pinecone/core-2020-05-10-deduplication huggingface dataset:
Resting CD4+ T Cells with CD38+CD62L+ Produce Interleukin-4 Which Contributes to Enhanced Replication of T-Tropic Human Immunodeficiency Virus Type 1

AbstractA significant increase in the CD38+ population among T lymphocytes has been observed in human immunodeficiency virus type 1 (HIV-1)-infected carriers. We previously reported a higher replication rate of T-tropic HIV-1 in the CD4+CD38+CD62L+ than CD38− subset under conditions of mitogen stimulation after infection. Here, we revealed a similarly high susceptibility in the CD38+ subset on culture with conditioned medium containing Th2 cytokine, interleukin (IL)-4 that was produced endogenously from this subset on stimulation with mitogen or anti-CD3 antibody for 3 days. The contribution of IL-4 to the upregulated production of virus in the CD38+ subset was confirmed by culture of this subset with recombinant human IL-4. In contrast, the rate of replication in the CD38− subset was not augmented in the conditioned medium from either subset or with IL-4. However, there were no differences in the surface expression of IL-4 receptor or HIV-1 receptors CD4 and CXCR4 between the two subsets. Thus, the CD4+CD38+CD62L+ subset comprises a specific cell population secreting endogenous Th2 cytokine that contributes to the efficient production of T-tropic HIV-1 through upregulation at a certain stage of the viral life cycle, probably after the adsorption step
Identification of a Nucleocapsid Protein (VP35) Gene of Shrimp White Spot Syndrome Virus and Characterization of the Motif Important for Targeting VP35 to the Nuclei of Transfected Insect Cells

AbstractTo identify the protein encoded by a 687-bp open reading frame (ORF) of a salI genomic DNA fragment of shrimp white spot syndrome virus (WSSV), we expressed the ORF in a baculovirus/insect cell expression system. The apparent molecular mass of the recombinant protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was 35 kDa in insect cells. Antibody raised against bacterially synthesized protein of the ORF identified a nucleocapsid protein (VP35) in the extracts of both the purified WSSV virions and the nucleocapsids which comigrated with the 35-kDa baculovirus-expressed recombinant protein on SDS-PAGE. We also show by transient expression in insect cells (Sf9) that VP35 targets the nucleus. Two potential nuclear localization signals (NLSs) were characterized, but only one of them was important for targeting VP35 to the nuclei of transfected insect cells. Replacement of a cluster of four positively charged residues (24KRKR27) at the N terminus of the protein with AAAA resulted in mutant proteins that were distributed only in the cytoplasm, thus confirming that this sequence is a critical part of the functionally active NLS of VP35
A Role for Human Cytomegalovirus Glycoprotein O (gO) in Cell Fusion and a New Hypervariable Locus

AbstractA cell fusion assay using fusion-from-without (FFWO) recombinant adenoviruses (RAds) and specific antibody showed a role in fusion modulation for glycoprotein gO, the recently identified third component of the gH/gL gCIII complex of human cytomegalovirus (HCMV). As in HCMV, RAd gO expressed multiple glycosylated species with a mature product of 125 kDa. Coexpression with gH/gL RAds showed gCIII reconstitution in the absence of other HCMV products and stabilisation by intermolecular disulfide bonds. Properties of HCMV clinical isolate, Pt, also implicated gO in cell spread. Compared to laboratory strain AD169, Pt was resistant to gH antibody plaque inhibition, but mature gH was identical. However, the gO sequences were highly divergent (20%), with further variation in laboratory strain Towne gO (34%). Thus, gO forms gCIII with gH/gL, performs in cell fusion, and is a newly identified HCMV hypervariable locus which may influence gCIII's function in mediating infection
The 3′-Untranslated Region of RNA1 as a Primary Determinant of Temperature Sensitivity of Red clover necrotic mosaic virus Canadian Strain

AbstractRed clover necrotic mosaic virus Canadian strain (RCNMV-Can) induces symptoms on host plants at 17°C, but not at 25°C. We investigated the temperature sensitivity of RCNMV-Can in Nicotiana benthamiana plants and protoplasts using infectious transcripts of genomic RNAs 1 and 2. Viral RNAs accumulated in both inoculated and noninoculated leaves at 17°C, whereas no viral RNAs were detected at 25°C in either inoculated or noninoculated leaves. Similar temperature sensitivity in RNA accumulation was observed in protoplasts, and no viral RNAs were detected at temperatures above 22°C. These results indicate that the temperature sensitivity of RCNMV-Can occurs at an early stage of infection, including during RNA replication. Using reassortant viruses and chimeric RNAs 1 between RCNMV-Can and the RCNMV Australian strain, which accumulates viral RNAs at nonpermissive temperatures for RCNMV-Can, we demonstrated that a viral determinant for the temperature sensitivity resides in the 3′-untranslated region of RNA1
Positional Effect of Deletions on Viability, Especially on Encapsidation, of Brome mosaic virus D-RNA in Barley Protoplasts

AbstractBrome mosaic virus (BMV), a tripartite RNA plant virus, accumulates RNA3-derived defective RNAs (D-RNAs) in which 477–500 nucleotides (nt) are deleted in the central region of the 3a protein open reading frame (ORF), after prolonged infection in barley. In the present study, six artificial D-RNAs (AD-RNAs), having deletions of the same size as the naturally occurring D-RNA but at different positions in the 3a ORF, were constructed and tested for their amplification and encapsidation in barley protoplasts by coinoculation with BMV RNA1 and 2, or RNA1, 2, and 3. Northern blot analysis of RNA accumulation in total and virion fractions showed that deletions of 492 nt in the 3′-proximal and the 5′-proximal regions of the 3a ORF decreased encapsidation efficiency of the AD-RNAs compared with that of RNA3, whereas deletions in the central region enhanced encapsidation efficiency. The present results also show that deletion positions affect competition with RNA3 in the amplification and encapsidation of AD-RNAs
The Immediate-Early Protein, ICP0, Is Essential for the Resistance of Herpes Simplex Virus to Interferon-α/β

AbstractHerpes simplex virus type 1 (HSV-1) is resistant to the antiviral effects of interferon (IFN)-α, -β, or -γ. The fact that ICP0− mutants replicate like wild-type virus in IFN-α/β receptor knockout mice (Leib et al., 1999, J. Exp. Med. 189, 663) suggested that ICP0 may serve a direct role in the resistance of HSV-1 to IFN. To test this hypothesis, the effects of IFN-α, -β, and -γ were compared against wild-type HSV-1 and an ICP0− mutant virus, 7134. In Vero cells, 7134 was more sensitive to inhibition by low doses of type I IFN (-α/β) or type II IFN (-γ) than vesicular stomatitis virus, a well-studied IFN-sensitive virus. At a concentration of 100 U/ml, IFN-α, -β, or -γ reduced the efficiency of 7134 plaque formation by 120-, 560-, and 45-fold, respectively. In contrast, none of the IFNs reduced wild-type HSV-1 plaque formation by more than 3-fold. Even when Vero cells were infected with 10 pfu per cell, IFN-α and -β inhibited 7134 replication by over 100-fold, but inhibition by IFN-γ decreased to less than 10-fold. While IFN-β efficiently inhibited 7134 replication in primary mouse kidney and SK-N-SH cells, IFN-γ did not inhibit 7134 to a comparable extent in these cells. ICP0 provided in trans from an adenovirus vector allowed 7134 to replicate efficiently in Vero cells in the presence of IFN-α, -β, or -γ. While IFN-β or -γ efficiently repressed the ICP0 promoter-lacZ reporter gene in 7134 (i.e., ∼60-fold reduction in β-galactosidase activity), ICP0 provided in trans almost completely reversed IFN-mediated repression of the lacZ gene in 7134. The results suggest that the rate of ICP0 expression in infected cells in vivo may be critical in determining whether host IFNs repress the HSV-1 genome. This concept is discussed in light of its potential relevance to the establishment of latent HSV-1 infections
Single Oral Immunization with Replication Deficient Recombinant Adenovirus Elicits Long-Lived Transgene-Specific Cellular and Humoral Immune Responses

AbstractOral–gastric delivery of vaccines is a preferred route of immunization and is particularly relevant to the development of vaccine-vector systems. We have investigated the ability of a replication deficient (E1-deleted) adenovirus construct (RAd68), which efficiently expresses the measles virus nucleocapsid (N) protein under the control of the strong HCMV IE promoter, to elicit antibody and cytotoxic T cell (CTL) responses in mice following intragastric administration. Measles virus N protein-specific CTL memory and serum antibody responses were analyzed in a total of 140 mice at time points 2–51 weeks after immunization either with a single dose of 108 pfu RAd68 or with a fivefold higher dose. Of the 20 animals analyzed in the first 4-week period following low-dose immunization, 6 mounted low-level splenic CTL responses while 13 animals had CTL in the mesenteric lymph nodes. Splenic CTL responses were largely undetectable at later times. Only 23% of low-dose-immunized mice made serum antibody responses and these were generally of low magnitude and frequently of short duration. In contrast, the majority of animals immunized orally with 5 × 108 pfu RAd68 mounted splenic CTL responses (70%) and/or antibody responses (89%). Notably, these responses were stronger and of greater duration than those seen following immunization at the lower dose. Gut mucosal immunization with replication deficient adenoviruses is a promising approach, not only for the development of complementary measles vaccine strategies which may be required for measles virus eradication, but also generally for vaccination against other infections
The Most Frequent Naturally Occurring Length Polymorphism in the HIV-1 LTR Has Little Effect on Proviral Transcription and Viral Replication

AbstractAbout 38% of primary HIV-1 long terminal repeats (LTRs) contain an insertion (consensus: 5′-ACYGCTGA-3′), termed the most frequent naturally occurring length polymorphism (MFNLP). The MFNLP binds several transcription factors and might affect HIV-1 replication and disease progression in infected individuals. However, its relevance for proviral transcription and for HIV-1 replication in primary cells is unclear. We utilized HIV-1 NL4-3 LTR variants to investigate the effect of the MFNLP on 5′LTR transcriptional activity in various cell types. Notably, viral promoter activity was studied in primary cells in the context of the integrated provirus, using both single cycle assays with pseudotyped Luciferase reporter viruses and replication-competent HIV-1 mutants. Our results demonstrate that the presence, absence, or duplication of the 5′-ACYGCTGA-3′ motif has little effect on viral promoter activity in T cell lines, peripheral blood mononuclear cells (PBMC), and monocyte-derived macrophages (MDM). Furthermore, all HIV-1 LTR variants showed efficient induction upon stimulation with TPA and/or ionomycin and replicated with comparable efficiency in a human T cell line and in PBMC. Thus, the MFNLP does not significantly affect HIV-1 5′LTR transcriptional activity and viral replication in primary cells, suggesting that this common sequence variation has little impact on the clinical course of HIV-1 infection
Human Immunodeficiency Virus Type 1 Preferentially Encapsidates Genomic RNAs That Encode Pr55Gag: Functional Linkage between Translation and RNA Packaging

AbstractFull-length retroviral RNA serves as both messenger and genomic RNA. Therefore, an unspliced RNA could play both roles: viral mRNA could be bound in cis by the same Gag polyprotein that it produced, becoming a packaged genomic RNA. To test this possibility, we used in vivo packaging experiments which coexpressed wild-type NL4-3 RNA and NL4-3-based mutant RNA that, ideally, could not translate Gag. However, mutating the gag initiator produced a mutant (pNLX) that expressed a truncated Gag, Gag*, initiated at methionine 10 in the CA region (142 of Pr55Gag). Gag* can be rescued into virions by Gag and, as it contains the NC domain, could package RNA in cis. To eliminate NC and the CA dimerization domain, a nonsense mutation in CA at residue 99 was introduced into pNLX to produce pNLXX, which expresses an RNA that should only be packaged in trans. Cotransfection packaging experiments revealed that wild-type genomic RNA was packaged at an 8-fold greater level than NLXX RNA given equal expression of both RNAs. Experiments that varied the relative amounts of these RNAs in the cell found that the wild-type RNA was encapsidated with a packaging preference (i.e., the relative amount of this RNA in virions versus cells) of 6- to 13-fold over the NLXX RNA, showing that the NLXX RNA did not efficiently compete with NL4-3 RNA. These data suggest that the wild-type RNA's ability to express Pr55Gag and, by inference, actively translate Gag confers an advantage in packaging over the nearly identical NLXX RNA. In contrast, the NLX RNA competed with wild-type RNA at a 1-to-3 preference. This ratio is similar to the amounts of Gag* rescued by Gag, suggesting that the presence of Gag* assists in the encapsidation of NLX RNA. Together, our data link translation and particle formation to the packaging of viral RNA and support a model of cis packaging where nascent Gag proteins encapsidate their cognate RNA
Efficient Gene Transfer into Spleen Cells of Newborn Mice by a Replication-Competent Retroviral Vector

AbstractA murine leukemia virus-derived replication-competent retroviral vector with a translational cassette for the enhanced green fluorescence protein (EGFP) was previously found to function efficiently in cell culture (Jespersen et al., 1999, Gene 239, 227–235). We here report that infection of newborn NIH Swiss mice gives rise to EGFP expression in a majority of spleen cells within the first days after infection. Among the nonadherent spleen cells, B, T, and NK lymphocytes were found to be efficiently marked by EGFP by flow cytometry analysis, whereas the adherent spleen cells were negative in most animals. Analysis at time points up to 60 days after infection reveals a decline in EGFP-positive spleen cells over time. Viremia analysis and PCR analysis of spleen DNA indicate that viruses that have lost the translation cassette predominate at later stages. The results provide a model for efficient gene delivery to spleen cells in a time window of 1 to 2 weeks after infection of newborns. Although this type of vector will not in itself be applicable in the clinic, we envision that efficient in vivo gene delivery will be valuable by analysis of differentiation and proliferation of hematopoietic cells in animal models and by development and evaluation of effectors interfering with these processes
NS1 Protein of Parvovirus B19 Interacts Directly with DNA Sequences of the p6 Promoter and with the Cellular Transcription Factors Sp1/Sp3

AbstractThe nonstructural proteins of parvovirus exert a variety of disparate functions during viral infection ranging from promoter regulation, involvement in DNA replication, and induction of apoptosis. Our interest was focused on the possible mechanism by which the NS1 protein mediates its effects on the p6 promoter of parvovirus B19. It is known that the p6 promoter is highly active in different cell lines and interaction with the viral NS1 protein results in a further increase of the activity. The protein may function by binding directly to the viral DNA or via an indirect binding through interaction with cellular transcription factors bound to the promoter. We examined the interaction of the NS1 protein with cellular transcription factors which are involved in regulating the promoter activity. After purified baculovirus-expressed NS1 protein in gel retardation assays was added, an altered complex formation was observed, indicating that NS1 protein interacts with Sp1/Sp3 transcription factors. Enzyme-linked immunosorbent assays verified these findings. The direct interaction of NS1 protein with p6 promoter elements was analyzed by a coprecipitation assay whereby labeled oligonucleotides spanning the entire promoter region were incubated with NS1 protein followed by an immunoprecipitation with NS1-specific antibodies. An eight-nucleotide-long, almost palindromic sequence (AGGGCGGA) was found as potential NS1-binding motif. Footprint analysis with oligonucleotides containing this DNA motif confirmed this result. Thus, transcriptional regulation by the NS1 protein may involve both the interaction with Sp1/Sp3 that binds to the promoter region and direct binding of NS1 to the promoter DNA
Characterization of Adeno-Associated Virus Rep Protein Inhibition of Adenovirus E2a Gene Expression

AbstractAdeno-associated virus (AAV) replication (Rep) proteins are pleiotropic effectors of viral DNA replication, RNA transcription, and site-specific integration into chromosome 19. In addition to regulating AAV gene expression, the Rep proteins modulate expression of a variety of cellular and viral genes. In this report we investigate Rep-mediated effects on expression of the adenovirus (Ad) E2a gene and the Ad major late promoter. We have found that all four Rep proteins repress E2a expression at the protein level, with Rep40 showing the weakest repression. Mutations in the purine nucleotide binding (PNB) site weakened each of the protein's abilities to repress expression. Analysis of steady-state E2a mRNA showed that Rep proteins decreased mRNA levels, but to a lesser extent than E2a protein levels. Analysis of mRNA stability demonstrated that neither Rep78 nor Rep52 affected E2a mRNA stability, suggesting that the decrease in mRNA is due to Rep-mediated inhibition of Ad E2a transcription. To determine if Rep68 proteins could directly inhibit RNA transcription, we performed in vitro transcription assays using HeLa nuclear extracts supplemented with Rep68 and Rep68PNB. We demonstrate that Rep68, but not mutant Rep68PNB, blocked in vitro transcription of a template containing the Ad major late promoter. These results provide insight into how AAV and its encoded Rep proteins interact with Ad and provide a model system for the study of AAV and host-cell interactions
In Vivo Tissue-Specific Regulation of the Human Papillomavirus Type 18 Early Promoter by Estrogen, Progesterone, and Their Antagonists

AbstractHuman papillomavirus type 18 is a causative agent of epithelial cancers in the uterine cervix. We show here that estrogen and progesterone activate β-galactosidase expression from the early promoter of this virus in the genital epithelia of transgenic mice. Ovariectomy caused suppression of transgene expression exclusively in vagina and cervix epithelia. β-Galactosidase expression could be restored in ovariectomized females by administration of estrogen, alone or in combination with progesterone. Further, rescue of transgene expression was inhibited by the estrogen antagonist tamoxifen and the anti-progesterone RU486, suggesting that this was a specific effect
Characterization of φ12, a Bacteriophage Related to φ6: Nucleotide Sequence of the Small and Middle Double-Stranded RNA

AbstractThe isolation of additional bacteriophages containing segmented double-stranded RNA genomes has expanded the Cystoviridae family to nine members. Comparing the genomic sequences of these viruses has allowed evaluation of important genetic as well as structural motifs. These comparative studies are resulting in greater understanding of viral evolution and the role played by genetic and structural variation in the assembly mechanisms of the cystoviruses. In this regard, the small and middle double-stranded RNA genomic segments of bacteriophage φ12 were copied as cDNA and their nucleotide sequences determined. This genome's organization is similar to that of the small and middle segments of bacteriophages φ6, φ8, and φ13. Although there is little similarity in the nucleotide sequences, similarity exists in the amino acid sequence of the lysis cassette proteins to those of φ6. The host cell attachment proteins are found to have marked similarity to the φ13 attachment proteins
Yeast Coexpression of Human Papillomavirus Types 6 and 16 Capsid Proteins

AbstractThe L1 and L2 capsid proteins of animal and human papillomaviruses (HPVs) can self-assemble into virus-like particles (VLPs) that closely resemble native virions. The use of different animal models shows that VLPs can be very efficient at inducing a protective immune response. However, studies with infectious HPV virions and VLPs of different HPV types indicate that the immune response is predominantly type-specific. We have generated a diploid yeast strain that coexpresses the L1 and L2 capsid proteins of both HPV-6b and HPV-16, and we have purified fully assembled VLPs banding in a cesium chloride gradient at the expected density of 1.29–1.3 mg/ml. Experimental evidence strongly indicated that the four proteins coassembled into VLPs. Western blot analysis, using anti-HPV-6 and anti-HPV-16 L1-specific monoclonal antibodies and type-specific L2 antisera, demonstrated that all four proteins copurified. Most importantly, immunoprecipitation experiments, carried out using type-specific anti-L1 monoclonals and either total yeast cell extracts or purified VLPs, confirmed the interaction and the formation of covalent disulfide bonds between the two L1 proteins. Finally, HPV-6/16 VLPs administered to mice induced conformational antibodies against both L1 protein types. These results suggest that coexpression of different capsid proteins may provide new tools for the induction of antibodies directed against multiple HPV types
Evidence of Nucleotidyl Phosphatase Activity Associated with Core Protein σA of Avian Reovirus S1133

AbstractBoth avian reovirus core protein σA purified from virus-infected cell extracts and the purified bacterially expressed protein σA (eσA) were characterized for their nucleoside triphosphate (NTP) hydrolysis activity by thin-layer chromotography. Protein σA from both preparations has a nonspecific nucleotidyl phosphatase activity that hydrolyzes four types of NTP to their corresponding nucleoside di- and monophosphates and free phosphate. The divalent cation requirement for this activity of eσA was further examined by the addition of Mn2+, Mg2+, Ca2+, and Zn2+ ions. NTP hydrolysis by eσA was maximal when Mn2+, Mg2+, or Ca2+ concentrations were 5, 4, or 1 mM, respectively. Addition of Mn2+ or Mg2+ stimulated the reactions up to 4- or 3-fold, respectively, higher than Ca2+ (2.2-fold). However, Zn2+ ion inhibited this activity of eσA. The results suggest that nucleotidyl phosphatase activity of eσA is absolutely dependent on the divalent cations Mn2+, Mg2+, or Ca2+, but not Zn2+. Similar results were obtained from the analysis of divalent cation requirements for the protein σA nucleotidyl phosphatase activity. Optimal pH for nucleotidyl phosphatase activity of protein σA from both preparations was determined using reaction mixtures buffered at different pH. The results show that the optimal activities of both proteins were similar and were achieved between pH 7.5 and 8.5