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PubMed1981

Immune reactive measles virus polypeptides on the cell's surface: turnover and relationship of the glycoproteins to each other and to HLA determinants.

To better understand the mechanism(s) whereby antibody and complement and cytotoxic lymphocytes lyse infected cells, we studied the structure, interrelationship and turnover of measles virus polypeptides expressed on the cell's surface. Of the 6 major viral structural polypeptides, L, HA, P, NC, F, and M, found in purified virions or infected cells, only 2, the HA and F, resided on the surface of infected cells. The HA was present primarily in the form of a 160k dimer, and F was identified as a 64k polypeptide migrating distinct from other viral polypeptides. With reduction, the HA migrated as a 80k monomer, and F0, after cleavage, was found to be composed of a 42k nonglycosylated polypeptide, F1, and a 24k glycosylated protein, F2. The relationship between F0 and F1 and between the HA dimer and monomer was verified by tryptic peptide mapping. The turnover of HA and F from the cell's surface was 10 and 9 hr, respectively. However, in the presence of specific antibody after a marked loss of viral antigen from the surface, the turnover for HA and F was 15 and 12 hr, respectively. Despite being independent molecules, HA and F were closely linked, as they moved together (co-capped) over the plasma membrane when incubated with monospecific or monoclonal antibody. In contrast, neither HA nor F co-capped with the major histocompatibility antigens or with other host cell proteins, which indicates a separation between these host cell proteins and measles viral glycoproteins on the cell's surface.

Antibodies, Viral↗

PubMed1999

Virus particles of cucumber green mottle mosaic tobamovirus move systemically in the phloem of infected cucumber plants.

Systemic movement through the phloem of infected host plants is a key process in the life cycle of plant viruses, knowledge of which is scant. A main point to be elucidated is the structural form in which virus infection moves within the phloem. Indirect evidence suggests that virions might be the viral structure that moves in the phloem, but data from direct analysis in phloem sap have not been reported. We have done such analysis in the system cucumber (from which phloem exudate can be collected)/cucumber green mottle mosaic tobamovirus (CGMMV). CGMMV has structurally well-characterized particles. Both CGMMV coat protein and RNA were found in phloem exudate from infected cucumbers. Analysis of the accessibility of CGMMV RNA in phloem exudate to RNase A indicates that it is protected within a ribonucleoprotein structure. The accessibility to RNase A of the RNA in these structures was as in virus particles. Centrifugation analyses showed that the ribonucleoprotein structures in the phloem exudate have the same mass and isopycnic density as virions. Virus particles indistinguishable from purified virions were detected by electron microscopy in phloem exudate. No evidence of free RNA or other CGMMV-related structure was found in phloem exudate of infected plants. These results indicate that CGMMV movement in the phloem occurs mainly, if not exclusively, in the form of virus particles.

Capsid↗

PubMed1999

Insulin-like growth factor-II/mannose 6 phosphate receptors facilitate the matrix effects of latent transforming growth factor-beta1 released from genetically modified keratinocytes in a fibroblast/keratinocyte co-culture system.

This study was conducted to explore the mechanism of activation of transforming growth factor-beta1 (TGF-beta1) which is critical to its role in many physiological and pathological conditions. To date, almost all reports concerning TGF-beta1 activation delineated that release of mature TGF-beta1 from latency associated protein (LAP) is required for its activation. We report that latent TGF-beta1 (LTGF-beta1) released from TGF-beta1 genetically modified keratinocytes grown in the top chamber of a co-culture system functions as a fibrogenic factor through interaction with insulin-like growth factor-II/mannose-6-phosphate (IGF-II/M6P) receptors of human dermal fibroblasts grown in the lower chamber of this system. Following successful transduction, the pLin-LTGF-beta1 vector was amplified in PA31 7 packaging cells which possess viral structural proteins for vector in the presence of neomycin. Conditioned medium derived from packaging cells containing competent viral particles was then used to transduce either keratinocytes or fibroblasts grown in the upper chamber of a co-culture system, in which a 0.4 microm porous membrane separates the two chambers. In this way, LTGF-beta1 produced by transduced cells in the upper chamber is released and diffuses into the lower chambers where dermal fibroblasts are grown. Conditioned medium from the lower chamber was removed 3 days later and used to evaluate the latency and bioactivity of TGF-beta1 using enzyme-linked immunosorbent assay (ELISA) and mink lung (Mv1 Lu) epithelial growth inhibition assay. Cells were also harvested and used for RNA extraction. The results of these experiments showed that 1) the TGF-beta1-LAP complex, which was latent in traditionally used mink lung growth inhibition assay, directly modulated the expression of collagenase, type I, and type III collagen mRNA by dermal fibroblasts; 2) this stimulation was inhibited by M6P in a dose-dependent manner; 3) the TGF-beta1-LAP inhibits Mv1Lu epithelial cells only when this complex was incubated with cell membranes isolated from dermal fibroblasts; and 4) LTGF-beta1 activation seems to occur through a conformational alteration rather than by release of the mature TGF-beta1 from LAP in our co-cultured system. This conformational alteration seems to occur through the interaction of the TGF-beta1-LAP complex with the IGF-II/M6P receptors. Thus, the quantity of IGF-II/M6P receptors is important in cellular response to LTGF-beta1 in any physiological and pathological conditions.

Autocrine Communication↗

PubMed1984

The cytolytic T lymphocyte response to the murine cytomegalovirus. II. Detection of virus replication stage-specific antigens by separate populations of in vivo active cytolytic T lymphocyte precursors.

During the acute cytolytic T lymphocyte (CTL) response of mice to infection with the murine cytomegalovirus two independent populations of activated interleukin-receptive CTL precursors can be demonstrated. One population is specific for cell membrane-incorporated viral structural antigens, whereas the second population detects an antigen, whose appearance is correlated with the synthesis of viral immediate early proteins. Since this new type of antigen is only defined by lymphocyte recognition, it is referred to as the lymphocyte-detected immediate early antigen (LYDIEA). Expression of immediate early antigen precedes the production of viral progeny and, therefore, it is possible that LYDIEA-specific CTL could serve as indicator cells for the very first activities of the viral genome, even during nonproductive infection.

Animals↗

PubMed1994

A functional interaction between Rev and yeast pre-mRNA is related to splicing complex formation.

The HIV-1 Rev protein regulates viral gene expression by enhancing the export of partially spliced or nonspliced viral transcripts to the cytoplasm. This activity is mediated by the interaction of Rev with a highly structured viral RNA sequence, the Rev response element (RRE). In this paper, Rev regulation has been recapitulated in the yeast Saccharomyces cerevisiae. The data show that Rev enhances yeast pre-mRNA translation in an RRE-dependent manner and requires intact RNA binding and effector domains. In addition, reporter gene mutations in the 5' splice site and in the branch point are epistatic to and eliminate Rev responsiveness, i.e. some spliceosome assembly pathway steps precede a functional interaction with Rev. The results indicate that some of the Rev-interacting components are conserved between yeast and mammals and suggest that Rev acts on yeast pre-mRNA in the context of the spliceosome.

Biological Transport↗

PubMed1984

A mutant of sindbis virus with a host-dependent defect in maturation associated with hyperglycosylation of E2.

Following serial passage of Sindbis virus (SV) on Aedes albopictus mosquito cells a mutant (SVap15/21) was isolated which in chick cells produced small plaques and was temperature sensitive (ts). At 34.5 degrees this mutant replicated normally in mosquito cells, but only poorly in chick or BHK cells. In the vertebrate cells SVap15/21 was RNA+ at both 34.5 and 40 degrees and on the basis of complementation tests carried out at 40 degrees, was assigned to complementation group E. The block in the replication of this mutant, like that of ts20, the prototype mutant of complementation group E, was at the level of nucleocapsid envelopment. The PE2 and E2 glycoproteins of SVap15/21 were found to be hyperglycosylated relative to the corresponding glycoproteins of the parent virus (SVstd). Analysis of revertants of SVap15/21 suggests a causal relationship between PE2 and E2 hyperglycosylation and the host-specific defect in virus maturation. The association of a host-specific defect in virion assembly with hyperglycosylation of a viral structural protein points to the potential importance of host-specific glycosylation patterns in the determination of viral host range.

Aedes↗

PubMed2026

HMGB1 as a convergent host factor in virus-induced carcinogenesis.

High-mobility group box 1 (HMGB1) is a chromatin-associated protein and a prototypical damage-associated molecular pattern whose dual intracellular and extracellular functions are increasingly implicated in cancer progression. Because viral proteins can harness HMGB1 to facilitate their own replication and remodel the microenvironment of transformed cells, human oncogenic viruses provide an instructive model for examining this duality. In this conceptual review, we organized the available evidence around two functional nodes. At the first node, intracellular HMGB1 supports viral replication, acting on viral chromatin in Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus, and on structured viral RNA in hepatitis C virus. At the second node, viral infection or specific viral oncoproteins induce HMGB1 secretion, which promotes infected-cell survival and remodels the tumor microenvironment, as reported for KSHV, hepatitis B virus, and human T-cell leukemia virus type 1. Human papillomavirus engage a receptor-level variant of this node through the HMGB1-TLR4 axis. Only KSHV currently supports both nodes in matched experimental systems. Therefore, we present a sequential two-node arrangement as a hypothesis, instead of an established property of oncogenic viruses. We further considered how viruses reverse the tumor-suppressive, genome-stabilizing functions of nuclear HMGB1, with conserved and divergent strategies apparent across viral families; why the absence of HMGB1 data for Merkel cell polyomavirus is a tractable and informative gap; and which HMGB1- and RAGE-directed agents are realistically positioned for evaluation in virus-associated cancers.

Damage-associated molecular pattern↗

PubMed1998

The recombinant nucleocapsid protein of classical swine fever virus can act as a transcriptional regulator.

The cDNA of the nucleocapsid (core) protein of classical swine fever virus (CSFV) was generated by reverse transcription-polymerase chain reaction (RT-PCR) and cloned into a eukaryotic expression vector. The effect of the recombinant core protein on the transcriptional regulation of cellular as well as viral promoters was studied. Using transient transfection assay, our results demonstrated that the core protein can activate the promoter of human heat shock protein 70 gene, and suppressed the SV40 early promoter. These findings indicate that the core protein appears to function not only as a viral structural protein but also as a regulator of gene expression. The implications of core proteins on the viral maturation are discussed.

Animals↗

PubMed2000

A leptomycin B-sensitive homologue of human CRM1 promotes nuclear export of nuclear export sequence-containing proteins in Drosophila cells.

The Rev protein of human immunodeficiency virus is a nuclear shuttling protein that promotes nuclear export of mRNAs that encode the viral structural proteins Gag, Pol, and Env. Rev binds to a highly structured RNA motif, the Rev-responsive element (RRE), that is present in all Rev-responsive viral transcripts and facilitates their entry into a nuclear export pathway by recruiting cellular export factors. In mammalian and yeast cells, the principal export receptor engaged by Rev has been identified as the importin/transportin family member CRM1/exportin 1. CRM1 binds directly to a leucine-rich nuclear export sequence (NES) present in Rev, and similar motifs have been identified in a variety of cellular nuclear shuttling proteins. We and our colleagues previously demonstrated that, in transfected Drosophila cells, HIV-1 Rev is fully functional and promotes expression of the viral envelope glycoprotein. We now demonstrate that the fundamental mechanism of Rev action in insect cells is identical to that observed in the mammalian systems. In particular, we show that Drosophila cells express a leptomycin B-sensitive homologue of human CRM1 that supports Rev-dependent gene expression and is required for nuclear export of NES-containing proteins in insect cells.

Amino Acid Sequence↗

PubMed1974

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.

Animals↗

PubMed1979

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 Line↗

PubMed2001

Monoclonal antibodies with broad specificity for hepatitis C virus hypervariable region 1 variants can recognize viral particles.

The hypervariable region 1 (HVR1) of the E2 protein of hepatitis C virus (HCV) is a highly heterogeneous sequence that is promiscuously recognized by human sera via binding to amino acid residues with conserved physicochemical properties. We generated a panel of mAbs from mice immunized with HVR1 surrogate peptides (mimotopes) affinity-selected with sera from HCV-infected patients from a phage display library. A high number of specific clones was obtained after immunization with a pool of nine mimotopes, and the resulting mAbs were shown to recognize several 16- and 27-mer peptides derived from natural HVR1 sequences isolated from patients with acute and chronic HCV infection, suggesting that HVR1 mimotopes were efficient antigenic and immunogenic mimics of naturally occurring HCV variants. Moreover, most mAbs were shown to bind HVR1 in the context of a complete soluble form of the E2 glycoprotein, indicating recognition of correctly folded HVR1. In addition, a highly promiscuous mAb was able to specifically capture bona fide viral particles (circulating HCV RNA) as well as rHCV-like particles assembled in insect cells expressing structural viral polypeptides derived from an HCV 1a isolate. These findings demonstrate that it is possible to induce a broadly cross-reactive clonal Ab response to multiple HCV variants. In consideration of the potentially important role of HVR1 in virus binding to cellular receptor(s), such a mechanism could be exploited for induction of neutralizing Abs specific for a large repertoire of viral variants.

Antibodies, Monoclonal↗

PubMed1984

Morphology of bovine viral diarrhea virus.

The morphology of bovine viral diarrhea virus (BVDV) was studied by electron microscopy. The NADL strain of BVDV was plaque purified 3 times, concentrated by polyethylene glycol precipitation, and purified by centrifugation to equilibrium in continuous potassium tartrate density-gradients. The virus was examined by negative-stain electron microscopy in the presence or absence of specific antiserum. The density of BVDV was between 1.101 g/cm3 and 1.174 g/cm3, with the peak at maximum infectivity at 1.122 g/cm3. Oval to pleomorphic viral particles, 120 ( +/- 30) nm in diameter, were enriched in the peak of maximum infectivity. The detailed structure of virions was revealed: a 5- to 7-microns thick unit membrane-like envelope layer with numerous projecting knobs, 4 to 5 nm in diameter, surrounding an interior core-like structure. Viral particles measuring 120 ( +/- 30) nm were found in large aggregates in the presence of specific antiserum.

Animals↗

PubMed2006

Structure of complex viruses and virus-infected cells by electron cryo tomography.

In microbiology, and in particular in virus research, electron microscopy (EM) is an important tool, offering a broad approach for investigating viral structure throughout their intracellular and extracellular life cycles. Currently, molecular tools and rapid developments in advanced light microscopy dominate the field and supply an enormous amount of information concerning virus biology. In recent years, numerous fascinating high-resolution EM structures obtained by single-particle electron cryo microscopy (cryo-EM) were revealed for viral particles that possess icosahedral symmetry. However, no comprehensive three-dimensional analysis of complex viruses or viruses within cells has yet been achieved using EM. Recent developments in electron cryo-tomography render this a proficient tool for the analysis of complex viruses and viruses within cells in greater detail.

Animals↗

PubMed1984

Ion etching of human adenovirus 2: structure of the core.

The surface of human adenovirus 2 was etched by irradiating intact virions with low-energy (1-keV) Ar+ ions in a Technics Hummer V sputter coater . Viral structures exposed by the etching process were shadowed and then examined in the electron microscope. Periods of etching that were sufficient to reduce the viral diameter by 20 to 30 nm revealed distinct substructural elements in the virion core. Cores were found to consist of a cluster of 12 large, uniformly size spheres which abutted one another in the intact virion. The spheres, for which we suggest the name " adenosomes ," had a diameter of 23.0 +/- 2.3 nm, and they were related to each other by two-, three-, and fivefold axes of rotational symmetry. The results support the view, originally suggested by Brown et al. (J. Virol. 16:366-387, 1975) that the adenovirus 2 core is composed of 12 large spheres packed tightly together in such a way that each is directed toward the vertex of an icosahedron . Such a structure, constructed of 23.0-nm-diameter spheres, would have an outside diameter (vertex-to-vertex distance) of 67.0 nm and a face-to-face distance of 58.2 nm. It could be accommodated inside the icosahedral adenovirus capsid if each large sphere were located beneath a capsid vertex.

Adenoviruses, Human↗

PubMed1969

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.

Amino Acids↗

PubMed1989

Passive transfer of antiviral antibodies restricts replication of Aleutian mink disease parvovirus in vivo.

When mink kits were infected neonatally with a highly virulent strain of Aleutian disease virus (ADV), 100% of both Aleutian and non-Aleutian genotype mink died of interstitial pneumonia characterized by permissive ADV infection of alveolar type II cells. Treatment of infected kits with either mink anti-ADV gamma globulin or mouse monoclonal antibodies against ADV structural proteins reduced mortality by 50 to 75% and drastically reduced the severity of clinical signs. Interestingly, mink kits that survived the acute pulmonary disease all developed the chronic form of immune complex-mediated Aleutian disease. Thus, the antibodies directed against ADV structural proteins were capable of modulating the in vivo pathogenicity from an acute fulminant disease to a chronic immune complex-mediated disorder. The mechanism of this modulation was examined by strand-specific in situ hybridization. We found that the number of ADV-infected type II cells was the same in both untreated and antibody-treated kits. However, in the treated kits, viral replication and transcription were restricted at the cellular level. These data suggested that antibodies prevented acute viral pneumonia by restricting the intracellular level of viral replication and that the relevant antigenic determinants were contained within the viral structural proteins. The restricted levels of viral replication and transcription seen in antibody-treated mink kits resembled the levels observed in infected adult mink and suggested a role of antiviral antibodies in development of persistent infection and chronic immune complex disease.

Acute Disease↗

PubMed1992

Molecular cloning and nucleotide sequence of a pestivirus genome, noncytopathic bovine viral diarrhea virus strain SD-1.

Genomic RNA of noncytopathic (NCP) bovine viral diarrhea virus (BVDV) strain SD-1 was extracted directly from serum obtained from a persistently infected animal. cDNA was synthesized and amplified by polymerase chain reaction (PCR) before cloning. The complete genomic nucleotide sequence was determined by sequencing at least two different clones from independent PCR reactions. The 5' and 3' end sequences of the SD-1 genome was determined from 5'-3' ligation clones. The complete genome sequence was comprised of 12,308 nucleotides containing one large open reading frame which encodes an amino acid sequence of 3898 residues with a calculated molecular weight of 438 kDa. In contrast to cytopathic (CP) BVDV strain NADL, which contains a cellular RNA insert of 270 nucleotides and CP BVDV strain Osloss, which has an inserted ubiquitin RNA sequence of 228 nucleotides, the NCP strain SD-1 had no insertion along the genome. Sequence comparison with other pestiviruses revealed that the overall nucleotide sequence homologies of SD-1 are 88.6% with NADL, 78.3% with Osloss, 67.1% with HoCV Alfort, and 67.2% with HoCV Brescia. The overall deduced amino acid sequence homologies of SD-1 are 92.7% with NADL, 86.2% with Osloss, 72.5% with HoCV Alfort, and 71.2% with HoCV Brescia. The most conserved nucleotide and amino acid sequences are located in the 5' untranslated region (5'UTR) and nonstructural protein p80 region, respectively. The viral glycoproteins, particularly gp53, and nonstructural proteins p54 and p58 have the lowest homology comparing both nucleotide and amino acid sequences between SD-1 and other pestiviruses. Extensive analyses of amino acid sequences for the viral structural proteins and nonstructural protein p54 regions from five pestiviruses led to the identification of four conserved domains (designated as C1, C2, C3, C4) and three highly variable domains (designated as V1, V2, V3) within this region. The C1, C2, and C3 domains are located in the capsid protein p14, glycoprotein gp48, and gp25, respectively. The C4 domain is located in the junction between gp53 and p54. Interestingly, out of three variable domains, two (V1, V2) are located in the same glycoprotein gp53. The third variable domain is located in the nonstructural protein p54.

Amino Acid Sequence↗