pinecone/core-2020-05-10-deduplication huggingface dataset:
Characterization of Avian H5N1 Influenza Viruses from Poultry in Hong Kong

AbstractThe transmission of avian H5N1 influenza viruses to 18 humans in Hong Kong in 1997 with six deaths established that avian influenza viruses can transmit to and cause lethal infection in humans. This report characterizes the antigenic and biological properties of the H5N1 influenza viruses isolated from chickens, ducks, and geese from farms and poultry markets in Hong Kong during 1997 and compares them with those of virus isolated from the index human case. Each of the H5N1 viruses from Hong Kong poultry markets that were tested were lethal in chickens, possessed polybasic amino acids at the carboxy-terminus of HA1, and by definition were highly pathogenic in poultry. The available nonpathogenic H5 influenza viruses and the pathogenic H5N1 virus from Hong Kong were analyzed with monoclonal antibodies prepared to A/chicken/Pennsylvania/1370/83 (H5N2). The analysis revealed limited antigenic drift in 15 years and established that monoclonal antibodies are useful reagents for identification and antigenic analysis of avian strains that may transmit to humans in the future. One of the monoclonal antibodies permitted separation of the H5N1 influenza viruses from poultry into two groups that correlated with the presence or absence of a carbohydrate at residue 158 adjacent to the receptor binding site on HA. The H5N1 viruses examined replicated in geese, pigs, rats, and mice, but to only a very limited extent in ducks. It is noteworthy that all infected geese shed virus and that the H5N1 viruses caused disease signs and death in a portion (3 of 16) of the geese, with evidence of systemic spread to the brain. The tropism for geese is unusual and may provide insight into the origin of these viruses. In mice, the H5N1 virus caused lethal pneumonia and spread systemically to the brain. Mice would thus provide an ideal model system for studying immune responses and pathogenesis. Transmission experiments in chickens revealed that the H5N1 viruses are spread by fecal–oral transmission rather than by aerosol, and that the viruses are inactivated by drying of feces at ambient temperature. However, infectivity is maintained for at least 4 days in wet feces at 25°C. There were differences in the morphology of the H5N1 viruses isolated from birds and humans. The perpetuation of H5N1 influenza viruses in the poultry markets in Hong Kong and the transmission of these viruses to humans emphasize the importance of these markets in the epidemiology of influenza. The poultry markets are of critical importance in the perpetuation and transmission of influenza viruses to other avian species and to mammals, including humans
Identification of Viral Genes Required for Cell-to-Cell Movement of Southern Bean Mosaic Virus

AbstractInoculation ofVigna unguiculata(cowpea) with transcripts synthesizedin vitrofrom a genome-length cDNA clone of the cowpea strain of southern bean mosaic virus (SBMV-C) resulted in a systemic SBMV-C infection of this host. Capped RNA was about five times more infectious than uncapped RNA as determined by a local lesion assay. The SBMV-C cDNA clone was also used for mutagenesis of the four SBMV-C open reading frames (ORFs). ORF1, ORF3, and coat protein (CP) mutants were not infectious in cowpea. Electroporation of cowpea protoplasts with mutant transcripts demonstrated that the ORF1, ORF3, and CP gene products were not required for SBMV-C RNA synthesis, and the ORF1 and ORF3 gene products were not required for SBMV-C assembly. From these results, it was concluded that the ORF1 and ORF3 proteins and the CP are required for SBMV-C cell-to-cell movement. One of the ORF3 mutants pSBMV2-UAA1833 contained a nonsense codon between the predicted −1 ribosomal frameshift site (SBMV-C nucleotides 1796–1802) and a potential ORF3 translation initiation codon at SBMV-C nucleotide 1895. The lack of infectivity of this mutant suggested that ORF3 was expressed by a −1 ribosomal frameshift in ORF2 rather than by initiation of translation at nucleotide 1895
Membrane Targeting Sequences in Tombusvirus Infections

AbstractInfection ofNicotiana benthamianacells with cymbidium ringspot (CymRSV) and carnation Italian ringspot (CIRV) viruses results in the formation of conspicuous membranous bodies [multivesicular bodies (MVBs)], which develop from modified peroxisomes or mitochondria, respectively. The organelle targeting signal is located in the proteins of 33 kDa (CymRSV) or 36 kDa (CIRV) encoded by ORF 1, which contain an N-terminal hydrophilic portion followed by two predicted hydrophobic transmembrane segments. Biochemical analysis showed that the 33- and 36-kDa proteins are integral membrane proteins. By exchanging small portions of the ORF 1 sequence between the infectious full-length clones of the two viruses, hybrid constructs were obtained of which thein vitrosynthesized RNA was inoculated toN. benthamianaplants and protoplasts. The structure of infectious clones suggested that both the N-terminal hydrophilic region and the transmembrane segments of the ORF 1-encoded proteins specify which organelle is involved in the synthesis of MVBs. Mutational analysis of the CIRV 36-kDa protein also suggested the presence of an internal mitochondrial targeting sequence similar to that found in several normal host proteins that are synthesized in the cytoplasm and transported to mitochondria. The CymRSV 33-kDa protein did not contain the obvious consensus signals thought to be characteristic of proteins targeted to peroxisomes, and an mitochondrial targeting sequence motif was not evident
DNA and Protein Interactions of the Small Subunit of Herpes Simplex Virus Type 1 DNA Polymerase

AbstractHerpes simplex virus DNA polymerase (HSV pol) holoenzyme consists of a large catalytic (UL30 gene product) and a small auxiliary subunit (UL42 gene product). The DNA binding of HSV pol, its cofactor, and the assembled holoenzyme complex was studied by bandshift analysis using purified proteins expressed via recombinant baculovirus. The functional activity of the recombinant UL42, purified by phenyl-Sepharose chromatography, was confirmed by its ability (1) to convert the salt sensitivity of both, 3′-5′ exonuclease and polymerase, activities of HSV pol and (2) to enhance the processivity of polymerization. Bandshift analyses revealed that the HSV pol holoenzyme–DNA complex was stably formed up to a salt concentration of 50 mM ammonium sulfate, indicating that the restricted DNA and protein interactions of both HSV pol and UL42 are responsible for the observed salt preference of the HSV pol holoenzyme under standard assay conditionsin vitro. Studies of the assembly of the holoenzyme complex demonstrated that initial DNA binding of HSV pol was advantageous for the formation of the HSV pol holoenzyme–DNA complex
Expression of the First 811 Nucleotides of the Herpes Simplex Virus Type 1 Latency-Associated Transcript (LAT) Partially Restores Wild-Type Spontaneous Reactivation to a LAT-Null Mutant

AbstractThe herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) gene is required for efficient spontaneous reactivation in the rabbit ocular model. We recently showed that insertion of 1.8 kb of the LAT promoter and the first 1.5 kb of the 8.3-kb primary LAT transcript into a novel, ectopic location in the virus unique long (UL) region restored wild-type spontaneous reactivation to a LAT-null mutant. To further map the LAT spontaneous reactivation function within the first 1.5 kb of LAT, we rescued the same LAT-null mutant by inserting 1.8 kb of the LAT promoter and just the first 811 nucleotides of LAT into the same location in the UL. In a series of three experiments, the resulting virus, designated LAT2.6A, had a spontaneous reactivation rate that was midway between the original LAT-null mutant and wild-type virus. Thus expression of the first 811 LAT nucleotides produced a spontaneous reactivation rate that was significantly higher than that of the LAT-null mutant but significantly less than that of wild type. This suggests that part, but not all, of the LAT function involved in efficient spontaneous reactivation is located within the first 811 nucleotides of the primary 8.3-kb LAT
An Analysis of the Role of the Target Membrane on the Gp64-induced Fusion Pore

AbstractInfluenza hemagglutinin (HA) and GP64 of the baculovirusAutographa californicamulticapsid nuclear polyhedrosis virus induce strikingly different initial fusion pores when mediating fusion between host cells that express these fusion proteins and target cells (Plonsky and Zimmerberg, 1996; Spruceet al.,1989, 1991; Zimmerberget al.,1994). However, in these experiments, variations in host and target membranes confounded the analysis of the role that major components of the fusion reaction play in determining initial pore characteristics. To determine the contribution of the target cell plasma membrane to the fusion pore phenotype, we studied GP64-induced fusion of stably transfected cells (Sf9Op1D) to either red blood cells (RBCs) or Sf9 cells. Initial fusion pores in Sf9Op1D/RBC and Sf9Op1D/Sf9 cell pairs exhibited the same conductance, and pore flickering was not observed in either combination of cells. This indicates that the target cell determines neither the size nor the reversibility of the initial pore. However, the target cell does influence the kinetics of pore formation. The waiting time between triggering and pore appearance was shorter for Sf9Op1D/RBC fusion than for Sf9Op1D/Sf9 pairs. No correlation between pore waiting time and conductance was found. This argues against a molecular model that assumes aggregation of the pore wall from a nonfixed number of components as the rate-limiting step in GP64 fusion pore formation
Mutational Analysis ofCis-Acting Sequences in the 3′- and 5′-Untranslated Regions of RNA2 of Red Clover Necrotic Mosaic Virus

AbstractMutational analysis has been used to investigatecis-acting sequences in the 3′- and 5′-untranslated regions of red clover necrotic mosaic virus RNA2 required for replication in the presence of wild-type RNA1. Deletion of a sequence near the 3′ end of RNA2, which is potentially capable of forming a stable stem-loop structure, abolished the ability of RNA2 to replicate inNicotiana clevelandiiprotoplasts, as judged by the failure to detect production of either the positive or negative RNA strand. A base-paired structure in the stem was shown to be essential for replication, but a mutant RNA2 in which every base of the stem had been altered was able to replicate inN. clevelandiiprotoplasts, to produce lesions on leaves ofVigna unguiculataplants, and to infectN. clevelandiiplants systemically. However, changing three bases in the loop of the potential stem-loop structure abolished the replication of RNA2. Upstream sequences in the 3′-untranslated region of RNA2 were also shown to be important for RNA2 replication. Deletions in the 5′-untranslated region of RNA2 showed that sequences across the whole of this region were needed for production of the positive strand but not for production of the negative strand. Some of thecis-acting sequences required for replication of RNA2 have almost identical counterparts in RNA1, but others appear to be unique to RNA2
Poxviral/Retroviral Chimeric Vectors Allow Cytoplasmic Production of Transducing Defective Retroviral Particles

AbstractDefective vaccinia viruses were constructed that express functional Moloney murine leukemia virus-based vector genomes, giving rise to substantial titers of transduction-competent retrovirus particles after infection of a retroviral packaging cell line. For this purpose, the proviral retrovirus genome, engineered into the vaccinia virus mutant, was subjected to several modifications, including the replacement of retroviral promoter sequences by vaccinia virus sequences and the precise fusion of the transcription stop signal downstream of and the removal of such signals within the transcription unit, allowing cytoplasmic transcription of distinct full-length retroviral transcripts. Vaccinia-mediated expression of retroviral vector particles could be observed as early as 3 h postinfection and resulted in stable transduction of NIH/3T3 target cells at higher titers than the control performed by conventional plasmid transfections. Thus at least part of the vaccinia life cycle and retroviral assembly can occur concomitantly. Due to the favorable properties of vaccinia vectors, including high coding capacity, stability, and wide host range, defective vaccinia viral/retroviral chimeric vectors are promising tools for gene therapy applications
Conditional Cell Transformation by Doxycycline-Controlled Expression of the MC29 v-mycAllele

AbstractTo investigate the molecular basis of oncogenesis induced by the v-myconcogene of avian myelocytomatosis virus MC29, we developed a conditional cell transformation system in which expression of the MC29 v-mycallele is dependent on a doxycycline-sensitive transactivator (tTA). Clonal lines of quail embryo fibroblasts transformed by doxycycline-controlled v-mycrevert to the normal phenotype and lose their ability to grow in soft agar after the addition of doxycycline. Repression of v-myccauses the cells to withdraw from the cell cycle, and long-term survival in culture requires reexpression of v-myc. Although complete repression of v-mycmRNA and v-Myc protein in these cells occurs within 14 h after the addition of doxycycline, the first morphological alterations are observed after 24 h, and after 3 days, the morphology changed entirely from small rounded cells showing a typicalmyc-transformed phenotype to large flat cells resembling normal fibroblasts. Cells exposed to doxycycline for 3 days reexpressed v-mycwithin 24 h after withdrawal of the drug from the culture medium, partial retransformation occurred after 2 days, and complete morphological transformation was reestablished after 6 days. Analogous results were obtained with a cell line in which expression of the v-mycallele is dependent on a reverse transactivator (rtTA) that is activated by doxycycline. The striking differential expression of known transformation-sensitive genes and of new candidate v-myctarget genes revealed the tightness of the doxycycline-controlled v-mycexpression system. The data also indicate that expression of v-mycin these cells is indispensable for enhanced proliferation, transformation, and immortalization
Trans-activator Tax of Human T-Cell Leukemia Virus Type 1 Enhances Mutation Frequency of the Cellular Genome

AbstractTax protein of human T-cell leukemia virus type 1 (HTLV-1) is a potent trans-activator of specific sets of target genes, and on the other hand, it is a trans-repressor of other sets of genes. It is also an inhibitor of the tumor suppressor protein p16INK4aand thus has been thought to contribute to induction of adult T-cell leukemia (ATL). We examined the mutagenic effects of Tax on a cellular gene, hypoxanthine guanine phosphoribosyltransferase (hprt), and LacI gene in lambda shuttle vector exogenously integrated in Big Blue Rat-2 (BBRat-2) cells. Expression of Tax in BBRat-2 cells enhanced the frequency of HPRT(−)phenotype severalfold. Tax-expressing cell clones, BBTax-1 and -2 established from BBRat-2 cells, gave rise to an average mutation frequency of 5.9 × 10−5in LacI gene, but Tax-negative cell clones, BBRat-C1 and -C2, showed 2.1 × 10−5. The 2.8-fold increase in mutation frequency in the presence of Tax indicates that Tax expression enhanced mutation frequency in chromosomal DNA. However, neither the mutation spectrum of base transitions, transversions, and deletions/insertions nor the loci of the mutations were significantly affected by Tax expression. These findings indicate that Tax has the capability to induce random mutations and suggest that Tax would be able to modulate cellular phenotypes through mutation of the cellular genome
Mutations in the Newcastle Disease Virus Hemagglutinin–Neuraminidase Protein That Interfere with Its Ability to Interact with the Homologous F Protein in the Promotion of Fusion

AbstractRecent evidence suggests that the attachment (HN) and fusion (F) glycoproteins of Newcastle disease virus interact at the cell surface in a virus-specific manner to promote syncytium formation. Consistent with the existence of such an interaction, we have shown that it is possible to coimmunoprecipitate (co-IP) the two proteins from the surface of transiently expressing cells using a monoclonal antibody to either protein. Further, we show that a point mutation in the globular domain of HN that abolishes its receptor recognition and neuraminidase (NA) and fusion activities also abolishes its ability to interact with F in the co-IP assay. The mechanism by which this mutation might interfere with the interaction between the two proteins is discussed in terms of the postulate that recognition by HN of cellular receptors triggers its interaction with F and the apparently conflicting evidence for an interaction between the two proteins in the endoplasmic reticulum. Also, characterization of a set of chimeric HN proteins, having short overlapping sequences from a heterologous HN protein in the F-specific domain in the protein stalk, reveals that a weakened interaction between HN and F is still sufficient to trigger fusion
Stable Ubiquitination of the ICP0R Protein of Herpes Simplex Virus Type 1 during Productive Infection

AbstractICP0R is the polypeptide product of an alternatively spliced transcript of the gene encoding the transactivator protein ICP0 of herpes simplex virus type 1 (HSV-1). Although it has been shown to act as a transrepressor of gene expression in transfection assays, overexpression of the ICP0R protein in the recombinant virus HSV–KST was previously found to have no detectable effect on virus replication, so that the role it plays in HSV-1 infection remains unclear. Analysis of HSV–KST-infected cell lysates by Western blotting revealed the presence of not only the 41-kDa ICP0R polypeptide but also a 64-kDa processed form of the protein. This processing event was the result of ubiquination of the ICP0R protein, as demonstrated by the reactivity of the 64-kDa species with antibody specific for the influenza virus hemagglutinin (HA) protein epitope in experiments where the gene encoding ICP0R was coexpressed with a gene encoding HA-tagged ubiquitin. Surprisingly, the 64-kDa form of ICP0R was found to be remarkably stable and persisted in infected cells for many hours after processing, despite the fact that ubiquitination normally functions as a means of tagging proteins for rapid degradation. Analyses of mutant polypeptides containing arginine substitutions at each of the lysine residues of ICP0R, which represent potential ubiquitin conjugation sites, revealed that a single lysine residue at codon 248 was both necessary and sufficient for the appearance of the 64-kDa processed form. However, a number of ICP0R mutants that retained the ubiquitination site at lysine 248 but contained disruptions of sequences at distant sites also lacked detectable 64-kDa protein, indicating that the integrity of the overall structure of ICP0R was an additional determinant for ubiquitination
In VivoDisturbance of Hematopoiesis in Mice Persistently Infected with Murine Cytomegalovirus: Impairment of Stromal Cell Function

AbstractAlthough the pathogenic effects of a primary cytomegalovirus (CMV) infection on hematopoiesis has been largely investigated so far, the effects of a persistent or latent infection have yet to be elucidated. The effects of persistent CMV infection on hematopoiesis thus were examined using BALB/c mice at 4 weeks postinfection with 0.2 LD50of murine CMV (MCMV) infection as a persistent infection model. The parameters of constitutive hematopoiesis of MCMV persistently infected mice were completely identical to those of the control. However, the inductive hematopoiesis, examined by the autologous marrow reconstitution after 5-fluorouracil administration, was significantly impaired in the MCMV persistently infected mice (P< 0.05). In a colony-forming unit-spleen assay and a long-term bone marrow culture system, a decreased capacity of bone marrow stromal cells to support hematopoiesis was observed in the MCMV-infected mice in comparison with the controls. The existence of MCMV DNA in the adherent cells of long-term bone marrow culture from the MCMV-infected mice were confirmed by a polymerase chain reaction but not in the nonadherent cells. Furthermore, the increased expression level of tumor necrosis factor-α by stromal cells was also observed by semiquantitative reverse transcriptase-polymerase chain reaction. These results therefore strongly suggest that MCMV remains to infect the stromal cells while also inhibiting inductive hematopoiesis through the impairment of the stromal cell functions in the MCMV persistently infected mice
Transmission of Human Immunodeficiency Type 1 Viruses with Intersubtype Recombinant Long Terminal Repeat Sequences

AbstractRetroviruses such as human immunodeficiency virus type 1 (HIV-1) contain two RNA strands per virion, and recombination can occur frequently during reverse transcription. Recombination may occur between HIV-1 genomes of the same subtype or among genomes of two or more distinct subtypes present in an individual. In the current study, we found that recombinatorial events were not limited to viral structural genes such asgagandenv,but rather, recombination could likewise occur within the 5′ long terminal repeat (LTR). Intersubtype recombinant LTRs among HIV-1 subtypes A, C, and D were found in Tanzanian infants. By introducing novel LTR sequences, these recombinant LTR viruses may further increase the adaptive potential and fitness of HIV-1
Subtype G and Multiple Forms of A/G Intersubtype Recombinant Human Immunodeficiency Virus Type 1 in Nigeria

AbstractMultiple human immunodeficiency virus type 1 (HIV-1) genetic subtypes, intersubtype recombinants, and group O have been found in west central Africa. In Nigeria, where HIV-1 prevalence is rising rapidly, characterization of HIV-1 strains has been limited. Each of three full-length genome sequences acquired to date shows evidence of recombination: two are largely subtype G with subtype A segments in the midgenome accessory region; the third, IbNG, is subtype G with the long terminal repeats and two segments of pol from subtype A. In this study, peripheral blood mononuclear cells obtained in 1994–1995 from 10 patients hospitalized in northeastern Nigeria were evaluated by sequencing of the complete envelope and, from 7 patients, a portion ofgag.Four patients harbored subtype G viruses and six patients had recombinant viruses. Two had strains sharing the A/G recombinant structure of IbNG. Two had a previously undescribed recombinant, mostly subtype A, whose carboxyl-terminal gp41 could not be classified. An A/G recombinant different from IbNG but similar to CA1, a Cameroonian strain, was found in one patient. The remaining patient had a strain that was otherwise subtype G but shared an unclassified carboxyl-terminal gp41 segment with the CA1-like strains. Other subtypes and group O were not found