pinecone/core-2020-05-10-deduplication huggingface dataset:
Productive Infection of CD34+-Cell-Derived Megakaryocytes by X4 and R5 HIV-1 Isolates

AbstractThe human immunodeficiency virus (HIV-1) causes various hematopoietic abnormalities, with thrombocytopenia (TP) occurring in 30% of infected individuals. In the present study, we aimed to determine whether HIV-1 in the bone marrow of TP patients can infect primary megakaryocytes in vitro, which may contribute to the development of thrombocytopenia. We amplified the V3 loop of HIV-1 envelope from the bone marrow of TP and non-TP patients and constructed recombinant viruses. We demonstrate that the bone marrow of TP and non-TP patients contained R5 strains, whereas X4 strains were present only in the bone marrow of TP patients. Furthermore, HIV-1 from the bone marrow of TP and non-TP patients infected megakaryocytes to similar levels, suggesting that the V3 loop of HIV-1 may not contain the viral determinants of HIV-associated TP. Chemokine receptor analysis determined that CD34+-cell-derived megakaryocytes express CD4, CXCR4, and CCR5 and are productively infected by both X4 and R5 HIV-1 isolates. Finally, we showed that CD34+-cell-derived megakaryocytes express the chemokine receptor CCR3
Enhancement of the Basal-Level Activity of HIV-1 Long Terminal Repeat by HIV-1 Nucleocapsid Protein

AbstractTwo HIV-1 proteins, Tat and NCp7 (NC), have zinc finger-like structures. NC is a virion protein and has been shown to accumulate in the nucleus 8 h postinfection. Since transcription factors with zinc fingers assist the transcriptional activity of both RNA polymerases II and III, we examined the effect of NC on HIV-1 LTR-directed gene expression. The HIV-1 NC binds to the HIV-1 LTR and results in a mobility shift in polyacrylamide gel electrophoresis. Competition assays with cold probes revealed that the binding of NC and formation of a DNA–protein complex could be prevented by the addition of excess unlabeled LTR self-probe, but not the HIV-1 V3 envelope gene. The DNase I footprint analysis showed that NC binds to six regions within HIV-1 LTR, four of which are near the transcription start site. The NC alone enhances LTR basal-level activity in RNA runoff experiments. When the general transcription factors (GTFs) were added in the assay, NC enhances NF-κB, Sp1, and TFIIB-induced HIV-1 LTR-directed RNA transcription. RNA transcription directed by the adenovirus major late promoter, however, is not significantly affected by NC in the cell-free system. Transient transfection of human T lymphocytes with the plasmids containing HIV-1 nc or gag showed enhancement of LTR-CAT activity. Moreover, transfection of HIV-1 provirus containing mutations in NC zinc-finger domains dramatically decreases the enhancement activity in human T cells, in which HIV-1 LTR is stably integrated into the cellular genome. These observations show that NC binds to HIV-1 LTR and cooperatively enhances GTFs and NF-κB induced HIV-1 LTR basal-level activity. NC may play the role of a nucleation protein, which binds to LTR and enhances basal-level transcription by recruiting cellular transcription factors to the HIV-1 promoter in competition with cellular promoters
Primate Cytomegaloviruses Encode and Express an IL-10-like Protein

AbstractAn open reading frame (ORF) with homology to interleukin-10 (IL-10) has been identified in rhesus cytomegalovirus (RhCMV). The IL-10-like protein is generated from a multispliced, polyadenylated early gene transcript encompassing part of the corresponding UL111A ORF of human CMV (HCMV). Immunological analyses confirm expression of the IL-10-like protein both in tissue culture and in RhCMV-infected rhesus macaques. Conserved ORFs were subsequently identified in human, baboon, and African green monkey CMV, and a fully processed transcript has been mapped in fibroblasts infected with the Towne strain of HCMV. The conservation of this previously unrecognized ORF suggests that the protein may play an essential role in primate CMV persistence and pathogenesis
Quantitation of Virus-Specific Classes of Antibodies Following Immunization of Mice with Attenuated Equine Herpesvirus 1 and Viral Glycoprotein D

AbstractThe antibody responses of CBA/J mice infected intranasally (i.n.) with either the attenuated KyA strain or the pathogenic RacL11 strain of equine herpesvirus 1 (EHV-1) or immunized with recombinant glycoprotein D (rgD) were investigated using the ELISPOT assay to measure EHV-1-specific antibody-secreting cells (ASC) in the regional lymphoid tissue of the respiratory tract. IgG, IgA, and IgM ASC specific for EHV-1 were detected in the mediastinal lymph nodes (MLN) and lungs 2 weeks after i.n. infection with EHV-1 strain KyA or RacL11, or immunization with heat-killed KyA or rgD. EHV-1-specific ASC were present in the MLN and lungs at 4 and 8 weeks, but declined in frequency by fivefold in the lung at 8 weeks. However, i.n. immunized (2 × 106 pfu KyA or 50 μg rgD/mouse) mice infected at 8 weeks with pathogenic EHV-1 RacL11 resisted challenge and showed eight- and tenfold increases in MLN ASC and lung ASC, respectively, by 3 days after challenge. In contrast to the intranasal route of immunization, intraperitoneal immunization yielded ASC frequencies in the MLN and lungs that were only slightly above those of nonimmunized control mice. These data indicate that immunization with infectious or heat-killed EHV-1 KyA, or rgD, induces significant levels of virus-specific ASC both in the MLN and lungs, a specific memory B-cell response, and long-term protective immunity. The finding that the numbers of ASC induced by the pathogenic strain versus the attenuated strain of EHV-1, which were virtually identical, indicated that the ability to generate a B-cell response is independent of and does not contribute to EHV-1 virulence
Glycoprotein B of Human Herpesvirus 8 Is a Component of the Virion in a Cleaved Form Composed of Amino- and Carboxyl-Terminal Fragments

AbstractHuman herpesvirus 8 (HHV-8) or Kaposi's sarcoma-associated herpesvirus (KSHV) is the only known human member of the Rhadinovirus genus of the gammaherpesvirus subfamily. Antibodies against peptides representing portions of the amino- and carboxyl-termini of HHV-8 gB were produced and used to detect gB expression in Vero cells transfected with the gB gene, in the HHV-8-harboring cell line, BCBL-1, and in purified virions. Expression of gB was detected in approximately 3% of uninduced BCBL-1 cells, while up to 30% of the cells expressed gB after 12-O-tetradecanoylphorbol-13-acetate (TPA) induction of virus replication. Indirect immunofluorescence assays and confocal microscopy showed that gB was distributed throughout the cytoplasm of BCBL-1 cells and transfected Vero cells. Immunoblot analyses of virion preparations revealed the presence of full-length as well as two smaller than full-length gB-derived species corresponding to the amino- and carboxy-terminal portions of gB, respectively. Biochemical analysis of the gB carbohydrate moieties using glycosylation inhibitors revealed that gB contained N-linked oligosacharides of the high-mannose type, characteristic of precursor carbohydrate chains added in the endoplasmic reticulum
Effective Construction of DNA Vaccines Against Variable Influenza Genes by Homologous Recombination

AbstractWe demonstrate the potential of cloning by homologous recombination as a rapid method to construct DNA molecules encoding newly developing hemagglutinins (HA) of influenza A virus. The variable parts of the HA genes were cloned into a basic construct containing the HA gene from an H3N2 strain. The recombinant DNAs thus created encode different variable domains with neutralising epitopes from four recently circulating influenza A H3 strains. The technology allows rapid production of DNA constructs for vaccines that can induce antibody and, particularly, cellular immune responses. These new constructs were also capable of conferring protection to challenge in mice. The technology may hence be a valuable tool for rapid adaptation of influenza vaccines to changes in the circulating influenza strains
Identification of Regions in the Moloney Murine Leukemia Virus SU Protein That Tolerate the Insertion of an Integrin-Binding Peptide

AbstractTargeting of retroviral vectors to specific cells has been attempted through engineering of the surface (SU) protein of the murine leukemia viruses (MuLVs), but in many cases this has adversely affected protein function and targeted delivery has been difficult to achieve. In this study, we have inserted a 15-mer peptide that binds specifically to the αvβ3 integrin into the Moloney MuLV SU protein, including regions that are surface exposed in the crystal structure of the ecotropic receptor-binding domain. We have concentrated in particular on the variable regions VRA, VRB, and VRC, which are responsible for the use of distinct cellular receptors by different MuLV subtypes and therefore may be more likely to accommodate a heterologous binding moiety. Despite these considerations, only 8 of 26 insertion sites were tolerated, including two separate regions in VRA, a cluster of sites in VRC, and previously identified sites at the N-terminus of the protein and in the proline-rich region immediately downstream of the receptor-binding domain. When expressed on retroviral vector particles, all of the viable proteins retained the ability to bind to and transduce murine cells, although the VRC mutants and an insertion in VRA gave reduced binding and titer. Finally, although all of the viable chimeras could bind to αvβ3 in a solid-phase binding assay, we were unable to demonstrate expanded tropism for αvβ3-expressing human cells. This study highlights the difficulty of engineering the Moloney MuLV SU protein, even when structural information is available, and provides guidelines for the insertion of peptide ligands into the SU protein
Recombination within the Nonstructural Genes of the Parvovirus Minute Virus of Mice (MVM) Generates Functional Levels of Wild-type NS1, Which Can Be Detected in the Absence of Selective Pressure Following Transfection of Nonreplicating Plasmids

AbstractRecombination within the coding region of the nonstructural genes of minute virus of mice (MVM), which generates functional levels of wild-type NS1, was observed in the absence of selective pressure following cotransfection of nonreplicating plasmids. P38 activity was used as a measure of recombinant NS1 production, which, together with direct detection of recombinant-generated products by RT-PCR, allowed an estimation of recombination efficiency. In addition, we show that very low levels of wild-type NS1 were able to significantly transactivate P38. Given that recombination following cotransfection can generate NS1 at these levels, our observations have implications for the study of parvoviral genetics, the construction of recombinant parvoviral vectors for gene therapy applications, and perhaps other systems using cotransfection of plasmids that share homologous sequences
The E1 Helicase of Human Papillomavirus Type 11 Binds to the Origin of Replication with Low Sequence Specificity

AbstractExpression of the human papillomavirus type 11 E1 and E2 genes is necessary and sufficient to support viral DNA replication. The full-length E2 protein is a transcriptional modulator that also interacts with the E1 helicase to form an E1/E2 complex at the viral origin of replication. Previous studies indicated that efficient binding of this complex to the replication origin is site-specific and that the E2 homodimer was required for efficient E1 binding. Human papillomavirus type 11 E2 and E1 proteins have been purified and their cooperative binding to the HPV type 11 viral replication origin has been characterized. Low-affinity E1 binding to the HPV type 11 replication origin was demonstrated and found to be largely nonspecific. DNA binding by E1 does not require complex formation with E2 and appears to be independent of ATP binding or hydrolysis. E1 binding quantitatively increased with the addition of increasing amounts of E2 and mutations in the E2 binding site demonstrated that the E2BS site is required for E1 and E2 to specifically bind as a high-affinity complex at the replication origin. Analysis of the A/T-rich E1 binding site via mutation showed that it was nonessential for high-affinity E1/E2 complex formation. Thus, although the replication functions between the animal and the human papillomaviruses are well conserved, there are subtle differences in the DNA binding requirements for E1, which may portend mechanistic differences among the DNA replication systems of various papillomavirus types
The Interaction of Heparin Sulfate and Adeno-Associated Virus 2

AbstractRecently heparan sulfate was proposed as the host cell receptor for the dependovirus, adeno-associated virus type 2 (AAV2). We show that although heparan sulfate on the cell surface may contribute to the binding of AAV2 to permissive cells, the amount of heparan sulfate on the cell surface as determined by flow cytometry using four different monoclonal antibodies does not correlate with AAV2 binding to cells or recombinant AAV2 transduction efficiency. Experiments with either mutant CHO cells or cells treated with chlorate to remove sulfate groups showed that sulfation was not absolutely required for infection or binding: in the absence of cell surface sulfation, recombinant AAV2 was still able to be transduced in previously permissive cells. Heparin is commonly used as a substitute in studies of the interaction between heparan sulfate and ligand, and we demonstrate that the binding affinity of AAV2/heparin is low, with a Kd value of ∼2.0 nM. A study of the direct interaction between AAV2 and artificial glycosaminoglycans showed that a high degree of sulfation on heparin was critical for the ability to bind AAV2 and compete rAAV2 transduction and that both O- and N-sulfate groups are required. Overall, our data suggest that, as has been shown for other viruses, the presence of a high-affinity AAV2 receptor mediates AAV2 infection in addition to the low-affinity heparan sulfate binding
Human Interleukin-6 Facilitates Hepatitis B Virus Infection in Vitro and in Vivo

AbstractBackground and aim. Research on hepatitis B virus (HBV) infection in vivo has been limited due to the absence of a suitable animal model. We have developed a human–mouse radiation chimera in which normal mice, preconditioned by lethal total body irradiation and radioprotected with SCID mouse bone marrow cells, are permissive for engraftment of human hematopoietic cells and solid tissues. This resulting human–mouse model, which comprises three genetically disparate sources of tissue, is therefore termed Trimera. This study was aimed at assessing the effect of human IL-6 on HBV infection in vivo in Trimera mice. Methods. Trimera mice were transplanted with human liver tissue fragments or with HepG2-derived cell lines, which had been previously infected ex vivo with HBV in the presence or absence of human interleukin-6 (hIL-6) and in the presence of anti-IL-6-neutralizing antibodies. Results. HBV sequences appeared in the sera of animals in which the liver tissue was incubated with both HBV and hIL-6 prior to transplantation. A similar result was obtained when a human hepatoblastoma cell line (HepG2), expressing the hIL-6 receptor, was infected ex vivo with HBV in the presence of hIL-6 prior to their injection into spleens of Trimera mice. However, when liver fragments were infected ex vivo and simultaneously treated with neutralizing antibodies against hIL-6 or were incubated with HBV prior to transplantation without hIL-6, the rate of mice positive for HBV DNA in their sera was lower. Human mononuclear cells are also permissive for HBV infection in vitro: in the presence of hIL-6 the infection of these cells is enhanced; and this infection is suppressed by the chimeric protein named Hyper-IL-6, generated by the fusion of hIL-6 to the soluble hIL-6 receptor (sIL-6Rα, gp80). Conclusion. hIL-6 facilitates HBV infection in vitro and in vivo
Adoptive Transfer of Infectious Bronchitis Virus Primed αβ T Cells Bearing CD8 Antigen Protects Chicks from Acute Infection

AbstractInfectious bronchitis virus (IBV) infection and associated illness may be dramatically modified by passive transfer of immune T lymphocytes. Lymphocytes collected 10 days postinfection were transferred to naive chicks before challenge with virus. As determined by respiratory illness and viral load, transfer of syngeneic immune T lymphocytes protected chicks from challenge infection, whereas no protection was observed in the chicks receiving the MHC compatible lymphocytes from uninfected chicks. Protection following administration of T lymphocytes could be observed in chicks with three distinct MHC haplotypes: B8/B8, B12/B12, and B19/B19. Nearly complete elimination of viral infection and illness was observed in chicks receiving cells enriched in αβ lymphocytes. In contrast, removal of γδ T lymphocytes had only a small effect on their potential to protect chicks. The adoptive transfer of enriched CD8+ or CD4+ T lymphocytes indicated that protection was also a function primarily of CD8-bearing cells. These results indicated that αβ T lymphocytes bearing CD8+ antigens are critical in protecting chicks from IBV infection
Crosslink Analysis of N-Terminal, C-Terminal, and N/B Determining Regions of the Moloney Murine Leukemia Virus Capsid Protein

AbstractTo analyze contacts made by Moloney murine leukemia virus (M-MuLV) capsid (CA) proteins in immature and mature virus particles, we have employed a cysteine-specific crosslinking approach that permits the identification of retroviral Gag protein interactions at particular residues. For analysis, single cysteine creation mutations were made in the context of protease-deficient or protease-competent parental constructs. Cysteine creation mutations were chosen near the N- and C-termini of CA and at a site adjacent to the M-MuLV Glu-Ala Fv1 N/B host range determination sequence. Analysis of immature virions showed that PrGag proteins were crosslinked at C-terminal CA residues to form dimers while crosslinking of particle-associated N-terminal and N/B region mutant proteins did not yield dimers, but showed evidence of linking to an unknown 140- to 160-kDa partner. Analysis of mature virions demonstrated that both N- and C-terminal CA residues participated in dimer formation, suggesting that processed CA N- and C-termini are free to establish interprotein associations. Interestingly, N/B region mutant residues in mature virus particles did not crosslink to form dimers, but showed a novel crosslinked band, consistent with an interaction between the N/B tropism determining region and a cellular protein of 45–55 kDa
Dox-Dependent SIVmac with Tetracycline-Inducible Promoter in the U3 Promoter Region

AbstractAn attenuated live vaccine is a candidate in developing vaccines against human immunodeficiency virus type 1 (HIV-1). The study using macaques and simian immunodeficiency virus (SIVmac) showed an attenuated virus to be more effective than any other vaccine candidate. However, development of a safer vaccine is required for clinical application. In this study, we constructed pSIVmac Δ nef with tetracycline inducible promoter (pTet) and tried to control viral expression in a drug-dependent manner. Promoter/enhancer motifs in the U3 region of the long terminal repeats (LTRs) were serially deleted and replaced with pTet. In mutant LTRs, which lack NF-κB and Sp1 binding sites, TATA box motifs, and the 5′ half of the U3 region, promoter activity was stringently controlled by doxycycline (Dox). Their activities were similar to or higher than that of wild-type LTR in the presence of Dox, based on the transient chloramphenicol acetyltransferase reporter assay. Three of these mutant LTRs were introduced into the pSIVmac239 Δ nef genome. Viral protein from these viruses was efficiently expressed in a Dox-dependent manner after transfection to a HeLa cell, which expresses reverse tetracycline transactivator (rtTA). The 2-LTR-form viral DNA of these viruses could be detected in M8166 cells that had been infected with supernatants from the transfected rtTA HeLa cell. These results suggest that pSIVmac Δ nef containing mutant LTRs can proceed through one viral replication cycle consisting of transcription, formation of viral particles, infection to cells, and reverse transcription. Although continuous replication of these Dox-dependent viruses requires a supply of rtTA as a constituent for the pTet-On viral genome, the successful replacement of the original promoter with a drug-dependent promoter suggests a new possibility for developing a safer attenuated live virus
Virtually Full-Length Subtype F and F/D Recombinant HIV-1 from Africa and South America

AbstractFor reliable classification of HIV-1 strains appropriate reference sequences are needed. The HIV-1 genetic subtype F has a wide geographic spread, causing significant epidemics in South America, Africa, and some regions of Europe. Previously only two full-length sequences of each of the HIV-1 subtype F subclusters F1 and F2 have been described. To extend the knowledge of subtype F variation on a complete genome level, three new virtually full-length F1 sequences were cloned and sequenced, two from Africa and one from South America. Comparison of the new and previously described sequences showed that monophyletic clustering of the subcluster F1 of subtype F is consistent and highly supported in all genome regions. Two additional full-length strains were shown to be mosaics of subtypes F and D. These epidemiologically unrelated F/D sequences showed similar chimeric structure, suggesting that they may represent a previously undescribed circulating recombinant form (CRF). This was supported by partial sequences from three additional unlinked F/D recombinants. Genetic distances in the phylogenetic trees suggest that the recombination event leading to the putative CRF occurred relatively long ago, close to the divergence of the F1 and F2 subclusters. Furthermore, all five F/D recombinants are linked to the Democratic Republic of Congo, suggesting that the original recombination event took place in central Africa
Rhesus and Pig-Tailed Macaque Parvoviruses: Identification of Two New Members of the Erythrovirus Genus in Monkeys

AbstractWe have previously reported the identification of a novel simian parvovirus in cynomolgus monkeys, which causes severe anemia in immunosuppressed cynomolgus monkeys and is currently being studied as an animal model for human B19 infection. We now report two similar outbreaks of anemia in rhesus and pig-tailed macaques associated with two distinct but similar simian parvoviruses (pig-tailed macaque and rhesus parvovirus). Both viruses have been cloned and over 5000 nucleotides sequenced from each virus. The viruses show marked similarities to other members of the Erythrovirus genus in the Parvoviridae family
Stimulation of Dengue Virus Replication in Cultured Aedes albopictus (C6/36) Mosquito Cells by the Antifungal Imidazoles Ketoconazole and Miconazole

AbstractReplication of dengue type 3 virus in Aedes albopictus C6/36 cells was enhanced more than 50-fold by addition of the antifungal imidazole derivative ketoconazole within the first 4 h of infection. The stimulatory effect was reflected in the yield of infectious virus and in levels of viral RNA and protein synthesis. Enhanced yields were observed also for other flaviviruses, including dengue type 2 virus and Murray Valley encephalitis virus. Increased yields of dengue type 3 virus were not observed in African green monkey kidney (Vero) cells, human monocytic (U-937) cells, or cells of the mosquito Toxorhynchites amboinensis (TRA-171)