pinecone/core-2020-05-10-deduplication huggingface dataset:
Covalently Crosslinked Complexes of Human Immunodeficiency Virus Type 1 (HIV-1) gp120 and CD4 Receptor Elicit a Neutralizing Immune Response That Includes Antibodies Selective for Primary Virus Isolates

AbstractSpecific conformational changes in the envelope glycoprotein gp120 of the human immunodeficiency virus type-1 (HIV-1) may be critical for eliciting a broadly neutralizing immune response against primary virus isolates. Since the interaction of gp120 with its receptor, CD4, induces conformational perturbations in both molecules, gp120-CD4 complexes should present unique immunogenic features that may include novel epitopes for broadly neutralizing antibodies. To test this hypothesis, we raised polyclonal antiserum against covalently crosslinked gp120–CD4 complexes in a goat and examined the ability of the anti-complex antibodies to neutralize primary and laboratory-adapted HIV-1 isolates. In cell-free neutralization assays with HIV-1MN, the antiserum demonstrated the ability to neutralize primary virus more effectively than the laboratory-adapted isolate. The neutralizing capacity of the anti-complex serum extended to primary isolates from distant genetic clades A, D, and E, although the degree of neutralization was found to vary among the clades. The neutralizing activity of the serum was composed of two components. The first component included anti-CD4 antibodies that recognized epitopes outside the gp120 binding site; the second was independent of CD4 reactivity and was retained after removal of cell surface anti-CD4 reactivity by repeated absorption with CD4-positive cells. These results demonstrate that gp120–CD4 complexes can elicit a unique polyclonal antibody response that is relevant to the neutralization of primary isolates of HIV-1
Host and Viral Factors Determine the Dispensability of Coat Protein for Bipartite Geminivirus Systemic Movement

AbstractGeminiviruses have unique, twinned icosahedral particles which encapsidate circular single-stranded DNA. Their genomes are composed of either one or two DNA segments. Monopartite geminiviruses absolutely require a functional coat protein (CP) for infectivity, whereas bipartite geminivirus CP null mutants can infect plants systemically. However, we show here that a CP mutant of the bipartite tomato golden mosaic virus (TGMV), which can infectNicotiana benthamianasystemically, is confined to the inoculated leaves ofNicotiana tabacumorDatura stramonium.We also show that a CP mutant of the related bean golden mosaic virus (BGMV), which can infect beans systemically, is confined to the inoculated leaves ofN. benthamiana.In each case, the extent of viral DNA accumulation in inoculated leaves was unaffected by the absence of CP, which suggests that CP is required specifically for systemic movement. The dispensability of CP is correlated with the degree of virus–host adaptation. TGMV is well adapted toN. benthamianaand does not require CP to infect this host systemically, whereas BGMV is poorly adapted toN. benthamianaand requires CP. Analysis of TGMV–BGMV hybrid viruses revealed that the viral genetic background can also affect the dispensability of CP for systemic movement inN. benthamiana.Thus, bipartite geminivirus movementin plantacan be resolved genetically into three components: (i) local, cell-to-cell movement, which does not require CP; (ii) CP-dependent systemic movement, which occurs in all hosts tested; and (iii) CP-independent systemic movement, which occurs in hosts to which a given virus is well adapted
Glycoprotein Incorporation and HIV-1 Infectivity Despite Exchange of the gp160 Membrane-Spanning Domain

AbstractWe have examined the role of the membrane-anchoring domain of the HIV-1 glycoproteins in viral glycoprotein function, glycoprotein incorporation, and viral infectivity. For this purpose, we initially exchanged the entire membrane-spanning region with that from a cellular glycoprotein (CD22). Subsequently, the strictly conserved arginine in the central position of the transmembranal alpha-helix was replaced by a neutral residue (R696→ I696). We have further examined the requirements within the cytoplasmic C-terminus for glycoprotein incorporation and replaced this region of gp160 with the long cytoplasmic C-terminus (118 amino acids) from CD22. Our results show that the specific amino acid sequence of the membrane-spanning region of gp160 is not necessary for viral infectivity, thus making it unlikely that this region is specifically involved in membrane fusion, in glycoprotein incorporation, or in infectivity of the cell lines tested. In contrast, recombinant gp160 with the CD22 C-terminal region, although present at the cell surface and membrane fusion-competent, was excluded from incorporation into particles. This could indicate that steric exclusion, and no pseudotyping, occurs when the heterologous, cytoplasmic C-terminal region is too long and not fitting
No Intermolecular Interaction between the Large Hepatitis Delta Antigens Is Required for the Secretion with Hepatitis B Surface Antigen: A Model of Empty HDV Particle

AbstractThe large delta antigen (LDAg) of hepatitis D virus (HDV), which is similar to the small delta antigen (SDAg), except it has 19 additional amino acids and an isoprenylation signal at the C-terminus, is crucial for interacting with hepatitis B surface antigen (HBsAg) to form a mature virion of HDV. Previous studies indicated that the LDAg alone, but not SDAg, can interact with HBsAg to form an empty particle. However, no evidence yet shows whether the intermolecular interaction of LDAg is necessary for forming an empty HDV particle. By cotransfection of plasmids encoding deletion or isoprenylation-negative mutants of LDAg with a plasmid encoding HBsAg into human hepatoma cells, we demonstrated that (i) the isoprenylation-negative LDAg cannot be secreted, (ii) the coiled-coil domain-deleted LDAg retains the secretion capability, (iii) the isoprenylation-negative LDAg can neither cosecrete with isoprenylation-positive LDAg nor suppress its secretion, and (iv) an intermolecular interaction between LDAgs is unlikely required for secretion. A hypothetical model of empty HDV particle containing HBsAg with isoprenylated LDAgs, which are probably present in a singular form, was then proposed
Syncytium Formation by Recombinant HTLV-II Envelope Glycoprotein

AbstractInfection by human T-cell leukemia viruses (HTLV-I and HTLV-II) induces syncytium formation in certain cell typesin vitro.Recombinant vaccinia viruses expressing the HTLV-II envelope (Env) gp61-II were used to infect human cells, and syncytium formation was observed, demonstrating that HTLV-II Env expression was sufficient to mediate syncytium formation in appropriate cells. This syncytium formation could be blocked by sera from HTLV-II-infected individuals. Infection with these recombinant vaccinia viruses caused fusion in mouse/human hybrid cells containing only human chromosome 17, whereas there was no obvious syncytium formation in mouse cells. This fusogenic phenotype of recombinant gp61-II is likely a property of the specific interaction between HTLV-II envelope protein and the HTLV cellular receptor, encoded by a gene that was previously indicated to be on human chromosome 17. On this basis, we developed a sensitive syncytium formation assay, usinglacZgene activation, for detecting the presence of the HTLV receptor(s). We used this fusion assay to test for HTLV receptor activity on a variety of cells. Our findings indicate that the HTLV receptor is widely distributed among species and cell types, including, to a limited extent, murine cells
Functional and Immunological Properties of the Baculovirus-Expressed Hemagglutinin of African Swine Fever Virus

AbstractA recombinant baculovirus harboring the hemagglutinin (HA) gene of African swine fever virus, with homology to the T-lymphocyte surface antigen CD2, was constructed. The efficient expression of the HA gene was determined by immunofluorescence and Western blot studies on insect cells infected with the recombinant baculovirus. The baculovirus-expressed HA showed hemadsorption and erythrocyte-agglutinating activities characteristic of the CD2 homolog protein induced by the virus in infected macrophages. Pigs immunized with the recombinant HA developed hemagglutination-inhibition and temporary infection-inhibition antibodies that recognize a 75-kDa structural protein and were protected against lethal infection
A Novel Human Amphotropic Packaging Cell Line: High Titer, Complement Resistance, and Improved Safety

AbstractSuccessful retroviral-mediated gene therapy will depend on safe, efficient packaging cell lines for vector particle production. Existing packaging lines for murine leukemia virus (MLV)-based vectors are predominantly derived from NIH/3T3 cells which carry endogenous MLV sequences that could participate in recombination to form replication-competent retrovirus (RCR). To identify cells devoid of such sequences, we screened genomic DNA from eight cell lines. DNA from the human 293 cell line did not cross-hybridize with MLV sequences, and these cells were able to secrete Gag particles after transfection. We derived a stable amphotropic packaging cell line (called ProPak-A) in 293 cells in which the Gag-Pol and Env (packaging) functions are expressed separately from a heterologous (non-MLV) promoter, to maximally reduce homology between packaging and vector sequences. ProPak-A-based producer cells are efficient, yielding higher stable titers than PA317-based producers. In addition, a vector that consistently gave rise to RCR in PA317 cells never resulted in detectable RCR in ProPak-A-based producer cultures. We have also shown that ProPak-A-packaged particles are not inactivated by human serum. Thus, the packaging cells we describe are as efficient and safer than the amphotropic packaging cells most commonly used in clinical gene therapy work and are also more appropriate forin vivogene delivery
A Genomic Region of Lactococcal Temperate Bacteriophage TP901-1 Encoding Major Virion Proteins

AbstractTwo major structural proteins, MHP (major head protein) and MTP (major tail protein), from the lactococcal temperate phage TP901-1 were sequenced at their amino acid termini, and derived degenerate oligonucleotides were used to locate the corresponding genes in the phage genome. This genomic region was sequenced. The sequence characterized includes a total of 11 open reading frames (ORFs) showing an operon structure. Upstream of each ORF, except ORFb2and ORFx,potential ribosome-binding sites were found, suggesting independent translation. However, coupled translation is suggested for ORFxand as a possibility for ORFb3and ORFc2,which have ribosome-binding sites located more distant from their start codons. ORFb2may be translationally fused withmhpat a low frequency. Themhpandmtpgenes are transcribed as a 3.7-kb mRNA with at least six additional ORFs. The organization of the genomic region analyzed resembles that of other distantly related phages, providing possible roles for the uncharacterized ORFs
The HTLV-1 Rex Protein Induces Nuclear Accumulation of Unspliced Viral RNA by Avoiding Intron Excision and Degradation

AbstractThe human T-cell leukemia virus (HTLV-1) Rex protein is essential for the cytoplasmic accumulation of incompletely spliced transcripts that code for the viral structural proteins. In this study effects of Rex on the amounts of total, spliced, and unspliced RNA from HTLV-1 were determined. In transfected fibroblasts Rex production resulted in reduced amounts of spliced RNA and increased quantities of unspliced RNA in the nucleus. However, the total amount of viral RNA was not affected and the stability of spliced transcripts was not changed, thus indicating that only the rate of splicing was reduced. Rex action also reduced splicing in immortalized human cord blood T-cells. However, the total amount of viral transcripts and the stability of unspliced RNA in these cells were also increased in the presence of Rex. This indicates that Rex also prevents the degradation of unspliced transcripts in T-cells. The changes in the relative amounts of spliced and unspliced RNA induced by Rex were observed not only in the cytoplasm but also in the nucleus. Thus Rex affects the nucleocytoplasmic transport, splicing, and stability of HTLV-1 RNA in the nucleus. These observations may suggest that Rex directs the unspliced viral RNA to the cytoplasm via a nuclear compartment that is not accessible to splicing and degradation factors
Protein Stability Influences Human Immunodeficiency Virus Type 2 Vpr Virion Incorporation and Cell Cycle Effect

AbstractUsing recombinant proteins as standards, we calculated the amount of Vpr and Vpx present in HIV-2RODparticles. We find 2000–3000 copies of Vpx per particle but only 40–50 copies of Vpr. We investigated the reasons for this discrepancy between Vpx and Vpr and found that viral factors, including HIV-2Vpx, do not restrict its incorporation. Instead, the accumulation of HIV-2RODVpr during infection is restricted by a short protein half-life which acts to limit its virion incorporation. The half-life of HIV-2 Vpr was calculated to be about 90 min, while HIV-2 Vpx and HIV-1 Vpr had half-lifes of 36 and 20 hr, respectively. Moreover, while both HIV-1 Vpr and HIV-2 Vpr cause cells to accumulate in G2 of the cell cycle, the effect of HIV-2 Vpr is attenuated relative to HIV-1 Vpr. Thus, protein stability correlates with both the function of Vpr and its virion incorporation
Identification of a Set of Proteins (C′ and C) Encoded by the Bicistronic P Gene of the Indiana Serotype of Vesicular Stomatitis Virus and Analysis of Their Effect on Transcription by the Viral RNA Polymerase

AbstractPrevious work (C. F. Spiropoulou and S. T. Nichol, 1993,J. Virol.67, 3103–3110) has demonstrated the existence in cells infected with the New Jersey serotype of vesicular stomatitis virus (VSV) of two small carboxy-coterminal proteins encoded by the P mRNA. These proteins have been named C′ and C. We are interested in studying the function of these proteins in the virus life cycle. Toward this end, we have cloned the ORF encoding the potential C′ protein of the Indiana serotype as a histidine-tagged fusion protein, purified the expressed protein fromEscherichia coli,and used the fusion protein as an immunogen to raise antiserum in a rabbit. We have used this anti-C′ protein serum to demonstrate that both of the predicted C′ and C proteins are synthesized in cells infected with the Indiana serotype of VSV. In addition we have localized a portion of these proteins to nucleocapsids isolated from infected cells, suggesting that they may play a role in RNA synthesis. Reconstitution of the viral polymerase activity by expressing the L and P protein subunits with or without the C proteins failed to demonstrate any effect of the presence of these latter proteins on reconstituted transcription using purified nucleocapsids as templates. However, we have been able to show a dramatic stimulation of the polymerase activity in purified virions by the addition of purified C′ protein toin vitrotranscription reactions. Both the level and the fidelity of mRNA synthesis are stimulated by this protein. Evidence for the specificity of this effect comes from the fact that stimulation appears to be serotype specific; C′ protein of the Indiana serotype stimulates transcription by purified Indiana serotype virions but has a minimal effect on transcription by purified virions of the New Jersey serotype. We are continuing our studies to determine the mechanism of this stimulation
Genetic Conservation of Highlands J Viruses

AbstractWe studied molecular evolution of the mosquito-borne alphavirus Highlands J (HJ) virus by sequencing PCR products generated from 19 strains isolated between 1952 and 1994. Sequences of 1200 nucleotides including portions of the E1 gene and the 3′ untranslated region revealed a relatively slow evolutionary rate estimated at 0.9–1.6 × 10−4substitutions per nucleotide per year. Phylogenetic trees indicated that all HJ viruses descended from a common ancestor and suggested the presence of one dominant lineage in North America. However, two or more minor lineages probably circulated simultaneously for periods of years to a few decades. Strains isolated from a horse suffering encephalitis, and implicated in a recent turkey outbreak, were not phylogenetically distinct from strains isolated in other locations during the same time periods. Our findings are remarkably similar to those we obtained previously for another North American alphavirus, eastern equine encephalomyelitis virus, with which Highlands J shares primary mosquito and avian hosts, geographical distribution, and ecology. These results support the hypotheses that the duration of the transmission season affects arboviral evolutionary rates and vertebrate host mobility influences genetic diversity
Functional Significance of the Nuclear-Targeting and NTP-Binding Motifs of Semliki Forest Virus Nonstructural Protein nsP2

AbstractSemliki Forest virus-specific polypeptide nsP2 is a nonstructural protein involved in multiple steps during viral RNA replication. It was recently shown to possess single-stranded RNA-stimulated ATPase and GTPase activities. Replacement of the highly conserved lysine (Lys-192) within the classical nucleotide-binding motif A/GXXGXGKS/T with asparagine abolished its NTP-hydrolyzing activity. Also, about half of nsP2 is transported into the nucleus during viral infection. Substitution of the second arginine in its nuclear localization signal (P648RRRV) with aspartic acid rendered nsP2 totally cytoplasmic. To assess the functional importance of these sequence motifs, the same mutations were introduced into a cDNA clone of Semliki Forest virus, from which infectious RNA can be producedin vitro.Transfection of an RNA encoding Lys-192 → Asn mutation into BHK cells did not promote viral infection. However, revertants encoding the wild-type amino acid were obtained. Cells transfected with RNA coding for Arg-649 → Asp mutation gave rise to infectious virus termed SFV-RDR. Indirect immunofluorescence and subcellular fractionation of SFV-RDR-infected cells confirmed the cytoplasmic localization of nsP2. Measurement of host DNA synthesis late in infection revealed that infection with the parental virus inhibited DNA synthesis to 10% of control cells. In contrast, infection with SFV-RDR led only to a partial shutoff of cellular DNA synthesis. Mice experiments indicated that the pathogenicity of SFV-RDR was attenuated
Sequence Requirements of ATF2 and CREB Binding to the Human T-Cell Leukemia Virus Type 1 LTR R Region

AbstractWe have identified crucial transcription factor contact sites within the distal portion of the HTLV-I LTR R region. Single base substitutions within this region greatly reduce formation of a complex which we previously described as a 70-kDa nuclear protein interacting with a CREB-like protein. Comparison of published sequences of HTLV-1 isolates obtained from scattered geographic locations revealed that clustered mutations in an 8-base segment near the U5 junction do, in fact, occur naturally. A single base substitution corresponding to a common naturally occurring mutation was introduced into the R region of an HTLV-1-LTR Cat construct. This resulted in derepression of the promoter in a cell line expressing high levels of the R region binding complex when compared to the wild-type LTR promoter. Affinity purification and electrophoretic mobility super-shift analysis identified a dominant 70-kDa DNA binding protein as ATF-2. Phosphorylated ATF-2 apparently interacts with CREB to form this downstream complex
In VitroImmortalization of Old World Monkey T Lymphocytes withHerpesvirus Saimiri:Its Susceptibility to Infection with Simian Immunodeficiency Viruses

AbstractPeripheral blood T lymphocytes of Old World monkeys, rhesus and cynomolgus monkey (Macaca mulattaandMacaca fascicularis,respectively), were successfully immortalized by infection withHerpesvirus Saimirisubtype C. The T cell lines were stably cultured without addition of exogenous IL-2. TheSTP-C488protein, the oncogene product of subtype C strain 488-77, was detected in these cells by Western blotting. They also expressed some markers of activated or matured T cell phenotypes such as CD2+, monkey Pan-T+, CD25+, CD29+, and MHC-II DR+. Interestingly, not only CD4+CD8− or CD4−CD8+ single positive subpopulations but also CD4+CD8+ double positive ones were present in all of them. Furthermore, they were productively infected with both SIVmac and SIVagm. The levels of the viral replication were comparable to those in human T cell lines. Thus, Herpes Virus Saimiri-immortalized Old World monkey T lymphocytes will be suitable for further studies of immune system in Old World monkeys and cell–virus interactions in SIV infection
Inhibition of HIV-1 in Cell Culture by Synthetic Humate Analogues Derived from Hydroquinone: Mechanism of Inhibition

AbstractHumic acids are natural constituents of soil and ground water and mainly consist of mixtures of polycyclic phenolic compounds. A similar complex of compounds with a mean size of about 1000 Da, designated HS-1500, was synthesized by oxidation of hydroquinone. HS-1500 inhibited HIV-1 infection of MT-2 cells with an IC50of 50–300 ng/ml and showed a mean cell toxicity of about 600 μg/ml. Inhibition of HIV-induced syncytium formation was observed at 10–50 μg/ml. Treatment of free and cell-attached HIV with HS-1500 irreversibly reduced its infectivity, whereas the susceptibility of target cells for the virus was not impaired by treatment prior to infection. The HIV envelope protein gp120SU bound to sepharose-coupled HS-1500 and could be eluted by high salt and detergent. HS-1500 interfered with the CD4-induced proteolytic cleavage of the V3 loop of virion gp120SU. Furthermore, binding of V3 loop-specific antibodies was irreversibly inhibited, whereas binding of soluble CD4 to gp120SU on virus and infected cells was not affected. In conclusion, our data suggest, that the synthetic humic acid analogue inhibits the infectivity of HIV particles by interference with a V3 loop-mediated step of virus entry
Myristylation of the Large Surface Protein Is Required for Hepatitis B Virusin VitroInfectivity

AbstractThe large surface protein (L) of the enveloped hepatitis B virus (HBV) is myristylated at glycine 2. To investigate whether this fatty acid moiety is required for HBV infectivity we made use of a point mutant in which the myristyl acceptor was mutated to a nonfunctional alanine. The mutant virus and a wild-type control were expressed in a human hepatoma cell line by transfection of genomic DNA constructs. Comparable amounts of virions were secreted by both strains as measured by the endogenous polymerase activity of immunoprecipitated virions. The presentation of an N-terminal epitope of L on the virion surface was not influenced by the mutation. To test the infectivity of this mutant virus primary human hepatocytes were incubated with the media of transfected cells. The covalently circular closed HBV DNA molecules generated after infection were discriminated from the open circular DNA genomes of inoculated virions by a sensitive PCR-based technique. The experiments demonstrated that the wild type was infectious but not the myristate negative mutant. This reflects the phenotype of an homologous duck hepatitis B virus mutant although the N-terminal L protein domains of this virus and of HBV show no primary sequence homology