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PubMed2000

Efficient DNA transfection mediated by the C-terminal domain of human immunodeficiency virus type 1 viral protein R.

Viral protein R (Vpr) of human immunodeficiency virus type 1 is produced late in the virus life cycle and is assembled into the virion through binding to the Gag protein. It is known to play a significant role early in the viral life cycle by facilitating the nuclear import of the preintegration complex in nondividing cells. Vpr is also able to interact with nucleic acids, and we show here that it induces condensation of plasmid DNA. We have explored the possibility of using these properties in DNA transfection experiments. We report that the C-terminal half of the protein (Vpr(52-96)) mediates DNA transfection in a variety of human and nonhuman cell lines with efficiencies comparable to those of the best-known transfection agents. Compared with polylysine, a standard polycationic transfection reagent, Vpr(52-96) was 10- to 1,000-fold more active. Vpr(52-96)-DNA complexes were able to reach the cell nucleus through a pH-independent mechanism. These observations possibly identify an alternate pathway for DNA transfection.

Amino Acid Sequence↗
PubMed1979

The in vitro of Rous sarcoma virus RNA and function of the viral protein during the viral replication.

The gag gene and pol gene of the Rous sarcoma virus are translated in vitro from the 35S viral RNA. The env gene cannot be translated in vitro from the 35S RNA. For the in vitro translation of the src gene. 3' end fragments of the viral RNA are used. The gag protein p15 has a proteolytic activity and specifically processes its own protein precursor pr76. The gag protein p19 suppresses the in vitro translation of the pol gene.

Animals↗
PubMed1991

BVD monoclonal antibodies: relationship between viral protein specificity and viral strain specificity.

Seventeen monoclonal antibodies raised against bovine viral diarrhoea virus were divided into three groups on the basis of radioimmunoprecipitation results. Seven monoclonal antibodies precipitated a polypeptide of 80kD and defined four domains, all of which showed considerable conservation amongst the 180 pestivirus strains and isolates examined. Nine monoclonal antibodies, including six with virus neutralizing activity, precipitated a 53kD polypeptide and all appeared to be directed towards a single domain of clustered epitopes. Several of these epitopes were present in many ruminant virus strains and isolates, but not in hog cholera viruses. A single monoclonal antibody precipitated a 48kD polypeptide, defining an epitope that was also present on many ruminant viruses, but not hog cholera viruses. Most pestiviruses from cattle and some from sheep shared a number of epitopes located on three different proteins.

Animals↗
PubMed1989

A promoter of Epstein-Barr virus that can function during latent infection can be transactivated by EBNA-1, a viral protein required for viral DNA replication during latent infection.

A viral promoter that functions on recombinant plasmids in cells immortalized by Epstein-Barr virus was identified and characterized. It is identical to that mapped on the viral genome by Bodescot et al. (M. Bodescot, M. Perricaudet, and P.J. Farrell, J. Virol. 61:3424-3430, 1987) which functions during the latent phase of the viral life cycle in some but not all cells to encode several latent viral gene products. Experiments with these plasmids indicated that this promoter requires the enhancer within oriP of Epstein-Barr virus in cis to function efficiently. They also indicated that it requires the EBNA-1 gene in trans to function efficiently. The EBNA-1 gene therefore positively affects both viral DNA replication (J.L. Yates, N. Warren, and B. Sugden, Nature [London] 313:812-815, 1985) and viral transcription.

Antigens, Viral↗
PubMed1986

Herpes simplex virus suppression of human endothelial matrix protein synthesis is independent of viral protein synthesis.

Protein synthesis was determined in cultures of human umbilical cord vein endothelial cells (EC) infected with either herpes simplex type 1 (HSV-1) or type 2 (HSV-2). Monolayers were infected for 1 hour with either 5 or 20 infectious virus particles per EC (multiplicity of infection of 5 or 20). At different times after infection, infected and noninfected cultures were pulsed with either 5 mu Ci/ml of [14C]proline or 25 mu Ci/ml of [35S]methionine for 1 or 2 hours. Autoradiograms of sodium dodecyl sulfate-gel electrophoresis showed suppression of matrix protein synthesis by 4 hours postinfection which became almost complete at 6 hours. Multiplicity of infection of 20 was more effective than multiplicity of infection of 5 at suppressing matrix protein synthesis at 4 or 6 hours postinfection. Infection of EC with ultraviolet light-inactivated virus resulted in the shutoff of host-cell as well as virus protein synthesis. Similar results were observed when monolayers of EC were infected with intact virus in the presence of 2 micrograms/ml of actinomycin-D, an inhibitor of RNA synthesis. On the other hand, uninfected EC in the presence of actinomycin-D continued to synthesize protein from pre-existing mRNA. The time of shutoff of synthesis of specific matrix proteins varied with the protein i.e., shut-off of type IV collagen occurred first, followed by that of fibronectin, and then of thrombospondin. The data suggest that the degree of suppression of synthesis of EC matrix proteins after infection with HSV-1 or HSV-2 is dependent on the dose of the virus inoculum; it occurs at the translational level and is not dependent on new viral protein synthesis.

Cells, Cultured↗
PubMed2000

Heat-shock protein 70 can replace viral protein R of HIV-1 during nuclear import of the viral preintegration complex.

Heat-shock proteins (Hsp's) are a family of molecular chaperones that contribute to protection from environmental stress. In this report, we demonstrate that a member of this family, Hsp70, facilitates nuclear import of HIV-1 preintegration complexes (PICs). The mechanism of this activity appears to be similar to the one used by Vpr, an HIV-1 protein regulating viral nuclear import and replication in macrophages. Indeed Hsp70 stimulated binding of HIV-1 matrix antigen to GST-karyopherin alpha fusion protein and rescued nuclear import of a Vpr-defective HIV-1 strain in vitro. Binding studies with truncated forms of GST-karyopherin alpha demonstrated that both Vpr and Hsp70 bind to a region in the amino-terminal part of the karyopherin alpha molecule. This region appears to be distinct from the binding sites for two other karyopherin alpha cargoes, basic-type NLS-containing proteins and transcription factor STAT-1. Vpr competed with Hsp70 for binding to karyopherin alpha. These results suggest the presence of a novel regulatory site on karyopherin alpha which is used by Hsp70 and Vpr to stimulate interaction between the HIV-1 PIC and karyopherin alpha and thus promote viral nuclear import.

Antigens, Polyomavirus Transforming↗
PubMed2006

Rotavirus Nonstructural Protein NSP3 is not required for viral protein synthesis.

Initiation is the rate-limiting step in protein synthesis and therefore an important target for regulation. For the initiation of translation of most cellular mRNAs, the cap structure at the 5' end is bound by the translation factor eukaryotic initiation factor 4E (eIF4E), while the poly(A) tail, at the 3' end, is recognized by the poly(A)-binding protein (PABP). eIF4G is a scaffold protein that brings together eIF4E and PABP, causing the circularization of the mRNA that is thought to be important for an efficient initiation of translation. Early in infection, rotaviruses take over the host translation machinery, causing a severe shutoff of cell protein synthesis. Rotavirus mRNAs lack a poly(A) tail but have instead a consensus sequence at their 3' ends that is bound by the viral nonstructural protein NSP3, which also interacts with eIF4GI, using the same region employed by PABP. It is widely believed that these interactions lead to the translation of rotaviral mRNAs, impairing at the same time the translation of cellular mRNAs. In this work, the expression of NSP3 in infected cells was knocked down using RNA interference. Unexpectedly, under these conditions the synthesis of viral proteins was not decreased, while the cellular protein synthesis was restored. Also, the yield of viral progeny increased, which correlated with an increased synthesis of viral RNA. Silencing the expression of eIF4GI further confirmed that the interaction between eIF4GI and NSP3 is not required for viral protein synthesis. These results indicate that NSP3 is neither required for the translation of viral mRNAs nor essential for virus replication in cell culture.

Animals↗
PubMed1983

Rat cells transformed by simian virus 40 give rise to tumor cells which contain no viral proteins and often no viral DNA.

Rat 3T3 cells transformed by simian virus 40 were injected into rats to examine their capacity to develop into tumors. Both large T-dependent (N) transformants and large T-independent (A) transformants were used. All the transformed cell lines contained large T and small t and could multiply efficiently in agar. Only some transformants could develop into tumors. All tumor cells examined had lost both large T and small t. Tumor cells in which the viral genome could still be detected were found together with tumor cells in which the simian virus 40 DNA could no longer be detected. N transformants which displayed the transformed phenotype in a temperature-sensitive manner became temperature insensitive during tumor formation.

Animals↗
PubMed1991

Determination of viral proteins present in the human immunodeficiency virus type 1 preintegration complex.

Cytoplasmic extracts prepared from cells infected with metabolically radiolabeled virions of human immunodeficiency virus type 1 contain viral DNA in association with labeled viral proteins. Viral DNA can be purified from these extracts by gel filtration chromatography and sucrose gradient sedimentation as a part of a nucleoprotein complex containing integrase as the only viral protein detectable by immunoprecipitation and gel electrophoretic analysis. The purified complex contains no detectable gag gene products, including p17, p24, p7, or p6, and contains no additional pol gene products, including the p10 protease, p66 and p51 polymerase, or the p15 RNase H. Nearly all of the purified nucleoprotein complexes are capable of integrating into heterologous DNA targets in vitro. These observations demonstrate that integrase is a component of the human immunodeficiency virus type 1 preintegration complex and suggest that integrase may be the only viral protein necessary for the integration of retroviral DNA.

Attachment Sites, Microbiological↗
PubMed1997

The three subunits of the polymerase and the nucleoprotein of influenza B virus are the minimum set of viral proteins required for expression of a model RNA template.

The genes encoding the nucleoprotein, PB1, PB2, and PA proteins of the influenza virus strain B/Panamá/45/90 have been cloned under control of the T7 RNA polymerase promoter of plasmid pGEM-3. Transfection of the recombinant plasmids obtained into mammalian cells, which had been infected with a vaccinia virus encoding the T7 RNA polymerase, resulted in expression of the expected influenza B virus polypeptides. Moreover, it is shown that coexpression of the four recombinant core proteins in COS-1 cells reconstituted a functional polymerase capable of expressing a synthetic influenza B virus-like CAT RNA. By using the influenza B virus recombinant plasmids and a set of pGEM-derived plasmids encoding the homologous core proteins of the influenza A virus A/Victoria/3/75 (I. Mena et al. (1994). J. Gen. Virol. 75, 2109-2114), the capabilities of homo- and heterotypic mixtures of the four core proteins to express synthetic type A and B CAT RNAs were analyzed. Both the influenza A and B virus polymerases were active in expressing, albeit with reduced efficiencies, the heterotypic model CAT RNAs. However, none of all possible heterotypic mixtures of the core proteins reconstituted a functional polymerase. In order to fully characterize the recombinant plasmids obtained, the nucleotide sequences of the cloned genes were determined and compared to sequences of other type B virus isolates. The results obtained from these latter analyses are discussed in terms of the conservation and evolution of the influenza B virus core genes.

Amino Acid Sequence↗
PubMed1999

Evidence for 4-hydroxyproline in viral proteins. Characterization of a viral prolyl 4-hydroxylase and its peptide substrates.

4-Hydroxyproline, the characteristic amino acid of collagens and collagen-like proteins in animals, is also found in certain proline-rich proteins in plants but has been believed to be absent from viral and bacterial proteins. We report here on the cloning and characterization from a eukaryotic algal virus, Paramecium bursaria Chlorella virus-1, of a 242-residue polypeptide, which shows distinct sequence similarity to the C-terminal half of the catalytic alpha subunits of animal prolyl 4-hydroxylases. The recombinant polypeptide, expressed in Escherichia coli, was found to be a soluble monomer and to hydroxylate both (Pro-Pro-Gly)(10) and poly(L-proline), the standard substrates of animal and plant prolyl 4-hydroxylases, respectively. Synthetic peptides such as (Pro-Ala-Pro-Lys)(n), (Ser-Pro-Lys-Pro-Pro)(5), and (Pro-Glu-Pro-Pro-Ala)(5) corresponding to proline-rich repeats coded by the viral genome also served as substrates. (Pro-Ala-Pro-Lys)(10) was a particularly good substrate, with a K(m) of 20 microM. The prolines in both positions in this repeat were hydroxylated, those preceding the alanines being hydroxylated more efficiently. The data strongly suggest that P. bursaria Chlorella virus-1 expresses proteins in which many prolines become hydroxylated to 4-hydroxyproline by a novel viral prolyl 4-hydroxylase.

Amino Acid Sequence↗
PubMed2000

Posttranslational processing of infected cell protein 22 mediated by viral protein kinases is sensitive to amino acid substitutions at distant sites and can be cell-type specific.

Infected cell protein 22 (ICP22) is posttranslationally phosphorylated by the viral kinases encoded by U(S)3 and U(L)13 and nucleotidylylated by casein kinase II. In rabbit and rodent cells and in primary human fibroblasts infected with mutants from which the alpha 22 gene encoding ICP22 had been deleted, a subset of late (gamma(2)) gene products exemplified by U(L)38 and U(S)11 proteins are expressed at a reduced level, as measured by the accumulation of both mRNA and protein. The same phenotype was observed in cells infected with mutants lacking the U(L)13 gene. The focus of this report is on three serine- and threonine-rich domains of ICP22. Two of these domains are homologs located between residues 38 to 66 and 300 to 328. The third domain is near the carboxyl terminus and contains the sequence T374SS. The results were as follows. (i) Alanine substitutions in the amino-terminal homolog precluded the posttranslational processing of ICP22 in rabbit skin cells and in Vero cells but had no effect on the accumulation of either U(S)11 or U(L)38 protein. (ii) Alanine substitutions in the carboxyl-terminal homolog had no effect on posttranslational processing of ICP22 accumulating in Vero cells but precluded full processing of ICP22 accumulating in rabbit skin cells. The effect on accumulation of U(L)38 and U(S)11 proteins was insignificant in Vero cells and minimal in rabbit skin cells. (iii) Substitutions of alanine for the threonine and serines in the third domain precluded full processing of ICP22 and caused a reduction of accumulation of U(S)11 and U(L)38 proteins. These results indicate the following. (i) The posttranslational processing of ICP22 is sensitive to mutations within the domains of ICP22 tested and is cell-type dependent. (ii) Posttranslational processing of ICP22 is not required for accumulation of U(L)38 and U(S)11 proteins to the same level as that seen in cells infected with the wild-type virus. (iii) The T374SS sequence shared by ICP22 and the U(S)1.5 proteins is essential for the accumulation of a subset of gamma(2) proteins exemplified by U(S)11 and U(L)38 and is the first step in mapping of the sequences necessary for optimal accumulation of U(S)11 and U(L)38 proteins.

Animals↗
PubMed1973

Radioimmunoassay of mammalian type C viral proteins. 3. Detection of viral antigen in normal murine cells and tissues.

A radioimmunoassay specific for a murine leukemia virus structural protein, the gs antigen, detects an antigenic reactivity in normal murine cells in culture and natural tissues. The assay was shown to measure an antigen that is highly related to the virion protein as shown by absorption tests, immunoadsorbent chromatography, and by analysis of linearized dose-response curves. These findings combined with the finding of viral-specific RNA indicate that portions of the viral genome are being expressed with a much greater frequency than previously appreciated.

Animals↗
PubMed1989

[Amino acid sequence analysis of barley stripe mosaic viral proteins: tentative identification of viral serine proteases].

Analysis of amino acid sequences of barley stripe mosaic virus (BSMV) proteins revealed the pentapeptide GDSGG, the sequence unique for catalytic centers of serine chymotrypsin-like proteases, in protein p14 encoded by open reading frame 4 of RNA beta. Computer-assisted comparisons revealed a statistically significant similarity between amino acid sequences of p14 and chymotrypsin-like proteases. The catalytic His and Asp residues tentatively identified in p14 together with the Ser residue of the GDSGG sequence, presumably, constitute the "catalytic triad" characteristic of chymotrypsin-like proteases. Based on these observations and on the presence of a potential N-proximal transmembrane domain in p14, this protein may be suggested to be a serine protease involved in processing of the replicase precursor within a membrane-bound replication complex of BSMV.

Amino Acid Sequence↗
PubMed1986

A mutant herpesvirus protein leads to a block in nuclear localization of other viral proteins.

The herpes simplex virus mutants KOS1.1 ts756 and HFEM tsLB2 express temperature-sensitive ICP4 proteins that are not localized properly to the cell nucleus at the nonpermissive temperature. In these infected cells at the nonpermissive temperature, nuclear localization of at least two other viral proteins, ICP0 and ICP8, is impaired. Replacement of the mutated sequences in the ICP4 gene of tsLB2 restored proper nuclear localization of all of the proteins. The ICP0 and ICP8 proteins expressed in cells transfected with their individual genes were localized to the cell nucleus. Therefore, in infected cells, the mutant ICP4 gene product appears to be the primary defect which leads to the block in nuclear localization of the other proteins. One viral protein, ICP27, was not inhibited for nuclear localization in these cells. These data indicate that there are at least two pathways for nuclear localization of HSV proteins, one of which is inhibited by the mutant ICP4 protein. The mutant ICP4 protein may define a probe for one of the pathways of nuclear localization of proteins.

Animals↗
PubMed1997

Interaction of the viral protein genome linked of turnip mosaic potyvirus with the translational eukaryotic initiation factor (iso) 4E of Arabidopsis thaliana using the yeast two-hybrid system.

The yeast LexA interaction trap was used to screen a cDNA library from Arabidopsis thaliana in order to identify proteins that interact with the viral protein genome linked (VPg)-proteinase of turnip mosaic potyvirus. The screen allowed the isolation of four candidate cDNA clones. Clones pHC4, pHC21, and pHC40 were partially sequenced but no homologies to known proteins were found. However, the amino acid sequence deduced from the complete nucleotide sequence of pSW56 revealed that it was the eukaryotic initiation factor (iso) 4E [eIF(iso)4E]. Deletion analysis indicated that the VPg domain was involved in the interaction with the plant protein. Interaction between the viral protein and the cellular protein was confirmed by ELISA-based binding experiments. eIF(iso)4E plays an essential role in the initiation of the translation of capped mRNAs and its association with VPg would point to a role of the viral protein in the translation of the virus.

Amino Acid Sequence↗
PubMed1972

Proteins specified by herpes simplex virus. VI. Viral proteins in the plasma membrane.

Previous papers in the series have shown that the surface membranes of herpesvirus-infected cells acquire new immunological specificities and that purified infected cell membrane preparations, characterized by their physical properties rather than topology in the cell, contain new glycoproteins genetically determined by the virus. In this study, we prepared purified plasma membrane identified by its 5' nucleotidase, fucose, and reduced nicotinamide adenine dinucleotide-diaphorase content. Analysis of the membrane proteins and glycoproteins by electrophoresis in acrylamide gels indicated the following. (i) Purified plasma membranes from infected cells contained two sets of proteins, i.e., host proteins were present both before and after infection and viral proteins were present only after infection. (ii) After infection, no appreciable selective or nonselective loss of host proteins from membranes was demonstrable. However, no new host proteins were made. (iii) Electropherograms of plasma membrane proteins from infected cells indicated the presence of at least 12 virus-specific proteins ranging in molecular weight from 25 x 10(3) to 126 x 10(3) daltons. Of these, at least nine were glycosylated. Proteins and glycoproteins with similar electrophoretic mobilities but in somewhat different ratios were also present in preparations of highly purified virions.

Carbon Isotopes↗
PubMed1989

Replication of the genome RNAs of defective interfering particles of vesicular stomatitis and Sendai viruses using heterologous viral proteins.

We have tested the ability of heterologous viral proteins to support the in vivo and in vitro replication of the RNA of defective interfering (DI) particles of two serotypes of VSV and of Sendai virus. In all the combinations of heterologous coinfections in vivo, DI particle replication was observed only in the coinfection with the VSV-Indiana DI particle and wild-type VSV-New Jersey. By quantitating RNA synthesis in reconstitution experiments we showed that with DI nucleocapsids isolated from infected cells, however, the soluble protein fraction from heterologous wild-type virus-infected cells could substitute in vitro to varying degrees for the homologous proteins in the elongation reaction of RNA replication and encapsidation. In these cases successful replication was confirmed by demonstrating the specific association of the heterologous N protein with the product nucleocapsid RNA. The initiation step, that is, the initial binding of the nucleocapsid protein to the leader RNA, in contrast, requires the homologous protein, since heterologous viral proteins could not support RNA replication and encapsidation from purified DI particles.

Capsid↗