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PubMed1975

[New data on the structure and the budding of FV3 (frog virus 3) (author's transl)].

The freeze-etching technique when applied to FV3 suspensions revealed the perfectly icosahedral structure of the viral nucleocapsids. This allowed a fine substructure suggesting the existence of numerous subunits of small dimensions to be detected at their surface. Observations of the replicas of infected cells obtained by freeze-etching revealed modifications of the external face of the cytoplasmic membrane occurring at the level of the budding viral particles; in these zones parallel stripes appeared, whose elementary structure is constant (13,5 nm). Observations of the infected cells with the scanning microscope enabled the spreading of the budding process on all the cells of a same culture to be studied. By counting the number of budding particles per mum2, it was possible to demonstrate the heterogeneity of the budding process at the surface of the cells.

Animals↗
PubMed2006

Evolutionary genomics of archaeal viruses: unique viral genomes in the third domain of life.

In terms of virion morphology, the known viruses of archaea fall into two distinct classes: viruses of mesophilic and moderately thermophilic Eueryarchaeota closely resemble head-and-tail bacteriophages whereas viruses of hyperthermophilic Crenarchaeota show a variety of unique morphotypes. In accord with this distinction, the sequenced genomes of euryarchaeal viruses encode many proteins homologous to bacteriophage capsid proteins. In contrast, initial analysis of the crenarchaeal viral genomes revealed no relationships with bacteriophages and, generally, very few proteins with detectable homologs. Here we describe a re-analysis of the proteins encoded by archaeal viruses, with an emphasis on comparative genomics of the unique viruses of Crenarchaeota. Detailed examination of conserved domains and motifs uncovered a significant number of previously unnoticed homologous relationships among the proteins of crenarchaeal viruses and between viral proteins and those from cellular life forms and allowed functional predictions for some of these conserved genes. A small pool of genes is shared by overlapping subsets of crenarchaeal viruses, in a general analogy with the metagenome structure of bacteriophages. The proteins encoded by the genes belonging to this pool include predicted transcription regulators, ATPases implicated in viral DNA replication and packaging, enzymes of DNA precursor metabolism, RNA modification enzymes, and glycosylases. In addition, each of the crenarchaeal viruses encodes several proteins with prokaryotic but not viral homologs, some of which, predictably, seem to have been scavenged from the crenarchaeal hosts, but others might have been acquired from bacteria. We conclude that crenarchaeal viruses are, in general, evolutionarily unrelated to other known viruses and, probably, evolved via independent accretion of genes derived from the hosts and, through more complex routes of horizontal gene transfer, from other prokaryotes.

Amino Acid Sequence↗
PubMed1991

The effect of site and mode of expression of a heterologous antigenic determinant on the properties of poliovirus hybrids.

The antigenic site normally situated in the EF loop of VP2 (2EF) of poliovirus type 2 (Lansing) [PV2 (L)] was expressed in 2EF or in the BC loop of VP1 (1BC) of PV1 (Mahoney) [PV1 (M)]. A hybrid virus expressing the site in 2EF of PV1 (M) is known to be neutralizable by PV2 (L)-specific antisera and to induce neutralizing antibodies against PV-2 (L). In contrast, a hybrid expressing a related sequence in 1BC of PV1 (M), which maintained the length of the native BC loop, was not neutralizable by PV2 (L)-specific antisera and did not induce PV2 (L)-neutralizing antibodies. However, when 1BC was extended, so that it was longer than the native 1BC, the resulting hybrids induced low titers of PV2 (L)-neutralizing antibody although they were still not neutralizable by PV2 (L)-specific antisera. Synthetic peptides copying the extended sequences raised neutralizing antibodies which were able to distinguish between the sequence expressed in the extended 1BC, in the native-length 1BC and in 2EF. The growth rates of the hybrids with modifications to 1BC depended upon the nature of the modifications. The hybrid with the native length 1BC grew poorly, but extending 1BC tended to improve the growth rate, and extending 1BC with PV1 (M)-specific sequences nearly restored wild-type growth rates. Thus, the size, precise sequence and location of a heterologous antigenic site expressed on poliovirus have significant effects on the properties of that determinant and of the hybrid expressing it. Antigenicity of hybrids can be modified and their growth rate can be enhanced by appropriate choices of these parameters.

Amino Acid Sequence↗
PubMed1999

Modification of the genetic program of human alveolar macrophages by adenovirus vectors in vitro is feasible but inefficient, limited in part by the low level of expression of the coxsackie/adenovirus receptor.

Robust expression of genes transferred by adenovirus (Ad) vectors depends upon efficient entry of vectors into target cells. Cells deficient in the coxsackie/adenovirus receptor (CAR) are difficult targets for Ad-mediated gene transfer. We hypothesized that low levels of CAR expression may be responsible, in part, for the relative inefficiency of Ad-mediated gene transfer to human alveolar macrophages (AMs). CAR gene expression was detected in human AMs by reverse transcription-polymerase chain reaction and at low levels by Northern analysis. Indirect immunofluorescence showed specific, low-intensity surface staining for CAR, but at levels below those found on the positive-control A549 human lung epithelial cell line. Consistent with this, AMs expressed Ad vector transgenes 100 to 1,000-fold less efficiently than A549 cells, as assessed using the beta-galactosidase reporter (chemiluminescence assay) and green fluorescent protein (fluorescence microscopy and flow cytometry). At high multiplicity of infection, AMs from an HIV+ individual could be transduced with an AdIFNgamma vector to secrete detectable human interferon-gamma. Ad transgene expression by AMs was blocked by capsid fiber protein, suggesting that CAR is required in the pathway for productive Ad entry into alveolar macrophages. To confirm that Ad transgene expression by AMs is limited by low levels of CAR expression, cells were infected with an Ad vector containing the CAR complementary DNA (cDNA). Enhanced expression of CAR protein was demonstrated by indirect immunofluorescence, and the CAR cDNA-transduced cells showed 5-fold enhancement of subsequent Ad transgene expression. These observations demonstrate that human AMs can be targets for Ad-mediated gene transfer, but that efficiency of transgene expression is limited, at least in part, by low levels of CAR expression.

Adenoviridae↗
PubMed1997

Structure, biological functions and inhibition of the HIV-1 proteins Vpr and NCp7.

The Gag-encoded nucleocapsid protein NCp7 (72 amino acids) from HIV-1, the regulatory protein, Vpr (96 amino acids) and numerous derivatives have been synthesized by solid phase method and their structures determined by 2D NMR. In NCp7, the two highly folded zinc fingers of the Cx2Cx4Hx4C type are in close spacial proximity and the replacement of H by C in the first zinc finger or P by L in the short interdigital domain led to structural modifications evidenced by NMR. In vivo, these point mutations induced a complete loss of viral infectivity by interrupting critical step(s) of the retroviral life cycle. To account for these findings, a model of the complex between NCp7 and d (ACGCC) has been proposed from NMR data, showing the intercalation of Trp37 in the oligonucleotide. This model could also explain the role of NCp7 in the formation of viral particles and agrees with the modifications in morphology of the virions containing mutations in the NCp7 zinc fingers. Vpr is essentially constituted by two long helical domains at its N- and C-terminals and the side chains of Leu60 and Leu67 participate in a leucine-zipper mode of intramolecular interaction. The results obtained have been used to try to develop new antiviral agents inhibiting NCp7 functions and thus possibly devoid of the resistance effects found with inhibitors of HIV enzymes (reverse transcriptase and protease).

Amino Acid Sequence↗
PubMed1983

Structural proteins of Herpesvirus saimiri.

Herpesvirus saimiri particles were purified from productively infected owl monkey kidney cell cultures, and the virion polypeptides were analyzed by polyacrylamide gel electrophoresis. A total of 21 predominant proteins were found in lysates of H. saimiri 11 particles by Coomassie blue staining or by [35S]methionine labeling and autoradiography; all proteins were between 160,000 and 12,000 daltons in size. They are most probably virion constituents, as most of them were precipitated by immune sera, and no dominant proteins of equivalent sizes were found in mock-infected cultures. Four glycoproteins (gp 155/160, gp 128, gp 84/90, gp 55) and three polypeptides that appeared not to be glycosylated (p71, p35, p28) were assigned to the envelope or matrix of virions, whereas at least four phosphoproteins (pp132, pp118, pp55, pp13) and ten polypeptides without apparent secondary modification (p155/160, p106, p96, p67, p53, p36, p32, p15, p14, p12) were found in the nucleocapsid fraction. Analysis of virion proteins from different H. saimiri strains did not reveal appreciable differences in the migration behavior of most polypeptides, including all glycoproteins; however, determination of a strain-specific size pattern was possible for three of four phosphoproteins. The overall similarity in protein architecture of H. saimiri strains obviously does not reflect the variability in biology, such as oncogenic properties. In comparison, DNA sequence divergences appear to remain a better taxonomic criterion for strain distinction.

Capsid↗
PubMed1998

Identification and characterization of virus assembly intermediate complexes in HIV-1-infected CD4+ T cells.

For type-C and lentiviruses, including human immunodeficiency virus type 1 (HIV-1), the pathway of virus assembly remains poorly defined, and the assembly and budding of capsids are believed to occur simultaneously at the plasma membrane of the infected cell. We have now identified two putative HIV-1 assembly intermediate complexes in infected CD4+ T cells. The first of these intermediates, a detergent-resistant complex (DRC), was identified as a large oligomer that had a density of 1.10-1.13 g/ml and was primarily composed of Pr55Gag and Pr160Gag-Pol precursors. The other putative intermediate was a detergent-sensitive complex (DSC) with a density of 1.15-1.17 g/ml, which apparently represented the products of extensive proteolytic processing of both the Pr55Gag and Pr160Gag-Pol precursors. Both complexes could be distinguished from released mature virions as well as immature viral particles. Surprisingly, the formation of DRC was not dependent upon the myristylation at the N-terminus of the Gag proteins, a signal required for plasma membrane targeting and virus production. However, the myristic acid modification was essential for the formation of DSC. These data suggest that interactions between individual Gag molecules and between Gag and Gag-Pol precursors may occur before their targeting to the plasma membrane during HIV-1 assembly. However, formation of the late virus assembly complex and productive processing of Pr55Gag and Pr160Gag-Pol precursors apparently do not occur until these precursors are targeted to the plasma membrane.

CD4-Positive T-Lymphocytes↗
PubMed1999

Structural investigation on the requirement of CCHH zinc finger type in nucleocapsid protein of human immunodeficiency virus 1.

The nucleocapsid proteins (NCps) of lentiviruses play a key role during the retroviral replication cycle. NCps contain one or two highly conserved domains characterized by a CX(2)CX(4)HX(4)C sequence which binds zinc with a high affinity. The reasons of the high conservation of zinc fingers of CCHC type in lentiviruses were investigated by a structural study of mutants in which the zinc-coordinated ligands were exchanged. The HCHC form was unable to bind zinc tetrahedrally, whereas in His(28)(13-30)NCp7 corresponding to the CCHH motif, the zinc was tightly complexed. The mutant peptide exists in two interconverting conformations E and D [DeltaG(DE) (293K) = 0.1 kcal/mol] arising from the zinc coordination of His(28), by either its Nepsilon2 or its Ndelta1, respectively. As compared to the native CCHC zinc finger, the Cys(28) --> His mutation induces structural changes in the finger due to a modification in the coordination state of His(23) bound to zinc by Nepsilon2 in the wild-type finger by Ndelta1 in both conformers of the mutant. Introduction of these single mutations within the NCp7 proximal zinc finger in the HIV-1 genome was very recently shown to result in a loss of viral infection. This supports the hypothesis that structural changes of the zinc finger domain of NCp7 inhibit the recognition of one or several targets critically involved in the virus life cycle.

Amino Acid Sequence↗
PubMed1998

The annealing of tRNA3Lys to human immunodeficiency virus type 1 primer binding site is critically dependent on the NCp7 zinc fingers structure.

The nucleocapsid protein NCp7 of the human immunodeficiency virus type 1 contains two zinc fingers of the CX2CX4HX4C type, flanked by several basic residues, and plays a major role in viral infectivity. Thus, NCp7 was shown to promote annealing of the tRNA3Lys to the primer binding site, a key step in reverse transcription. However, previous in vitro experiments were unable to clarify the role of the zinc fingers in this process, due to nucleic acid aggregation induced by the basic N- and C-terminal domains of NCp7. We show here that deletion of these sequences in (12-53)NCp7 strongly reduces the formation of aggregates and allows a direct visualization of the binary or ternary complexes between NCp7 and nucleic acids by gel electrophoresis. (12-53)NCp7 is able to induce hybridization of the 33P tRNA3Lys and the human immunodeficiency virus type 1 viral RNA-(77-257), which contains the primer binding site. Modification of the proximal zinc finger conformation in Cys23(12-53)NCp7 led to a large reduction in this hybridization process, while replacement of Trp37 by Leu in the distal zinc fingers resulted in a complete absence of annealing activity. These data account for the in vivo loss of viral infectivity following these mutations and emphasize the critical role of the structure of the zinc finger domain of NCp7. This could facilitate a rational approach to new antiviral agents directed toward NCp7.

Amino Acid Sequence↗
PubMed1996

Release of virus-like particles from cells infected with poliovirus replicons which express human immunodeficiency virus type 1 Gag.

The effectiveness of attenuated poliovirus vaccines when given orally to induce both systemic and mucosal immune responses against poliovirus has resulted in an effort to develop poliovirus-based vectors to express foreign proteins. We have previously described the construction of poliovirus genomes (referred to as replicons) in which the complete human immunodeficiency virus type 1 (HIV-1) gag gene was substituted for the capsid gene (P1) (D.C. Porter, D.C. Ansardi, and C.D. Morrow, J. Virol. 69:1548-1555, 1995). Infection of cells with encapsidated replicons resulted in the expression of a 55-kDa protein. To further characterize the biological features of the HIV-1 Gag proteins expressed in cells infected with encapsidated replicons, we utilized biochemical analysis and electron microscopy. Expression of the 55-kDa protein in cells infected with encapsidated replicons resulted in myristylation of the Pr55gag protein. The Gag precursor protein was released from infected cells; analysis on sucrose density gradients revealed that the precursor sedimented at a density consistent with that of an HIV-1 virus-like particle. Analysis of replicon-infected cells by electron microscopy demonstrated the presence of condensed structures at the plasma membrane and the release of virus-like particles. These studies demonstrate that poliovirus-based vectors can be used to express foreign proteins which require posttranslational modifications, such as myristylation, and assemble into higher-order structures, providing a foundation for the future use of poliovirus replicons as vaccine vectors.

Amino Acid Sequence↗
PubMedDate not supplied

[Modification of tobacco mosaic virus envelope protein using trinitrobenzenesulfonic acid].

The process of modification of tobacco mosaic virus (TMV) coat protein with a lysine-specific reagent trinitrobenzenesulfonic acid (TNBS) was studied. TMV coat protein molecule is known to contain just two Lys residues (K53 and K68) localized in the same region of TMV coat protein subunit tertiary structure at a distance of about 70Ao from the virion axis. TNBS was used to modify the coat protein of wild type (U1) TMV and that of a coat protein ts-mutant ts21-66, bearing two amino-acid substitutions (I21 T and D66 G), both localized in the 70Ao region. In the mutant coat protein, TNBS modification rate for one of the two Lys residues was found to increase drastically (7 to 8 times) between 30 and 32o at pH 8.0. In U1 coat protein a similar increase was observed for one of Lys residues at 36o and pH 8.6. The results may indicate that the 70Ao region represents the labile section of TMV coat protein molecule, and gradual destruction of this site results first in loss of protein capacity to form well arranged two-cylindrical aggregates and then (after slight heating) in loss of capacity to form ordered helical aggregates.

Capsid Proteins↗
PubMed1997

Properties of the coat protein of a new tobacco mosaic virus coat protein ts-mutant.

Amino acid substitutions in a majority of tobacco mosaic virus (TMV) coat protein (CP) ts-mutants have previously been mapped to the same region of the CP molecule tertiary structure, located at a distance of about 70 A from TMV virion axis. In the present work some properties of a new TMV CP ts-mutant ts21-66 (two substitutions I21=>T and D66=>G, both in the 70-A region) were studied. Thermal inactivation characteristics, sedimentation properties, circular dichroism spectra, and modification by a lysine-specific reagent, trinitrobenzensulfonic acid, of ts21-66 CP were compared with those of wild-type (U1) TMV CP. It is concluded that the 70-A region represents the most labile portion of the TMV CP molecule. Partial disordering of this region in the mutant CP at permissive temperatures leads to loss of the capacity to form two-layer aggregates of the cylindrical type, while further disordering induced by mild heating results also in the loss of the ability to form ordered helical aggregates.

Capsid↗
PubMed1995

A combinatorial method for constructing libraries of long peptides displayed by filamentous phage.

We describe the construction and screening of a random peptide library displayed by filamentous phage. The peptides are expressed in multiple copies on the filamentous phage M13 as amino-terminal fusions with the major coat protein, the product of gene VIII. These libraries are efficiently screened for reactive peptides, using a combination of panning in solution followed by a plaque lift assay. Advantages of this system are that both high- and low-affinity phage clones are simultaneously identified and the analysis of non-reactive phage is minimized. The vector system utilized to construct this library enables it to be used for the construction of peptide libraries employing a combinatorial cloning strategy. This feature makes it especially suitable for construction of peptide libraries using codon-based oligonucleotide synthesis. The vectors also allow rapid optimization and modification of lead peptides by codon-based mutagenesis. A 20-amino acid long random peptide library of 1 x 10(9) members was constructed and screened for peptides that bound to (i) a monoclonal antibody recognizing the amino-terminus of beta-endorphin; (ii) a monoclonal antibody recognizing a peptide epitope derived from the v-ros oncogene product; and (iii) the constant region of murine IgG2b. The approach described here provides a means for the construction of customized libraries that can be screened with a variety of target molecules.

Amino Acid Sequence↗
PubMed1990

Specific inhibition of the synthesis of influenza virus late proteins and stimulation of early, M2, and NS2 protein synthesis by 3-deazaadenosine.

3-Deazaaristeromycin and 3-deazaadenosine (3DA-Ado) both interfere with the methylation of RNA, but only 3DA-Ado is metabolized to the corresponding homocysteine derivative. In contrast to 3-deazaaristeromycin, 3DA-Ado inhibits the synthesis of late influenza A virus proteins in chicken embryo cells (CEC), while it causes an overproduction of early proteins and of the nonstructural proteins NS2 and M2. Only the former effect of 3DA-Ado can be reversed by concomitant addition of adenosine, but not by guanosine. 3DA-Ado acts only early in the infectious cycle and, after removal of the drug, its effect on the yield of infectious virus is reversible. It can be significantly enhanced by homocysteine thiolactone. Except for the M gene, synthesis of viral mRNA is not significantly affected by 3DA-Ado. We conclude that 3DA-Ado acts via its homocysteine derivative by interfering with a specific post-transcriptional modification of viral mRNA and on splicing of specifically the M mRNA. In L-cells influenza viral protein synthesis is comparable to that in CEC in the presence of 3DA-Ado in that there is only little HA and M1 synthesized, and a severe overproduction of NS2 is observed. Under the experimental conditions 3DA-Ado has no inhibiting effect on the replication of other RNA viruses like Newcastle disease virus, Semliki Forest virus, or West Nile virus whose RNA is not methylated, since they do not have a nuclear phase during replication.

Adenosine↗
PubMed2000

The majority of duck hepatitis B virus reverse transcriptase in cells is nonencapsidated and is bound to a cytoplasmic structure.

The hepadnavirus reverse transcriptase binds cotranslationally to the viral pregenomic RNA. This ribonucleoprotein complex is then encapsidated into nascent viral core particles, where the reverse transcriptase copies the viral RNA into DNA. Here we report that 75% of the duck hepatitis B virus reverse transcriptase present in transfected LMH cells does not follow this well-known pathway but rather exists in the cell separate from the core protein or nucleocapsids. The nonencapsidated reverse transcriptase is also abundant in infected duck liver. The nonencapsidated reverse transcriptase exists as a complex set of isoforms that are most likely produced by posttranslational modification. Interestingly, only the smallest of these isoforms is encapsidated into viral core particles. The nonencapsidated reverse transcriptase is bound to a large cellular cytoplasmic structure(s) in a detergent-sensitive complex. The cellular distribution of the reverse transcriptase only partially overlaps that of the core protein, and this distribution is unaffected by blocking encapsidation. These observations raise the possibilities that the metabolic fate of the reverse transcriptase may be posttranscriptionally regulated and that the reverse transcriptase may have roles in the viral replication cycle beyond its well-known function in copying the viral genome.

Animals↗
PubMed1995

Response of foot-and-mouth disease virus C3 Resende to immunological pressure exerted in vitro by antiviral polyclonal sera.

The foot-and-mouth disease virus (FMDV) shows a remarkable antigenic variability. Like other RNA viruses, FMDV has a high mutation rate and it has been proposed that selection exerted by antibodies of the host could play a major role in its evolution. In this work, antiserum-resistant variants of FMDV (Nr variants) were selected upon 25 serial passages of a cloned C3 Resende strain on secondary monolayers of fetal bovine kidney (FBK-2) cells in the presence of subneutralizing levels of antiviral polyclonal sera (APS). After serial passage under immune selective pressure, the five Nr variant populations selected from five independent serial passages--their controls remaining unmodified--acquired the following characteristics: (i) increased resistance to neutralization by APS; (ii) five different antigenic specificities detected by enzyme-linked and neutralization assays using monoclonal antibodies; (iii) the same modification (residue 146, S to L) at the major antigenic site of VP1 (G-H loop, the 135-160 region); and (iv) specific changes for each Nr population outside the major antigenic site of VP1 at residues 46, 48 and 49 of the 40-60 region of VP1 (B-C loop). These results extend our previous work on selection of Nr variants using polyclonal sera, and add new information with regard to antigenic variation, mainly concerning the involvement of the 40-60 region of VP1 in the process of immune selection.

Amino Acid Sequence↗
PubMed1989

[Comparison of antigenic properties of the X-potato virus and its coat proteins. Effect of proteolytic cleavage on antigenic characteristics].

Antigenic properties of intact potato virus X (PVX) particles and of PVX coat protein (CP) preparations were compared using different modifications of ELISA test. In the competitive ELISA test (reaction in solution) antibodies to intact virus react much stronger with PVX than with CP while antibodies to CP react much stronger with CP than with PVX. In the direct ELISA test (reaction on the solid support) the difference in reactions of antiCP antibodies with PVX and CP is eliminated while the one in reactions of antiPVX antibodies with these antigens remains. No difference was registered in reactivity of PVX absorbed directly on polystyrene or on immunoglobulin-coated wells (sandwich ELISA) to antiCP antibodies.

Antibodies, Viral↗
PubMed2000

Complete nucleotide sequence, molecular analysis and genome structure of bacteriophage A118 of Listeria monocytogenes: implications for phage evolution.

A118 is a temperate phage isolated from Listeria monocytogenes. In this study, we report the entire nucleotide sequence and structural analysis of its 40 834 bp DNA. Electron microscopic and enzymatic analyses revealed that the A118 genome is a linear, circularly permuted, terminally redundant collection of double-stranded DNA molecules. No evidence for cohesive ends or for a terminase recognition (pac) site could be obtained, suggesting that A118 viral DNA is packaged via a headful mechanism. Partial denaturation mapping of DNA cross-linked to the tail shaft indicated that DNA packaging proceeds from left to right with respect to the arbitrary genomic map and the direction of genes necessary for lytic development. Seventy-two open reading frames (ORFs) were identified on the A118 genome, which are apparently organized in a life cycle-specific manner into at least three major transcriptional units. N-terminal amino acid sequencing, bioinformatic analyses and functional characterizations enabled the assignment of possible functions to 26 ORFs, which included DNA packaging proteins, morphopoetic proteins, lysis components, lysogeny control-associated functions and proteins necessary for DNA recombination, modification and replication. Comparative analysis of the A118 genome structure with other bacteriophages revealed local, but sometimes extensive, similarities to a number of phages spanning a broader phylogenetic range of various low G+C host bacteria, which implies relatively recent exchange of genes or genetic modules. We have also identified the A118 attachment site attP and the corresponding attB in Listeria monocytogenes, and show that site-specific integration of the A118 prophage by the A118 integrase occurs into a host gene homologous to comK of Bacillus subtilis, an autoregulatory gene specifying the major competence transcription factor.

Amino Acid Sequence↗