bluuebunny/crossref_metadata_2025_split huggingface dataset:
Heterogeneity of Envelope Molecules Expressed on Primary Human Immunodeficiency Virus Type 1 Particles as Probed by the Binding of Neutralizing and Nonneutralizing Antibodies
Authors: Poignard Pascal , Moulard Maxime , Golez Edwin , Vivona Veronique , Franti Michael , Venturini Sara , Wang Meng , Parren Paul W. H. I. , Burton Dennis R. 
Published: December 2002

ABSTRACTVirion capture assays, in which immobilized antibodies (Abs) capture virus particles, have been used to suggest that nonneutralizing Abs bind effectively to human immunodeficiency virus type 1 (HIV-1) primary viruses. Here, we show that virion capture assays, under conditions commonly reported in the literature, give a poor indication of epitope expression on the surface of infectious primary HIV-1. First, estimation of primary HIV-1 capture by p24 measurements shows a very poor correlation with an estimation based on infectivity measurements. Second, virion capture appears to require relatively low Ab affinity for the virion, as shown by the ability of a monoclonal Ab to capture a wild-type and a neutralization escape variant virus equally well. Nevertheless, in a more interpretable competition format, it is shown that nonneutralizing anti-CD4 binding site (CD4bs) Abs compete with a neutralizing anti-CD4bs Ab (b12) for virus capture, suggesting that the nonneutralizing anti-CD4bs Abs are able to bind to the envelope species that is involved in virion capture in these experiments. However, the nonneutralizing anti-CD4bs Abs do not inhibit neutralization by b12 even at considerable excess. This suggests that the nonneutralizing Abs are unable to bind effectively to the envelope species required for virus infectivity. The results were obtained for three different primary virus envelopes. The explanation that we favor is that infectious HIV-1 primary virions can express two forms of gp120, an accessible nonfunctional form and a functional form with limited access. Binding to the nonfunctional form, which needs only to be present at relatively low density on the virion, permits capture but does not lead to neutralization. The expression of a nonfunctional but accessible form of gp120 on virions may contribute to the general failure of HIV-1 infection to elicit cross-neutralizing Abs and may represent a significant problem for vaccines based on viruses or virus-like particles.
The Dimer Interfaces of Protease and Extra-Protease Domains Influence the Activation of Protease and the Specificity of GagPol Cleavage
Authors: Pettit Steven C. , Gulnik Sergei , Everitt Lori , Kaplan Andrew H. 
Published: December 2002

ABSTRACT

Activation of the human immunodeficiency virus type 1 (HIV-1) protease is an essential step in viral replication. As is the case for all retroviral proteases, enzyme activation requires the formation of protease homodimers. However, little is known about the mechanisms by which retroviral proteases become active within their precursors. Using an in vitro expression system, we have examined the determinants of activation efficiency and the order of cleavage site processing for the protease of HIV-1 within the full-length GagPol precursor. Following activation, initial cleavage occurs between the viral p2 and nucleocapsid proteins. This is followed by cleavage of a novel site located in the transframe domain. Mutational analysis of the dimer interface of the protease produced differential effects on activation and specificity. A subset of mutations produced enhanced cleavage at the amino terminus of the protease, suggesting that, in the wild-type precursor, cleavages that liberate the protease are a relatively late event. Replacement of the proline residue at position 1 of the protease dimer interface resulted in altered cleavage of distal sites and suggests that this residue functions as a
cis
-directed specificity determinant. In summary, our studies indicate that interactions within the protease dimer interface help determine the order of precursor cleavage and contribute to the formation of extended-protease intermediates. Assembly domains within GagPol outside the protease domain also influence enzyme activation.
Gris1
, a New Common Integration Site in Graffi Murine Leukemia Virus-Induced Leukemias: Overexpression of a Truncated Cyclin D2 due to Alternative Splicing
Authors: Denicourt Catherine , Kozak Christine A. , Rassart Eric 
Published: December 2002

ABSTRACT

The Graffi murine leukemia virus is a nondefective ecotropic retrovirus that was originally reported to induce myeloid leukemia in some strains of mice (A. Graffi, Ann. N.Y. Acad. Sci.
68:
540-558, 1957). Using provirus-flanking sequences as DNA probes, we identified a new common retroviral integration site called
Gris1
(for Graffi integration site 1). Viral integrations in
Gris1
were detected in 13% of the tumors analyzed. The
Gris1
locus was mapped to the distal region of mouse chromosome 6, 85 kb upstream of the cyclin D2 gene. Such viral integration in
Gris1
causes overexpression of the normal 6.5-kb major transcript of cyclin D2 but also induces the expression of a new, alternatively spliced 1.1-kb transcript from the cyclin D2 gene that encodes a truncated cyclin D2 of 17 kDa. The expression of this 1.1-kb transcript is specific to tumors in which
Gris1
is rearranged but is also detected at low levels in normal tissue.
Importance of B-Cell Responses for Immunological Control of Variant Strains of Simian Immunodeficiency Virus
Authors: Johnson Welkin E. , Lifson Jeffrey D. , Lang Sabine M. , Johnson R. Paul , Desrosiers Ronald C. 
Published: December 2002

ABSTRACT
The properties of three variants of cloned simian immunodeficiency virus strain 239 (SIV239) were compared. One strain (M5) lacked five sites for N-linked carbohydrate attachment in variable regions 1 and 2 (V1 and V2) of the gp120 envelope protein, one strain (ΔV1-V2) completely lacked V1 and V2 sequences, and another (316) had nine mutations in the envelope that impart high replicative capacity for tissue macrophages. All three strains were capable of significant levels of fusion independent of CD4, and all three were considerably more sensitive to antibody-mediated neutralization than the parent strain from which they were derived. Upon experimental infection of rhesus monkeys, these three variant strains replicated to viral loads at peak height around day 14 that were indistinguishable from or only slightly less than those observed in monkeys infected with the parental SIV239 strain. Viral loads at the set point 20 to 50 weeks after infection, however, were more than 400- to 10,000-fold lower with the variant strains. Depletion of B cells around the time of infection with M5 resulted in less effective immunological control and much higher viral loads at the set point in two of three monkeys. The differences between SIV239 infection, where there is not effective immunological control, and SIVM5 infection, where there is effective immunological control, cannot be easily explained by differences in the inherent replicative capacity of the viruses; rather, they are more readily explained by differences in the effectiveness of the antibody response. These results suggest that resistance of SIV239 to antibody-mediated neutralization is very important for evading effective immunological control, for allowing continuous viral replication, for maintenance of moderate-to-high viral loads at set point, and for disease progression.
DNA Immunization with Hepatitis C Virus (HCV) Polycistronic Genes or Immunization by HCV DNA Priming-Recombinant Canarypox Virus Boosting Induces Immune Responses and Protection from Recombinant HCV-Vaccinia Virus Infection in HLA-A2.1-Transgenic Mice
Authors: Pancholi Preeti , Perkus Marion , Tricoche Nancy , Liu Qingyan , Prince Alfred M. 
Published: December 2002

ABSTRACTWe studied immune responses to hepatitis C virus (HCV) genes delivered as DNA encoding the entire HCV protein coding genome in two polycistronic plasmids encoding HCV capsid-E1-E2-NS2-NS3 and HCV NS3-NS4-NS5 in HLA-A2.1-transgenic mice. Immune responses to HCV DNA prime and recombinant canarypox virus boost were also studied with the above constructs. At 8 weeks after a canarypox virus boost, the DNA prime/canarypox virus boosting regimen induced potent cellular immune responses to HCV structural and nonstructural proteins on target cells expressing the HLA-A2.1 allele. High frequencies of gamma interferon-secreting cells, as detected by enzyme-linked immunospot assay, were obtained in response to several endogenously expressed HCV proteins. We also observed cytotoxic-T-lymphocyte reactivity in response to endogenously expressed HCV proteins in fresh spleen cells without in vitro expansion. Upon challenge with a recombinant vaccinia virus expressing HCV proteins at 2 months postimmunization, the HCV DNA prime/canarypox virus-immunized mice showed a complete reduction in vaccinia virus titers compared to HCV DNA prime/boost- and mock-immunized controls. Immune responses were still detectable 4 months after canarypox virus boost in immunized mice. Interestingly, at 10 months postimmunization (8 months after canarypox virus boost), the protection in HCV DNA prime/boost-immunized mice against recombinant HCV-vaccinia virus challenge was higher than that observed in HCV DNA prime/canarypox virus boost-immunized mice.
Residues of VP26 of Herpes Simplex Virus Type 1 That Are Required for Its Interaction with Capsids
Authors: Desai Prashant , Akpa Jean-Claude , Person Stanley 
Published: December 2002

ABSTRACT
VP26 is the smallest capsid protein and decorates the outer surface of the capsid shell of herpes simplex virus. It is located on the hexons at equimolar amounts with VP5. Its small size (112 amino acids) and high copy number make it an attractive molecule to use as a probe to investigate the complex pattern of capsid protein interactions. An in vitro capsid binding assay and a green fluorescent protein (GFP) localization assay were used to identify VP26 residues important for its interaction with capsids. To test for regions of VP26 that may be essential for binding to capsids, three small in-frame deletion mutations were generated in VP26, Δ18-25, Δ54-60, and Δ93-100. Their designations refer to the amino acids deleted by the mutation. The mutation at the C terminus of the molecule, which encompasses a region of highly conserved residues, abolished binding to the capsid and the localization of GFP to the nucleus in characteristic large puncta. Additional mutations revealed that a region of VP26 spanning from residue 50 to 112 was sufficient for the localization of the fused protein (VP26-GFP) to the nucleus and for it to bind to capsids. Using site-directed mutagenesis of conserved residues in VP26, two key residues for protein-protein interaction, F79 and G93, were identified as judged by the localization of GFP to nuclear puncta. When these mutations were analyzed in the capsid binding assay, they were also found to eliminate binding of VP26 to the capsid structure. Surprisingly, additional mutations that affected the ability of VP26 to bind to capsids in vitro were uncovered. Mutations at residues A58 and L64 resulted in a reduced ability of VP26 to bind to capsids. Mutation of the hydrophobic residues M78 and A80, which are adjacent to the hydrophobic residue F79, abolished VP26 capsid binding. In addition, the block of conserved amino acids in the carboxy end of the molecule had the most profound effect on the ability of VP26 to interact with capsids. Mutation of amino acid G93, L94, R95, R96, or T97 resulted in a greatly diminished ability of VP26 to bind capsids. Yet, all of these residues other than G93 were able to efficiently translocate or concentrate GFP into the nucleus, giving rise to the punctate fluorescence. Thus, the interaction of VP26 with the capsid appears to occur through at least two separate mechanisms. The initial interaction of VP26 and VP5 may occur in the cytoplasm or when VP5 is localized in the nucleus. Residues F79 and G93 are important for this bi-molecular interaction, resulting in the accumulation of VP26 in the nucleus in concentrated foci. Subsequent to this association, additional amino acids of VP26, including those in the C-terminal conserved domain, are important for interaction of VP26 with the three-dimensional capsid structure.
Norwalk Virus-Like Particle Hemagglutination by Binding to H Histo-Blood Group Antigens
Authors: Hutson Anne M. , Atmar Robert L. , Marcus Donald M. , Estes Mary K. 
Published: December 2002

ABSTRACTNoroviruses are a major cause of epidemic acute nonbacterial gastroenteritis worldwide. Here we report our discovery that recombinant Norwalk virus virus-like particles (rNV VLPs) agglutinate red blood cells (RBCs). Since histo-blood group antigens are expressed on gut mucosa as well as RBCs, we used rNV VLP hemagglutination (HA) as a model system for studying NV attachment to cells in order to help identify a potential NV receptor(s). rNV VLP HA is dependent on low temperature (4°C) and acidic pH. Of the 13 species of RBCs tested, rNV VLPs hemagglutinated only chimpanzee and human RBCs. The rNV VLPs hemagglutinated all human type O (11 of 11), A (9 of 9), and AB (4 of 4) RBCs; however, few human type B RBC samples (4 of 14) were hemagglutinated. HA with periodate- and neuraminidase-treated RBCs indicated that rNV VLP binding was carbohydrate dependent and did not require sialic acid. The rNV VLPs did not hemagglutinate Bombay RBCs (zero of seven) that lack H type 2 antigen, and an anti-H type 2 antibody inhibited rNV VLP HA of human type O RBCs. These data indicated that the H type 2 antigen functions as the rNV VLP HA receptor on human type O RBCs. The rNV VLP HA was also inhibited by rNV VLP-specific monoclonal antibody 8812, an antibody that inhibits VLP binding to Caco-2 cells. Convalescent-phase sera from NV-infected individuals showed increased rNV VLP HA inhibition titers compared to prechallenge sera. In carbohydrate binding assays, the rNV VLPs bound to synthetic Lewis d (Led), Leb, H type 2, and Leyantigens, and these antigens also inhibited rNV VLP HA of human type O RBCs. Overall, our results indicate that carbohydrate antigens in the gut are a previously unrecognized factor in NV pathogenesis.
Mapping of Amino Acid Side Chains on the Surface of Hepatitis B Virus Capsids Required for Envelopment and Virion Formation
Authors: Ponsel Dirk , Bruss Volker 
Published: December 2002

ABSTRACT
The crystal structure of recombinant hepatitis B virus (HBV) capsids formed by 240 core proteins has recently been published. We wanted to map sites on the surface of the icosahedral 35-nm particle that are important for nucleocapsid envelopment by HBV surface proteins during virion morphogenesis. For this purpose, we individually mutated 52 amino acids (aa) within the N-terminal 140 aa of the 185-aa long core protein displaying their side chains to the external surface of the capsid to alanine residues. The phenotype of the mutations with respect to virion formation was tested by transcomplementation of a core gene-negative HBV genome in transiently cotransfected cells, immunoprecipitation of nucleocapsids from cells and secreted virions from culture media, and detection of the particles by radioactive endogenous polymerase reactions. Thirteen point mutations impeded nucleocapsid detection by endogenous polymerase reactions. Twenty-seven mutations were compatible with virion formation. Among these were all capsid-forming mutations in the upper half of the spike protruding from the particle shell and two additional triple mutations at tip of the spike. Eleven mutations (S17, F18, L60, L95, K96, F122, I126, R127, N136, A137, and I139) allowed nucleocapsid formation but blocked particle envelopment and virion formation to undetectable levels. These mutations map to a ring-like groove around the base of the spike and to a small area at the capsid surface close to the pores in the capsid shell. These residues are candidate sites for the interaction with envelope proteins during virion morphogenesis.
Marker Rescue of Adeno-Associated Virus (AAV) Capsid Mutants: a Novel Approach for Chimeric AAV Production
Authors: Bowles Dawn E. , Rabinowitz Joseph E. , Samulski R. Jude 
Published: December 2002

ABSTRACT

Marker rescue, the restoration of gene function by replacement of a defective gene with a normal one by recombination, has been utilized to produce novel adeno-associated virus (AAV) vectors. AAV serotype 2 (AAV2) clones containing wild-type terminal repeats, an intact
rep
gene, and a mutated
cap
gene, served as the template for marker rescue. When transfected alone in 293 cells, these AAV2 mutant plasmids produced noninfectious AAV virions that could not bind heparin sulfate after infection with adenovirus
dl
309 helper virus. However, the mutation in the
cap
gene was corrected after cotransfection with AAV serotype 3 (AAV3) capsid DNA fragments, resulting in the production of AAV2/AAV3 chimeric viruses. The
cap
genes from several independent marker rescue experiments were PCR amplified, cloned, and then sequenced. Sequencing results confirmed not only that homologous recombination occurred but, more importantly, that a mixed population of AAV chimeras carrying 16 to 2,200 bp throughout different regions of the type 3
cap
gene were generated in a single marker rescue experiment. A 100% correlation was observed between infectivity and the ability of the chimeric virus to bind heparin sulfate. In addition, many of the AAV2/AAV3 chimeras examined exhibited differences at both the nucleotide and amino acid levels, suggesting that these chimeras may also exhibit unique infectious properties. Furthermore, AAV helper plasmids containing these chimeric
cap
genes were able to function in the triple transfection method to generate recombinant AAV. Together, the results suggest that DNA from other AAV serotypes can rescue AAV capsid mutants and that marker rescue may be a powerful, yet simple, technique to map, as well as develop, chimeric AAV capsids that display different serotype-specific properties.
Regulation of Minute Virus of Mice NS1 Replicative Functions by Atypical PKCλ In Vivo
Authors: Nüesch Jürg P. F. , Lachmann Sylvie , Corbau Romuald , Rommelaere Jean 
Published: December 2002

ABSTRACT
Minute virus of mice NS1 protein is a multifunctional phosphoprotein endowed with a variety of enzymatic and regulatory activities necessary for progeny virus particle production. To regulate all of its different functions in the course of a viral infection, NS1 has been proposed to be modulated by posttranslational modifications, in particular, phosphorylation. Indeed, it was shown that the NS1 phosphorylation pattern is altered during the infectious cycle and that the biochemical profile of the protein is dependent on the phosphorylation state of the polypeptide. Moreover, in vitro approaches have identified members of the protein kinase C (PKC) family, in particular, atypical PKC, as regulators of viral DNA replication through the phosphorylation of NS1 residues T435 and S473, thereby activating the protein for DNA unwinding activities. In order to substantiate these findings in vivo, we produced NS1 in the presence of a dominant-negative PKCλ mutant and characterized the purified protein in vitro. The NS1 protein produced under these conditions was found to be only partially phosphorylated and as a consequence to be deficient for viral DNA replication. However, it could be rescued for this viral function by treatment with recombinant activated PKCλ. Our data clearly demonstrate that NS1 is a target for PKCλ phosphorylation in vivo and that this modification is essential for the helicase activity of the viral polypeptide. In addition, the phosphorylation of NS1 at residues T435 and S473 appeared to occur mainly in the nucleus, providing further evidence for the involvement of PKCλ which, unlike PKCζ, accumulates in the nuclear compartment of infected cells.
Spontaneous Mutations Restore the Viability of Tick-Borne Encephalitis Virus Mutants with Large Deletions in Protein C
Authors: Kofler Regina M. , Leitner Agnes , O'Riordain Gabriel , Heinz Franz X. , Mandl Christian W. 
Published: December 2002

ABSTRACT
The capsid protein, C, of tick-borne encephalitis virus has recently been found to tolerate deletions up to a length of 16 amino acid residues that partially removed the central hydrophobic domain, a sequence element conserved among flaviviruses which may be crucial for virion assembly. In this study, mutants with deletion lengths of 19, 21, 27, or 30 residues, removing more or all of this hydrophobic domain, were found to yield viable virus progeny, but this was without exception accompanied by the emergence of additional mutations within protein C. These point mutations or sequence duplications were located downstream of the engineered deletion and generally increased the hydrophobicity, suggesting that they may compensate for the loss of the central hydrophobic domain. Two of the second-site mutations, together with the corresponding deletion, were introduced into a wild-type genetic backbone, and the analysis of these “double mutants” provided direct evidence that the viability of the deletion mutant indeed depended on the presence of the second-site mutation. Our results corroborate the notion that hydrophobic interactions of protein C are essential for the assembly of infectious flavivirus particles but rule out the possibility that individual residues of the central hydrophobic domain are absolutely required for infectivity. Furthermore, the double mutants were found to be highly attenuated and capable of inducing a protective immune response in mice at even lower inoculation doses than the previously characterized 16-amino-acid-residue deletion mutant, suggesting that the combination of large deletions and second-site mutations may be a superior way to generate safe, attenuated flavivirus vaccine strains.
The Major Apoptotic Pathway Activated and Suppressed by Poliovirus
Authors: Belov George A. , Romanova Lyudmila I. , Tolskaya Elena A. , Kolesnikova Marina S. , Lazebnik Yuri A. , Agol Vadim I. 
Published: December 2002

ABSTRACT

Cells respond to poliovirus infection by switching on the apoptotic program, implementation of which is usually suppressed by viral antiapoptotic functions. We show here that poliovirus infection of HeLa cells or derivatives of MCF-7 cells was accompanied by the efflux of cytochrome
c
from mitochondria. This efflux occurred during both abortive infection (e.g., interrupted by guanidine-HCl and ending with apoptosis) and productive infection (leading to cytopathic effect). The former type of infection, but not the latter, was accompanied by truncation of the proapoptotic protein Bid. The virus-triggered cytochrome
c
efflux was suppressed by overexpression of Bcl-2. Both abortive and productive infections also resulted in a decreased level of procaspase-9, as revealed by Western blotting. In the former case, this decrease was accompanied by the accumulation of a protein with the electrophoretic mobility of active caspase-9. In contrast, in the productively infected cells, the latter protein was absent but caspase-9-related polypeptides with altered mobility could be detected. Both caspase-9 and caspase-3 were shown to be essential for the development of such hallmarks of virus-induced apoptosis as chromatin condensation, DNA degradation, and nuclear fragmentation. These and some other results suggest the following scenario. Poliovirus infection activates the apoptotic pathway, involving mitochondrial damage, cytochrome
c
efflux, and consecutive activation of caspase-9 and caspase-3. The apoptotic signal appears to be amplified by a loop which includes secondary processing of Bid. The implementation of the apoptotic program in productively infected cells may be suppressed, however, by the viral antiapoptotic functions, which act at a step(s) downstream of the cytochrome
c
efflux. The suppression appears to be caused, at least in part, by aberrant processing and degradation of procaspase-9.