pinecone/core-2020-05-10-deduplication huggingface dataset:
Paired DNA Three-Way Junctions as Scaffolds for Assembling Integrase Complexes

AbstractEarly steps of retroviral replication involve reverse transcription of the viral RNA genome and integration of the resulting cDNA copy into a chromosome of the host cell. The initial DNA breaking and joining steps of integration are carried out by the virus-encoded integrase enzyme. Integrases bind specifically to the ends of the unintegrated viral cDNA but nonspecifically to target DNA. Conventional assays in vitro reveal primarily the nonspecific DNA binding mode, complicating studies of integrase–DNA complexes. Here, we report an investigation of unconventional DNA structures useful for positioning integrase at predetermined sites. We find that paired DNA three-way junctions can be used to mimic branched DNAs normally formed as reaction intermediates. The three-way junctions differ from authentic intermediates in the connectivity of the DNAs, which, in contrast to the authentic intermediate, allow formation of stable DNA structures under physiological conditions. Assays in vitro showed that integrase can direct hydrolysis at sequences resembling the viral cDNA ends within the three-way junction, but not on junctions with mutant sequences. Changing the spacing between the paired three-way junctions disrupted the cleavage pattern, emphasizing the importance of the correct DNA scaffold. DNase I footprinting studies revealed protection of specific bases at the terminus of the LTR in the three-way junction complex, but not on control linear DNA, specifying the locations of tight interactions between integrase and DNA. Paired DNA three-way junctions are attractive reagents for structural studies of integrase–DNA complexes
In Vitro Analysis of an RNA Binding Site within the N-Terminal 30 Amino Acids of the Southern cowpea mosaic virus Coat Protein

AbstractSouthern cowpea mosaic virus (SCPMV) is a positive-sense RNA virus with T = 3 icosahedral symmetry. The coat protein (CP) has two domains, the random (R) domain and the shell (S) domain. The R domain is formed by the N-terminal 64 amino acids (aa) and is localized to the interior of the particle where it is expected to interact with the viral RNA. The R domain (aa 1–57) was expressed in Escherichia coli as a recombinant protein (rWTR) containing a nonviral C-terminal extension with two histidine tags. The RNA binding site of the R domain was identified by Northwestern blotting and electrophoretic mobility shift assay (EMSA) using recombinant wild-type and mutant R domain proteins. Deletions within the R domain revealed that the RNA binding site is localized to its N-terminal 30 aa. RNA binding by this element was found to be nonspecific with regard to RNA sequence and was sensitive to high salt concentrations, suggesting that electrostatic interactions are important for RNA binding by the R domain. The RNA binding site includes 11 basic residues, eight of which are located in the arginine-rich region between aa 22 and 30. It was demonstrated using alanine substitution mutants that the basic residues of the arginine-rich region but not those present at positions 3, 4, and 7 are necessary for RNA binding. None of the basic residues within the arginine-rich region are specifically required for RNA binding, but the overall charge of the N-terminal 30 aa is important. Proline substitution mutations within the N-terminal 30 aa, and alanine substitutions for prolines at positions 18, 20, and 21, did not affect the RNA binding activity of the R domain. However, it was demonstrated by circular dichroism (CD) that the conformation of the N-terminal 30 aa of the R domain changes from a random coil to an α-helix in the presence of 50% trifluoroethanol (TFE). The possible role for this structural change in RNA binding by the R domain is discussed
UDP-Galactose Transporter Is Required for Theiler's Virus Entry into Mammalian Cells

AbstractTheiler's murine encephalomyelitis viruses (TMEV) are divided into two groups: high-neurovirulence strains, such as GDVII, cause fatal encephalitis, while low-neurovirulence strains, such as BeAn and DA, cause persistent infection and demyelination in mice. Cell surface sialic acid is bound by the low-neurovirulence DA and BeAn viruses, but not by the high-neurovirulence GDVII virus. We have identified a clone from a BHK-21 cell cDNA library that mediates TMEV entry and infection by viruses of both TMEV groups in a receptor-negative BHK-21 cell variant (R26). The sequence of this clone is 96.4% identical to the human UDP-galactose transporter (UGT), which belongs to a family of nucleotide-sugar transporter proteins involved in the biosynthesis of complex carbohydrate structures in the trans-Golgi network. UGT mRNA from R26 cells was found to have a 490-nucleotide deletion involving the C-terminal amino acids 255 to 392 and 81 nucleotides of the 3′ noncoding region. These results suggest two possibilities by which UGT may mediate TMEV entry and infection. The most likely one relates to the transporter function of adding galactose to another receptor protein. This possibility suggests the requirement for a specific glycoprotein interaction for GDVII virus cell binding and entry, e.g., galactose for GDVII and sialic acid for BeAn. Alternatively, UGT might be a TMEV receptor itself, acting via UGT cycling to the cell surface
Umbravirus Gene Expression Helps Potato leafroll virus to Invade Mesophyll Tissues and to be Transmitted Mechanically between Plants

AbstractPotato leafroll virus (PLRV) was mechanically transmissible when inocula also contained the umbravirus Pea enation mosaic virus-2 (PEMV-2). In plants infected with PLRV and PEMV-2, PLRV accumulated in clusters of mesophyll cells in both inoculated and systemically infected leaves. No transmissions were obtained by coinoculation with Potato virus Y, Potato virus X (PVX), Tobacco mosaic virus, or Cucumber mosaic virus (CMV), although PLRV was transmissible from mixtures with CMV(ORF4) (a recombinant that contained the movement protein (MP) gene of the umbravirus Groundnut rosette virus (GRV) in place of the CMV MP gene). In contrast, neither a recombinant PVX that expressed GRV MP nor a mutant of CMV(ORF4), in which the CMV 2b gene was untranslatable, was able to help PLRV transmission. Possibly both a cell-to-cell movement function and counterdefense mechanisms such as those that block posttranscriptional gene silencing are involved in movement of PLRV within plants and its mechanical transmission between plants
Genetic Identification and Characterization of Limestone Canyon Virus, a Unique Peromyscus-Borne Hantavirus

AbstractHantaviruses, family Bunyaviridae, are rodent-borne RNA viruses that can cause hantavirus pulmonary syndrome (HPS) in various regions of the Americas. A coevolutionary relationship exists between hantaviruses and their specific rodent reservoir hosts; the phylogeny of the viruses generally matches that of the rodents. There are several Peromyscus-borne hantaviruses, including Sin Nombre virus, the most common cause of HPS in North America. This report describes the genetic detection and characterization of a newly discovered Peromyscus boylii-borne virus, Limestone Canyon (LSC) virus, the most divergent member of the Peromyscus-borne hantaviruses to date. Analysis of a 1209-nucleotide region of the S segment of LSC virus showed it to be more closely related to hantaviruses found in harvest mice (Reithrodontomys megalotis and R. mexicanus) than to other Peromyscus-associated hantaviruses (Sin Nombre, New York, and Monongahela). Phylogenetic analysis of virtually the entire M genome segment (3489 nucleotides) of LSC virus revealed a similar picture in which LSC virus was found to be very distinct from other Peromyscus-associated viruses, but its exact relationship to the other Peromyscus-borne and the Reithrodontomys-borne viruses was not resolved. These results indicate that hantavirus host species-jumping events can occur by which a hantavirus may switch to, and become established in, a rodent host belonging to a different genus. P. boylii are present throughout the southwestern United States and central Mexico. More extensive screening of HPS patients by using RT–PCR assays will be necessary to determine if LSC virus can cause human disease
Nef Is Required for Efficient HIV-1 Replication in Cocultures of Dendritic Cells and Lymphocytes

AbstractDendritic cells (DCs) are thought to play a crucial role in the pathogenesis of HIV-1 infection. DCs are believed to transport virus particles to lymph nodes before transfer to CD4+ lymphocytes. We have investigated the role of Nef in these processes. HIV-1 replication was examined in human immature DC-lymphocyte cocultures and in DCs or lymphocytes separately. Using various R5-tropic and X4-tropic HIV-1 strains and their nef-deleted (Δnef) counterparts, we show that Nef is required for optimal viral replication in immature DC-T cells clusters and in T lymphocytes. Nef exerts only a marginal role on viral replication in immature DCs alone as well as on virion capture by DCs, long-term intracellular accumulation and transmission of X4 strains to lymphocytes. We also show that wild-type and Δnef virions are similarly processed for MHC-I restricted exogenous presentation by DCs. Taken together, these results help explain how HIV-1 Nef may affect viral spread and immune responses in the infected host
Highly Sulfated Forms of Heparin Sulfate Are Involved in Japanese Encephalitis Virus Infection

AbstractJapanese encephalitis virus (JEV) infects a broad range of cell types in vitro, though little is known about the initial events of JEV infection. In the present study, we found that highly sulfated glycosaminoglycans (GAGs) are involved in infection of both neurovirulent (RP-9) and attenuated (RP-2ms) JEV strains. Competition experiments using highly sulfated GAGs, heparin and dextran sulfate, demonstrated an inhibition of JEV's attachment and subsequent infection of BHK-21 cells. Treatment of target cells by a potent sulfation inhibitor, sodium chlorate, greatly reduced viral binding ability as well as infection, suggesting a critical role of GAGs' sulfation status on the cellular surface in JEV infection. This phenomenon was confirmed by the manifestation of a distinct binding efficiency of JEV to the wild-type CHO cell line and its mutants with defects in GAG biosynthesis. We also demonstrated the binding of JEV particles and virus envelope glycoprotein to immobilized heparin beads. Furthermore, the addition of heparin suppressed the cytopathic effects induced by JEV infection in cultured cells. Our results establish that the highly sulfated form of GAGs on cell surfaces plays a determining role in the early stage of in vitro JEV infection
Characterization of the cis-Acting Elements Controlling Subgenomic mRNAs of Citrus tristeza virus: Production of Positive- and Negative-Stranded 3′-Terminal and Positive-Stranded 5′-Terminal RNAs

AbstractCitrus tristeza virus (CTV), a member of the Closteroviridae, has an ∼20-kb positive-sense RNA genome with two 5′ ORFs translated from the genomic RNA and 10 3′ genes expressed via nine or ten 3′-terminal subgenomic (sg) RNAs. The expression of the 3′ genes appears to have properties intermediate between the smaller viruses of the “alphavirus supergroup” and the larger viruses of the Coronaviridae. The sgRNAs are contiguous with the genome, without a common 5′ leader, and are associated with large amounts of complementary sgRNAs. Production of the different sgRNAs is regulated temporally and quantitatively, with the highly expressed genes having noncoding regions (NCR) 5′ of the ORFs. The cis-acting elements that control the highly expressed major coat protein (CP) gene and the intermediately expressed minor coat protein (CPm) gene were mapped and compared. Mutational analysis showed that the CP sgRNA controller element mapped within nts −47 to −5 upstream of the transcription start site, entirely within the NCR, while the CPm control region mapped within a 57 nt sequence within the upstream ORF. Although both regions were predicted to fold into two stem-loop structures, mutagenesis suggested that primary structure might be more important than the secondary structure. Because each controller element produced large amounts of 3′-terminal positive- and negative-stranded sgRNAs, we could not differentiate whether the cis-acting element functioned as a promoter or terminator, or both. Reversal of the control element unexpectedly produced large amounts of a negative-stranded sgRNA apparently by termination of negative-stranded genomic RNA synthesis. Further examination of controller elements in their native orientation showed normal production of abundant amounts of positive-stranded sgRNAs extending to near the 5′-terminus, corresponding to termination at each controller element. Thus, each controller element produced three sgRNAs, a 5′-terminal positive strand and both positive- and negative-stranded 3′-terminal RNAs. Therefore, theoretically CTV could produce 30–33 species of RNAs in infected cells
Molecular Characterizations of the Equine Herpesvirus 1 ETIF Promoter Region and Translation Initiation Site

AbstractThe equine herpesvirus 1 (EHV-1) homolog of the herpes simplex virus type 1 (HSV-1) tegument phosphoprotein, αTIF (Vmw65; VP16), was identified previously as the product of open reading frame 12 (ORF12), was shown to trans-activate immediate-early (IE) gene promoters, and was described as a 60-kDa virion component designated ETIF. However, the ETIF promoter region and transcription initiation site were not identified. The poly(A) signal of the gene 11 (UL49 homolog) lies just upstream of the first ETIF translation initiation codon, indicating that the first ATG may not be used for initiating ETIF translation. Another in-frame translation initiation codon (ATG2) is located 88 bp downstream of the first ETIF initiation codon (ATG1). Western blot analysis showed that the expressed ETIF protein migrated in SDS–PAGE with an apparent molecular mass of approximately 56 kDa, the same molecular weight identified in SDS–PAGE analysis of the KyD EHV-1 virion preparations. The ETIF expression vector pCETIF, which contains ATG2, trans-activated the IE promoter more efficiently than the pC12 containing both ATG1 and ATG2. S1 nuclease analyses mapped the 5′ initiation site of the 1.4-kb transcript approximately 17 to 21 nt downstream of the ATG1. The nucleotide sequence upstream of the ATG1 did not have any promoter activity, while the nucleotide sequence upstream of the ATG2 had promoter activity. In transient transfection assays, the pETIFM2 vector, which was mutated in the ATG2, did not trans-activate the IE promoter; however, the pETIFM1 vector, which was mutated in the ATG1, trans-activated the IE promoter. These results demonstrated that the ATG2 of the ETIF ORF is the ETIF translation initiation codon. ETIF trans-activated only the IE promoter, not early (EICP0, EICP22, EICP27, and thymidine kinase) or late (IR5) promoters, confirming that EICP0, EICP22, and EICP27 are early genes
Use of Chimeric Nectin-1(HveC)-Related Receptors to Demonstrate That Ability to Bind Alphaherpesvirus gD Is Not Necessarily Sufficient for Viral Entry

AbstractHuman nectin-1 (HveC, Prr1), a member of the immunoglobulin superfamily and a receptor for the entry of herpes simplex viruses 1 and 2 (HSV-1, HSV-2), pseudorabies virus (PRV), and bovine herpesvirus 1 (BHV-1), binds to viral gD. For HSV-1, HSV-2, and PRV, the gD-binding region of nectin-1 has been localized to the N-terminal V-like domain. To determine whether the two C-like domains of nectin-1 influenced gD binding and entry activity, genes encoding chimeric proteins were constructed. Portions of nectin-1 were replaced with homologous regions from nectin-2 (HveB, Prr2), a related protein with ability to mediate the entry of PRV, HSV-2, and Rid mutants of HSV-1, but not HSV-1 or BHV-1. Also, one or more domains of nectin-1 were fused to the two membrane-proximal Ig domains of CD4, a protein with no herpesvirus entry or gD-binding activity. The chimeric proteins were expressed in Chinese hamster ovary cells, which normally lack alphaherpesvirus entry receptors, and detected on the cell surface by one or more anti-nectin-1 monoclonal antibodies. One chimeric protein (nectin-1 amino acids 1–124 fused to CD4) failed to bind to soluble forms of HSV-1, HSV-2, PRV, and BHV-1 gD and, as expected, also failed to mediate entry of the viruses from which these gDs were derived. The other chimeric receptors bound all forms of gD. Some mediated the entry of all the viruses tested but others mediated entry of some but not all the viruses. We conclude that binding of gD to the nectin-1 V domain is not sufficient for entry activity, that there are structural requirements for entry activity independent of gD binding, and that these requirements are different for the several alphaherpesviruses that can use nectin-1 as a receptor
Cytoplasmic Localization Is Important for Transcription Factor Nuclear Factor-κB Activation by Hepatitis C Virus Core Protein through Its Amino Terminal Region

AbstractWe previously reported that hepatitis C virus core protein (core) activates the transcription factor nuclear factor-κB (NF-κB) when expressed transiently. In the present study, we investigated the relationship between the NF-κB activation capacity and subcellular localization of the core. By changing the subcellular localization of the C-terminally truncated core from the nucleus to the cytoplasm, NF-κB was activated. In addition, NF-κB activity was augmented by forcing the mutated core to move to the endoplasmic reticulum. It was also suggested that the region from aa 21 to 80 of the core is involved in the activation of NF-κB
B-Cell-Mediated Lysis of Cells Infected with the Neurotropic JHM Strain of Mouse Hepatitis Virus

AbstractCells expressing the spike (S) glycoprotein of the neurotropic JHM strain (JHMV) of mouse hepatitis virus (MHV) are susceptible to lysis by B cells derived from naı̈ve mice, including B cells from perforin-deficient mice. Cytolysis requires interaction of the virus receptor and the viral S glycoprotein, is independent of other viral-induced components, and is not a unique property of B cells. Neutralizing anti-S-protein monoclonal antibodies (mAb) and a mAb specific for the viral receptor inhibit lysis. However, cells infected with an MHV strain unable to induce cell–cell fusion are resistant to lysis and lysis of JHMV-infected cells is inhibited by an anti-S-protein nonneutralizing mAb which prevents S-protein-mediated cell fusion. These data suggest that B cells may function as antibody-independent innate immune response during JHMV infection in vivo
Viral Neuraminidase Treatment of Dendritic Cells Enhances Antigen-Specific CD8+ T Cell Proliferation, but Does Not Account for the CD4+ T Cell Independence of the CD8+ T Cell Response during Influenza Virus Infection

AbstractIn vitro studies demonstrate that the increased alloreactive T cell response to dendritic cells (DC) that are treated with either live or inactivated influenza virus A/PR/8/34 is due to viral neuraminidase (NA) activity. Since virus-specific cytotoxic T lymphocytes (CTL) play an important role in immunity to heterologous influenza strains, we compared the activation of CD8+ T cells by untreated and NA-treated DC. Increased CTL activity was induced by NA-treated DC both in vitro and in vivo. Since the generation of CTL in response to influenza virus infection does not require prior “activation” of DC by CD4+ T cells (as is the case for many antigens), we asked whether NA activity contributed to this unconditional CD8+ T cell response. This was not the case. Future studies will determine the factors that are responsible for the CD4+ T-cell-independent influenza virus-specific CTL response
HIV-1 NL4-3, but Not IIIB, Inhibits JAK3/STAT5 Activation in CD4+ T Cells

AbstractHIV-1 infection leads to T cell dysfunction and apoptosis in vivo and in vitro. The shared common γ chain of IL-2R and its associated Janus kinase, JAK3, are indispensable for normal T cell function and survival. We have reported that CD4 ligation with HIV gp120 inhibits T cell receptor-induced activation and expression of JAK3. We have also shown that while some strains of HIV-1, such as NL4-3, induce apoptosis of infected CD4+ T cells, other strains, such as HIV-1 IIIB, do not. Interestingly, we show here that infection of CD4+ T cells with HIV-1 NL4-3, but not IIIB, inhibited activation and expression of JAK3. NL4-3-infected T cells were unable to upregulate JAK3 expression following stimulation through TCR/CD3. In addition, NL4-3, but not IIIB, inhibited tyrosine phosphorylation and expression of STAT5, a downstream target of JAK3. These data suggest a correlation between apoptosis of HIV-1-infected T cells and inhibition of the JAK3/STAT5 activation pathway
Reverse Genetics and Recombination in Φ8, a dsRNA Bacteriophage

AbstractBacteriophage Φ8 has a genome of three dsRNA segments. It is able to acquire plasmid transcripts of cDNA copies of the genomic segments as replacements of its resident chromosomes. It is also able to effect recombination between the plasmid transcripts and the resident chromosomes. Depending upon the extent of sequence identity between the plasmid transcript and the resident chromosome, the recombination can be homologous or heterologous. Homologous recombination has not previously been reported for viruses with double-stranded RNA genomes
The Porcine Endogenous Retrovirus Long Terminal Repeat Contains a Single Nucleotide Polymorphism That Confers Distinct Differences in Estrogen Receptor Binding Affinity between PERV A and PERV B/C Subtypes

AbstractPorcine endogenous retroviruses (PERV) have been shown to have zoonotic potential, both in vitro and in vivo. Once integrated into the host cell genome activation of the proviral genes is ultimately dependent upon transactivation of the long terminal repeat (LTR). Currently there is no direct evidence of host cell transcription factors interacting with PERV LTRs. Using comparative genomics we discovered a potentially functional single nucleotide polymorphism (SNP) within the U5 region downstream of the TATA box in the PERV LTR that distinguishes PERV A from PERV B and PERV C subtypes. We demonstrated that the SNP occurs within a potential hormone-responsive region where it has a profound effect, not only upon estrogen receptor binding but also upon the binding of other transcription factors at this site. These results suggest that differences in transcriptional regulation between PERV subtypes are subtle and, as for other retroviruses, transcription can be mediated by steroid hormone receptors
HIV Tat Activates c-Jun Amino-terminal Kinase through an Oxidant-Dependent Mechanism

AbstractThe HIV-1 accessory protein Tat has been found to exert profound effects on vascular cell behavior. Recently, Tat has been found to activate the c-Jun amino-terminal kinase (JNK1, SAPK) MAP kinase in lymphoid cells. We found that purified Tat rapidly activated JNK1 in human umbilical vein endothelial cells and ECV-304 cells, and coculture of ECV-304 cells with Tat-transfected HeLa cells resulted in persistent activation of JNK1. In addition, lower doses of Tat potentiated TNFα-induced JNK1 activation, although higher doses paradoxically diminished JNK1 activation by TNFα. Treatment of ECV-304 cells with Tat acutely increased intracellular oxidant levels, and Tat-induced oxidant activity was decreased by two structurally distinct NADPH oxidase inhibitors, diphenylene iodonium and apocynin. Both oxidase inhibitors and the thiol antioxidant N-acetyl cysteine decreased Tat-induced JNK1 activation in parallel with reduction in oxidant levels. Activation of JNK1 by Tat was also inhibited by cytochalasin B, suggesting that Tat signaling was dependent upon intact cytoskeletal function. Indeed, JNK1 activation by Tat was associated with actin microfilament rearrangement. We conclude that HIV Tat may cause acute and persistent activation of the JNK MAP kinase through activation of a specific oxidase