the virion 175-kDa protein either can autophosphorylate or is a major substrate in vitro for virion-associated protein kinase activity.     

Herpes simplex virus phosphoproteins. II. Characterization of the virion protein kinase and of the polypeptides phosphorylated in the virion.

The protein kinase associated with purified herpes simplex virus 1 and 2 virions partitioned with the capsid-tegument structures and was not solubilized by non-ionic detergents and low, non-inhibitory concentrations of urea. The enzyme required Mg2+ or Mn2+ and utilized ATP or GTP. The activity was enhanced by non-ionic detergents and by Na+ even in the presence of high concentrations of of Mg2+, but not by cyclic nucleotides. The enzyme associated with capsid-tegument structures phosphorylated virion polypeptides only; exogenously added substrates (acidic and basic histones, casein, phosphovitin, protamine, and bovine serum albumin) were not phosphorylated. The major phosphorylated species were virion polypeptides (VP) 1-2, 4, 11-12, 13-14, 18.7, 18.8 and 23. VP 18.7 and VP 18.8 have not been previously detected, but may be phosphorylated forms of polypeptides co-migrating with VP 19. Of the remainder, only VP 23 has been previously identified as a capsid protein; the others are constituents of the tegument or of the under surface of the virion envelope. The distribution of the phosphate bound to viral polypeptides varied depending on the Mg2+ concentration and pH. In the absence of dithiothreitol, in vitro phosphate exchange was demonstrable in VP 23 and to a lesser extent in two other polypeptides on sequential phosphorylation frist with saturating amounts off unlabeled ATP and then with [gamma-32P]ATP. Analysis of the virion polypeptides specified by herpes simplex virus 1 X herpes simplex virus 2 recombinants indicates that the genes specifying the polypeptides which serve as a substrate for the protein kinase map in the unique sequences near the left and right reinterated DNA sequences of the L component.     

Human cytomegalovirus virion-associated protein with kinase activity.

Protein kinase activity was detected in immunoprecipitates of human cytomegalovirus virions and infected cells by using a monoclonal antibody directed against an abundant 68,000-dalton virion structural protein. Purification of this protein by electrophoresis confirmed that the kinase activity was associated with this protein. The kinase activity was dependent on divalent cations (Mg2+, Mn2+) and cyclic nucleotide independent and exhibited optimal activity at pH 7 to 8. The kinase phosphorylated threonine and serine but not tyrosine.     

Vaccinia virus penetration requires cholesterol and results in specific viral envelope proteins associated with lipid rafts

Vaccinia virus infects a wide variety of mammalian cells from different hosts, but the mechanism of virus entry is not clearly defined. The mature intracellular vaccinia virus contains several envelope proteins mediating virion adsorption to cell surface glycosaminoglycans; however, it is not known how the bound virions initiate virion penetration into cells. For this study, we investigated the importance of plasma membrane lipid rafts in the mature intracellular vaccinia virus infection process by using biochemical and fluorescence imaging techniques. A raft-disrupting drug, methyl-beta-cyclodextrin, inhibited vaccinia virus uncoating without affecting virion attachment, indicating that cholesterol-containing lipid rafts are essential for virion penetration into mammalian cells. To provide direct evidence of a virus and lipid raft association, we isolated detergent-insoluble glycolipid-enriched membranes from cells immediately after virus infection and demonstrated that several viral envelope proteins, A14, A17L, and D8L, were present in the cell membrane lipid raft fractions, whereas the envelope H3L protein was not. Such an association did not occur after virions attached to cells at 4 degrees C and was only observed when virion penetration occurred at 37 degrees C. Immunofluorescence microscopy also revealed that cell surface staining of viral envelope proteins was colocalized with GM1, a lipid raft marker on the plasma membrane, consistent with biochemical analyses. Finally, mutant viruses lacking the H3L, D8L, or A27L protein remained associated with lipid rafts, indicating that the initial attachment of vaccinia virions through glycosaminoglycans is not required for lipid raft formation.