pinecone/core-2020-05-10-deduplication huggingface dataset:
DNA-Binding Activity of the Streptococcus thermophilus Phage Sfi21 Repressor

AbstractThe cloned Streptococcus thermophilus phage Sfi21 repressor open reading frame (orf) 127 gp protects a cell against superinfection with the homologous temperate, but not against virulent phages. As demonstrated by DNase protection assay and gel shift experiments, the repressor binds to a 25-bp operator site located upstream of the repressor gene. A second sequence-related operator was identified 265 bp apart at the 3′-end of orf 75, the topological equivalent of a cro repressor gene. The replacement of a bp at the middle or at the right side of the operator decreased substantially the affinity of the repressor for the operator. In gel shift assays, the 75 gp did not bind DNA from the genetic switch region. However, when increasing amounts of orf 75 gp containing cell extracts were added to orf 127 gp containing cell extracts, the repressor could no longer bind its operator site
Differential Receptor Usage of Small Ruminant Lentiviruses in Ovine and Caprine Cells: Host Range but not Cytopathic Phenotype Is Determined by Receptor Usage

AbstractThe ovine maedi-visna (MVV) and caprine arthritis-encephalitis (CAEV) small ruminant lentiviruses (SRLV) exhibit differential species tropism and cytopathic effects in vitro. Icelandic MVV-K1514 is a lytic SRLV which can infect cells from many species in addition to ruminants, whereas a lytic North American MVV strain (85/34) as well as nonlytic MVV strain S93 and CAEV can infect only ruminant cells. In the present study, we determined if differential receptor usage in sheep and goat cells is the basis of differential species tropism or cytopathic phenotype of SRLV. Infection interference assays in sheep and goat synovial membrane cells using pseudotyped CAEV vectors showed that North American MVV strains 85/34 and S93 and CAEV use a common receptor (SRLV receptor A), whereas MVV-K1514 uses a different receptor (SRLV receptor B). In addition, human 293T cells expressing CAEV but not MVV-K1514 envelope glycoproteins fused with a goat cell line persistently infected with MVV-K1514, indicating that MVV-K1514 does not use SRLV receptor A for cell-to-cell fusion. Therefore, our results indicate that the differential species tropism of SRLV is determined by receptor usage. However, receptor usage is unrelated to cytopathic phenotype
HIV-1 Vpr Displays Natural Protein-Transducing Properties: Implications for Viral Pathogenesis

AbstractThe 14-kDa Vpr protein of human immunodeficiency virus type 1 (HIV-1) serves multiple functions in the retroviral life cycle, including the enhancement of viral replication in nondividing macrophages, the induction of G2 cell-cycle arrest in proliferating T lymphocytes, and the modulation of HIV-1-induced apoptosis. Extracellular Vpr has been detected in the sera and cerebral spinal fluid of HIV-infected patients. However, it is not known whether such forms of Vpr are biologically active. Vpr contains a carboxy-terminal basic amino acid rich segment stretch that is homologous to domains that mediate the energy- and receptor-independent cellular uptake of polypeptides by a process termed protein transduction. Similar functional protein-transducing domains are present in HIV-1 Tat, herpes simplex virus-1 DNA-binding protein VP22, and the Drosophila antennapedia homeotic transcription factor. We now demonstrate effective transduction of biologically active, synthetic Vpr (sVpr) as well as the Vpr-β-galactosidase fusion protein. However, in contrast to other transducing proteins, Vpr transduction is not enhanced by protein denaturation, and Vpr's carboxy-terminal basic domain alone is not sufficient for its transduction across biological membranes. In contrast, the full-length Vpr protein effectively transduces a broad array of cells, leading to dose-dependent G2 cell-cycle arrest and apoptosis. Addition of Vpr into the extracellular medium also rescues the replication of Vpr-deficient strains of HIV-1 in human macrophage cultures. Native Vpr may thus be optimized for protein transduction, a feature that might enhance and extend the pathological effects of HIV infection
Generation of a Consensus Sequence from Prevalent and Incident HIV-1 Infections in West Africa to Guide AIDS Vaccine Development

AbstractWe considered several key issues regarding the development of a DNA-based human immunodeficiency virus type 1 (HIV-1) vaccine: (1) should the candidate vaccine construct be derived from incident or prevalent HIV-1 strains; and (2) should circulating plasma virus, archived HIV-1 provirus recovered from peripheral blood mononuclear cells, or both be included? To address these questions, we collected circulating HIV-1 strains from infected individuals residing in Abidjan, Côte d'Ivoire. From a panel of 23 strains, 22 were HIV-1 subtype A in gag, 19 of which phylogenetically clustered with the recombinant HIV-1, CRF02-AG strains from West Africa. The mosaic genome of CRF02-AG was confirmed by sequencing the protease gene. A consensus gag p24 protein sequence was generated and 147 of 148 codons were identical to CRF02-AG (IbNG). Regardless of the sequence origin (RNA, provirus, incident, or prevalent), the gag p24 consensus sequences were highly representative of these distinct virologic compartments. These data suggest that the consensus sequence generated from incident and prevalent infections may provide an appropriate sequence for a DNA vaccine and is largely representative of the major circulating viral strain
The Primary Nucleotide Sequence of the Bovine Leukemia Virus RNA Packaging Signal Can Influence Efficient RNA Packaging and Virus Replication

AbstractTwo RNA stem-loop structures in the gag gene have been implicated as representing the primary encapsidation (packaging) signal for bovine leukemia virus (BLV), a member of the Deltaretrovirus of the Retroviridae. In this study, we conducted an analysis of these RNA structures, stem loop 1 (SL1) and stem loop 2 (SL2), to determine if both the loop and the stem nucleotide bases are important for RNA encapsidation. We have found that the primary sequence of the unpaired bases located in the loop regions of both SL1 and SL2 are important for efficient RNA encapsidation and virus replication. The primary sequence of the bases that form the stems for both SL1 and SL2 was observed to aid in efficient encapsidation and replication. We also observed that the order of SL1 and SL2 is important for RNA encapsidation and virus replication efficiency. A viral RNA with two copies of either SL1 or SL2 was found to replicate and package RNA as efficiently as a viral RNA with only one copy of SL1 or SL2. This provides evidence that SL1 and SL2 are not functionally equivalent. Sequences from human T cell leukemia virus type 1 (HTLV-1) that are located in the same region of HTLV-1 as the SL1 and SL2 of BLV were used to replace the BLV SL1, SL2, or both in a BLV RNA. These BLV RNAs were still encapsidated and replicated, suggesting that these sequences may function as an encapsidation signal in HTLV-1. The chimeric RNAs did not replicate as well as the parental, indicating that the primary nucleotide sequence along with the secondary and tertiary structure of the RNA plays a role in efficient RNA encapsidation and replication
Rescue of AAV by Antibody-induced Fas-mediated Apoptosis from Viral DNA Integrated in HeLa Chromosome

AbstractAdenoassociated virus (AAV) provirus in latently infected cells is rescued by superinfection with adenovirus. We examined helper-independent rescue of AAV by Fas-mediated apoptosis from the HeLa lines with AAV DNA integrated in chromosome (HeLa/AAV). In HeLa/AAV, anti-Fas antibody was found to induce low-level production of AAV virions, as detected by the presence of AAV DNA protected from DNase digestion. The antibody induced higher virion production in Fas-enriched HeLa/AAV, which was generated by transfecting HeLa/AAV with an expression plasmid for Fas, than in HeLa/AAV, and the rescued virions were shown to be infectious as assayed along with adenovirus. The antibody also induced apoptotic DNA fragmentation, as detected by staining intracellular fragmented DNA and electrophoresis, in HeLa/AAV and, more markedly, in Fas-enriched HeLa/AAV. Furthermore, an inhibitor for caspase-8 suppressed both the AAV virion production and the DNA fragmentation. Thus, the integrated AAV DNA is likely to be induced to initiate a low level of replication when Fas-mediated apoptosis is activated in HeLa cells
Construction and Characterization of E1-Minus Replication-Defective Adenovirus Vectors that Express E3 Proteins from the E1 Region

AbstractPrevious research has indicated that the adenovirus protein complex named RID, derived from the E3 transcription unit, functions to remove the receptors named Fas/Apo1/CD95 (Fas) and epidermal growth factor receptor (EGFR) from the surface of cells. (The RID complex is composed of the RIDα and RIDβ polypeptides, previously named 10.4K and 14.5K, respectively.) In response to RID, Fas and EGFR appear to be internalized into endosomes and degraded in lysosomes. Fas is a death receptor in the tumor necrosis factor (TNF) receptor superfamily. RID inhibits apoptosis via the Fas pathway, presumably because RID gets rid of Fas. Earlier work further showed that another adenovirus E3-coded protein, E3-14.7K, inhibits apoptosis induced by TNF. Most of the above studies have been conducted using viable virus mutants that lack one or more of the genes for RID, E3-14.7K, or E1B-19K (this protein, coded by the E1B transcription unit, also inhibits apoptosis via the TNF and Fas pathways). Some studies have also been conducted with the genes for RID or E3-14.7K transiently or stably transfected into cells. We now report a new approach to studying the E3 genes. We have constructed four E1-minus replication-defective vectors that have all the E3 genes deleted from their natural position and then reinserted, in different permutations, into the deleted E1 region under control of the cytomegalovirus immediate early promoter. Vector Ad/RID only has the genes for RIDα and RIDβ. Vector Ad/14.7K only has the gene for E3-14.7K. Vector Ad/RID/14.7K only has the genes for RIDα, RIDβ, and E3-14.7K. Vector Ad/E3 has all E3 genes, but there are two missense mutations in the gene for Adenovirus Death Protein. These vectors expressed RID and/or E3-14.7K, as expected. The RID-expressing vectors forced the internalization and degradation of Fas and EGFR, and they inhibited apoptosis induced through the Fas pathway. These vectors should be useful reagents to study the E3 proteins
Activation of Cellular and Heterologous Promoters by the Human Herpesvirus 8 Replication and Transcription Activator

AbstractThe key regulator of the switch from latent to lytic replication of the human herpesvirus 8 (HHV-8; KSHV) is the replication and transcription activator (Rta). The ability of Rta to regulate cellular gene expression was examined by transient transfection into cells that were not infected with HHV-8. Rta induced some, but not all, NF-κB-responsive reporters through mechanisms that did not involve activation of classic forms of NF-κB. Furthermore, transfection of the NF-κB subunit Rel A inhibited the ability of Rta to transactivate some but not all reporters. For example, Rel A inhibited the ability of Rta to transactivate the IL-6 promoter, but only when sequences upstream of the NF-κB site were present. The ability of Rel A to inhibit Rta-mediated transactivation was not dependent on a functional NF-κB site within the promoter, suggesting an indirect mechanism for inhibition. These studies suggest that Rta expression during lytic reactivation of HHV-8 would lead to expression of some cellular genes, including IL-6, whereas activation of NF-κB could inhibit some responses to Rta
Codon Modified Human Papillomavirus Type 16 E7 DNA Vaccine Enhances Cytotoxic T-Lymphocyte Induction and Anti-tumour Activity

AbstractPolynucleotide immunisation with the E7 gene of human papillomavirus (HPV) type 16 induces only moderate levels of immune response, which may in part be due to limitation in E7 gene expression influenced by biased HPV codon usage. Here we compare for expression and immunogenicity polynucleotide expression plasmids encoding wild-type (pWE7) or synthetic codon optimised (pHE7) HPV16 E7 DNA. Cos-1 cells transfected with pHE7 expressed higher levels of E7 protein than similar cells transfected with pW7. C57BL/6 mice and F1 (C57× FVB) E7 transgenic mice immunised intradermally with E7 plasmids produced high levels of anti-E7 antibody. pHE7 induced a significantly stronger E7-specific cytotoxic T-lymphocyte response than pWE7 and 100% tumour protection in C57BL/6 mice, but neither vaccine induced CTL in partially E7 tolerant K14E7 transgenic mice. The data indicate that immunogenicity of an E7 polynucleotide vaccine can be enhanced by codon modification. However, this may be insufficient for priming E7 responses in animals with split tolerance to E7 as a consequence of expression of E7 in somatic cells
Identification and Characterization of a Second Novel Human Erythrovirus Variant, A6

AbstractParvovirus B19 (B19), currently the only accepted member of the Erythrovirus genus, is the only parvovirus known to be pathogenic in humans. Recently a viral sequence, tentatively termed V9 which showed 11% variability from the published B19 sequences, was described from a patient with aplastic crisis. To search for additional parvovirus variants, we used the new NS1/7.5EC PCR assay whose primers were designed from a conserved region of the B19/V9 sequence and encompasses an MfeI restriction enzyme site that would allow differentiation between B19- and V9-like sequences. Screening of 225 serum and bone marrow samples and 62 plasma pools identified one new atypical parvovirus sequence, A6, from an anemic HIV-positive patient. A6 exhibited 88% similarity to B19 and 92% to V9, compared to >98% correspondence between reported B19 isolates. Based on the genome similarity to B19, an RT-PCR for A6 capsid transcripts was developed and used to test for A6 infectivity of UT7/Epo/S1 cells. Despite high viral titers, A6 viral transcripts were not detected. Thus, although the prevalence of B19 variants probably is low, the true clinical significance remains unknown. Current PCR analyses are unlikely to detect novel variants without the design of specific primers to the A6/V9/B19 common sequences
Caspase-Mediated Cleavage of Adenovirus Early Region 1A Proteins

AbstractAdenovirus 2 and 12 early region 1A (Ad2 and Ad12 E1A) proteins were cleaved during cisplatin-induced apoptosis of Ad-transformed rat and human cells. Cleavage was inhibited in the presence of caspase inhibitors such as Z-VAD-FMK. In Ad12 transformants both 13S and 12S E1A proteins were cleaved at a similar rate. In Ad2 transformants the E1A 13S component was appreciably less stable than the 12S component. In in vitro studies Ad2 and Ad12 E1A 13S and Ad2 12S proteins were rapidly cleaved by caspase 3 whereas Ad12 12S E1A and Ad12 13S E1A were rapidly degraded by caspase 7. Cleavage sites in Ad12 13S proteins for caspase 3 have been determined. Initial cleavage occurred at D24 and D150; this was followed by cleavage at D204 and D242. Caspase-3-mediated cleavage of Ad12 13S E1A destroyed its ability to bind to CBP and TBP but interaction between C terminal E1A polypeptides and CtBP was observed. During viral infection Ad5 and Ad12 E1A 12S proteins were markedly more stable than 13S proteins but no difference was observed in Ad E1A levels in the absence or presence of the caspase inhibitors Z-VAD-FMK or Z-D(OMe)-E(OMe)-V-D(OMe)-CH2F. Limited caspase 3 and 10 activation occurred during infection with the E1B 19K− virus Ad2 pm1722 but little or no activation of caspase 3 was observed during wt virus infection. Examination of protein cleavage during viral infection of A549 cells showed proteolysis of lamin B and PARP in response to Ad5 wt and Ad2 pm1722. Protein degradation in response to both viruses was partially inhibited by Z-VAD-FMK. Following infection of human skin fibroblasts lamin B was degraded, although only limited changes in PARP levels were observed. We have concluded that Ad E1A is cleaved by caspases during apoptosis but not during viral infection. However, some of the processes commonly associated with apoptosis occur during viral infection, particularly with E1B 19K− mutants, although apoptosis per se is not evident
Bidirectional Infection and Release of Rift Valley Fever Virus in Polarized Epithelial Cells

AbstractRift Valley Fever (RVF) virus is an arbovirus and is responsible for large outbreaks of disease predominantly in sub-Saharan Africa. However, several aspects of RVF virus transmission, such as high viremia, multiple vector species, and broad host range, result in a pathogen with high likelihood of geographic spread. RVF virus infection in humans and livestock is characterized by broad dissemination of RVF virus antigens throughout the body. We sought insight into the high pathogenicity and broad tropism of this virus through a characterization of its interaction with polarized epithelial cells. Our results indicate that infection and release of RVF virus in polarized epithelial cells occurs at both apical and basolateral membranes and hence is bidirectional. Furthermore, our results indicate that RVF virus causes disruptions in both the microfilament and the microtubule networks. These disruptions may provide a mechanism for bidirectional release of RVF virions
Chimeric Human Papilloma Virus–Simian/Human Immunodeficiency Virus Virus-like-Particle Vaccines: Immunogenicity and Protective Efficacy in Macaques

AbstractVaccines to efficiently block or limit sexual transmission of both HIV and human papilloma virus (HPV) are urgently needed. Chimeric virus-like-particle (VLP) vaccines consisting of both multimerized HPV L1 proteins and fragments of SIV gag p27, HIV-1 tat, and HIV-1 rev proteins (HPV–SHIV VLPs) were constructed and administered to macaques both systemically and mucosally. An additional group of macaques first received a priming vaccination with DNA vaccines expressing the same SIV and HIV-1 antigens prior to chimeric HPV–SHIV VLP boosting vaccinations. Although HPV L1 antibodies were induced in all immunized macaques, weak antibody or T cell responses to the chimeric SHIV antigens were detected only in animals receiving the DNA prime/HPV–SHIV VLP boost vaccine regimen. Significant but partial protection from a virulent mucosal SHIV challenge was also detected only in the prime/boosted macaques and not in animals receiving the HPV–SHIV VLP vaccines alone, with three of five prime/boosted animals retaining some CD4+ T cells following challenge. Thus, although some immunogenicity and partial protection was observed in non-human primates receiving both DNA and chimeric HPV–SHIV VLP vaccines, significant improvements in vaccine design are required before we can confidently proceed with this approach to clinical trials
Deficient Dimerization of Human Immunodeficiency Virus Type 1 RNA Caused by Mutations of the U5 RNA Sequences

AbstractThe human immunodeficiency virus type 1 (HIV-1) virion contains two copies of genomic RNA that are noncovalently attached along a region at their 5′ ends, in which two contact sites have been observed by electron microscopy. One of these sites is believed to be the stem-loop 1 (SL1) sequence which serves as the dimerization initiation site (DIS), and the other site, closer to the 5′ end of the viral RNA, may involve the R or U5 RNA sequences. In this study, we present biochemical evidence showing that alteration of the U5 RNA sequence in the context of full-length viral RNA leads to diminished dimerization of virion RNA. In particular, two stretches of GU-rich sequences, which are located at nucleotides (nt) 99 to 108 and nt 112 to 123 within U5, were either deleted or substituted with exogenous sequences. The mutated viruses thus generated all exhibited deficient RNA dimerization. This dimerization deficit was not corrected by second-site mutations that preserved local RNA structures, such as the poly(A) hairpin, and was overcome to only a limited extent by compensatory mutations within Gag; these mutations were identified after long-term culture of the relevant mutant viruses in permissive cell lines and were able to restore viral infectiousness and RNA packaging to wild-type levels. Therefore, these GU sequences do not regulate RNA dimerization by the formation of local secondary structures nor by the maintenance of efficient viral RNA packaging; instead, they may mediate direct RNA–RNA interactions in the dimer structure. In contrast, mutation of palindrome 5′-AAGCUU-3′, which resides within R and crowns the poly(A) hairpin, did not affect either RNA dimerization or RNA packaging
The Mechanism of Inhibition of HIV-1 Env-Mediated Cell–Cell Fusion by Recombinant Cores of gp41 Ectodomain

AbstractN36(L6)C34 is a recombinant protein that forms a six-helix bundle with high thermal stability. It consists of the N-terminal heptad-repeat region (N36 peptide) and the C-terminal heptad-repeat region (C34) of HIV-1 gp41, connected by six polar amino acids. The protein inhibits HIV-1 envelope-induced membrane fusion. Whether inhibition occurs while N36(L6)C34 is in its six-helix bundle configuration was investigated. Mutating a critical residue within the N36 region to promote dissociation of C34 from the grooves of the N36 coiled coil reduced bundle stability and increased the inhibition of fusion. In contrast, mutating a key residue within the C34 region to reduce bundle stability decreased inhibitory potency. The data provide strong evidence that the proteins inhibit fusion while they expose their C34 segments, rather than as six-helix bundles. Thus, despite high thermal stability of the bundle, the recombinants' less folded structures are present in sufficient concentration to inhibit fusion at physiological temperatures
Regulation of Fusion Activity by the Cytoplasmic Domain of a Paramyxovirus F Protein

AbstractSER virus is a member of the family Paramyxoviridae, genus Rubulavirus, which has been isolated from pigs. It is very closely related to SV5 virus serologically, in protein profile, and in nucleotide sequence. However, unlike SV5, SER induces minimal syncytium formation in infected CV-1 or BHK cells. Fluorescence transfer experiments between labeled erythrocytes and infected MDBK cells revealed that SER also induces hemifusion and pore formation with reduced efficiency. The virion polypeptide profiles of SER and SV5 are very similar, except that the SER F1 subunit shows an apparent molecular weight that is about 2 kDa higher than that of SV5. Comparison of the deduced amino acid sequences revealed the SER F (551 aa) to be longer than SV5 F (529 aa) by 22 residues in the cytoplasmic tail (CT) domain. The HN and M gene sequences of the viruses were found to be very similar. The SER F showed minimal fusion activity when coexpressed with either SV5 or SER HN. In contrast, SV5 F was highly fusogenic when coexpressed with either HN protein, indicating that the restricted fusion capacity of SER virus is a property of its F protein. Truncation in the CT of SER F by 22 residues completely rescued its ability to cause syncytium formation, whereas other truncations rescued syncytium formation partially. These results demonstrate that an elongated CT of a paramyxovirus F protein suppresses its membrane fusion activity