pinecone/core-2020-05-10-deduplication huggingface dataset:
Recombinant Hepatitis Delta Antigen from E. coli Promotes Hepatitis Delta Virus RNA Replication Only from the Genomic Strand but Not the Antigenomic Strand

AbstractHepatitis delta antigen (HDAg) of hepatitis delta virus (HDV) typically consists of two related protein species. The small HDAg (S-HDAg) is a 24-kDa protein of 195 amino acids and the large HDAg (L-HDAg) is a 27-kDa protein with an additional 19 amino acids at its C-terminus. These two proteins have distinct functions in the HDV life cycle. We have developed conditions for expressing S-HDAg and L-HDAg in E. coli as soluble proteins to facilitate large-scale purification. These proteins were purified to homogeneity and shown to be biologically active. Transfection of the purified recombinant S-HDAg together with HDV genomic RNA resulted in viral RNA replication. Surprisingly, the purified S-HDAg could not initiate replication from the antigenomic-sense HDV RNA, even though the latter led to RNA replication when transfected with an mRNA encoding the S-HDAg. These results suggest that initiation of HDV RNA synthesis from the antigenomic RNA may require a form of HDAg that is modified in mammalian cells; in contrast, RNA synthesis from the genomic RNA could be initiated by the recombinant S-HDAg from E. coli. Interestingly, the purified L-HDAg appeared as multiple protein species, including one corresponding to S-HDAg, probably as a result of degradation. The partially proteolyzed L-HDAg also initiated HDV RNA replication under the same conditions. These results add to the mounting evidence that genomic- and antigenomic-strand HDV RNA syntheses are carried out by different mechanisms
Varied Tropism of HIV-1 Isolates Derived from Different Regions of Adult Brain Cortex Discriminate between Patients with and without AIDS Dementia Complex (ADC): Evidence for Neurotropic HIV Variants

AbstractA number of infected individuals develop neuropathological disorders, such as AIDS dementia complex (ADC), as a consequence of HIV/AIDS. The biological features governing HIV entry and tropism in different brain cell types remain unclear, as do the genetics of the virus regulating these events and the neuropathogenic processes within the brain tissues. HIV-1 was isolated from the right and left parietal, occipital, and frontal lobes of the brain cortex of three HIV-1-infected patients: two with ADC and one without. The viral strains were studied from the innate tissues and various primary cell cultures. The kinetics and tropism of viral strains from different brain regions showed clear differences on various primary cell types (monocytes, monocyte-derived macrophages, and T cells), which could discriminate between biological behavior of HIV-1 strains from patients with and without ADC. The variable effect of different donor cells on tropism was also clearly evident. The majority (with a few exceptions) of isolates from different brain regions of all three patients used CCR5 as coreceptor for entry. The consistent CCR5 use, macrophage tropism, and non-syncytium-inducing phenotype were the main characteristics of the brain-derived HIV-1 strains from all three patients. Importantly, viral strains derived directly from innate brain tissue of the patient without ADC showed some differences from the cultured variants of the same patient, whereas those from brain tissue of the patients with ADC were more similar to the culture-adapted strains. This suggests that the emergence of primary cell type-adapted isolates during ADC may play a crucial role in the development and progression of the neuropathology associated with ADC. The different genotypes residing in different areas of brain combined with their differential tropism and coreceptor use suggest that neurotropic variants exist that may be governing the neurological manifestation of HIV disease in infected patients
Molecular Characterization of Serotype G9 Rotavirus Strains from a Global Collection

AbstractBetween 1992 and 1998, serotype G9 human rotavirus (RV) strains have been detected in 10 countries, including Thailand, India, Brazil, Bangladesh, Malawi, Italy, France, the United States, the United Kingdom, and Australia, suggesting the possible emergence of the fifth common serotype worldwide. Unlike the previously characterized reference G9 strains (i.e., WI61 and F45), the recent G9 isolates had a variety of gene combinations, raising questions concerning their origin and evolution. To identify the progenitor strain and examine the on-going evolution of the recent G9 strains, we characterized by genetic and antigenic analyses 16 isolates obtained from children with diarrhea in India, Bangladesh, the United States, and Malawi. Specifically, we sequenced their VP7 and NSP4 genes and compared the nucleotide (nt) and deduced amino acid sequences with the reference G9 strains. To identify reassortment, we examined the products of five gene segments; VP4, VP7, and NSP4 genotypes (genes 4, 9, and 10); subgroups (gene 6); electropherotypes (gene 11); and the genogroup profiles of all of the recent G9 isolates. Sequence analysis of the VP7 gene indicated that the recent U.S. P[6],G9 strains were closely related to the Malawian G9 strains (>99% nt identity) but distinct from G9 strains of India (∼97% nt identity), Bangladesh (∼98% nt identity), and the reference strains (∼97% nt identity). Phylogenetic analysis identified a single cluster for the U.S. P[6],G9 strains that may have common progenitors with Malawian P[6],G9 strains whereas separate lineages were defined for the Indian, Bangladeshi, and reference G9 strains. Northern hybridization results indicated that all 11 gene segments of the Malawian P[6],G9 strains hybridized with a probe derived from a U.S. strain of the same genotype and may have the same progenitor, different from the Indian G9 strains, whereas the Bangladesh strains may have evolved from the U.S. G9 progenitors. Overall, our findings suggest that much greater diversity among the newly identified G9 strains has been generated by reassortment between gene segments than through the accumulation of mutations in a single gene
Alleviation of Murine Leukemia Virus Repression in Embryonic Carcinoma Cells by Genetically Engineered Primer Binding Sites and Artificial tRNA Primers

AbstractThe primer binding site (PBS) plays pivotal roles during reverse transcription of retroviruses and also is the target of a cellular host defense impeding the transcription of murine leukemia virus (MLV) harboring a proline (pro) PBS in embryonic cells. Both the PBS and the tRNA primer are copied during reverse transcription and anneal as complementary DNA sequences creating the PBS of the integrated provirus. The pro PBS of MLV can be exchanged by PBS sequences matching endogenous or engineered tRNAs to allow replication of Akv MLV-derived vectors in fibroblasts. Here we use the PBS escape mutant B2 to demonstrate the capacity of the synthetic tRNAB2 to function in reverse transcription in competition with endogenous tRNAs in fibroblasts and embryonic carcinoma (EC) cells. We further show symmetry between PBS and the primer by the ability of the synthetic tRNAB2 to confer escape from EC repression of a PBS-Pro vector. Of a panel of vectors with the repressed pro PBS substituted for other natural or artificial PBS sequences, all except one efficiently expressed the neo marker gene when transferred to NIH/3T3 and EC cells, hence avoiding PBS-mediated silencing in EC cells. A non-natural PBS matching an artificially designed tRNA molecule conferred no further relief from repression than that attained with the B2 escape mutant or the natural alternative PBSs. Interestingly, a vector harboring a PBS matching tRNALys1.2 suffered repression similar to the wild-type PBS-Pro but was partially rescued by a single point mutation of the PBS
In Vitro Induction of HIV-1 Replication in Resting CD4+ T Cells Derived from Individuals with Undetectable Plasma Viremia upon Stimulation with Human T-Cell Leukemia Virus Type I

AbstractMicrobial coinfections have been associated with transient bursts of human immunodeficiency virus (HIV) viremia in patients. In this study we investigated whether human T-cell leukemia virus type I (HTLV-I), another human retrovirus that is prevalent among certain HIV-infected populations, can induce HIV-1 replication in patients who had been successfully treated with highly active antiretroviral therapy. We demonstrate that supernatants from HTLV-I-producing MT-2 cells can induce in vitro replication of HIV-1 from highly purified, resting CD4+ T cells obtained from individuals with undetectable plasma viremia. Depletion of proinflammatory cytokines from the supernatants reduced, but did not abrogate, the ability to induce HIV-1 replication, indicating that other factors such as HTLV-I Tax or Env also have a role. The HTLV-I-mediated effect does not require productive infection: exposure to heat-inactivated HTLV-I virions, purified Tax protein, or HTLV-I Env glycoprotein also induced expression of HIV-1. Furthermore, we demonstrate that coculture of resting CD4+ T cells with autologous CD8+ T cells markedly inhibits the HTLV-I-induced virus replication. Our results suggest that coinfection with HTLV-I may induce viral replication in the latent viral reservoirs; however, CD8+ T cells may play an important role in controlling the spread of virus upon microbial stimulation
Variability of Human Immunodeficiency Virus Type 2 (HIV-2) Infecting Patients Living in France

AbstractTo determine the prevalence of human immunodeficiency virus type 2 (HIV-2) subtypes circulating in France and to identify possible relationships between these subtypes and pathogenesis, we studied 33 HIV-2-infected patients living in France. HIV-2 DNA was directly amplified from peripheral blood mononuclear cells by nested PCR with specific HIV-2 env primers, and the env gene was sequenced. The serological consequences of antigenic variability were studied by using a panel of peptides and by Western blotting. Phylogenetic analysis classified the 33 HIV-2 strains as subtype A (n = 23) or B (n = 10). There were no significant clinical or epidemiological differences between patients infected with either of these two subtypes. There was some evidence for geographical clustering. Subtype A strains from patients originating from the Cape Verde Islands and Guinea Bissau clustered together. The majority of patients infected with subtype B strains originated from the Ivory Coast or Mali. Strains from patients originating in Mali also clustered in subtype A but distinctly from the Cape Verde or Guinea Bissau strains. The subtype B strains showed greater diversity and included some highly divergent strains relative to those previously characterized. The V3 loop of HIV-2 subtypes A and B was found to be quite conserved in comparison with HIV-1. A strong HIV-2 subtype B serological cross-reactivity was found on HIV-1 env antigen by Western blot mostly in the gp41 transmembrane glycoprotein. This could partly explain the double HIV-1 and HIV-2 reactive profiles found in countries where HIV-2 subtype B is prevalent
In Vitro Concatemer Formation Catalyzed by Vaccinia Virus DNA Polymerase

AbstractDuring poxvirus infection, both viral genomes and transfected DNAs are converted into high-molecular-weight concatemers by the replicative machinery. However, aside from the fact that concatemer formation coincides with viral replication, the mechanism and protein(s) catalyzing the reaction are unknown. Here we show that vaccinia virus DNA polymerase can catalyze single-stranded annealing reactions in vitro, converting linear duplex substrates into linear or circular concatemers, in a manner directed by sequences located at the DNA ends. The reaction required ≥12 bp of shared sequence and was stimulated by vaccinia single-stranded DNA-binding protein (gpI3L). Varying the structures at the cleaved ends of the molecules had no effect on efficiency. These duplex-joining reactions are dependent on nucleolytic processing of the molecules by the 3′-to-5′ proofreading exonuclease, as judged by the fact that only a 5′-32P-end label is retained in the joint molecules and the reaction is inhibited by dNTPs. The resulting concatemers are joined only through noncovalent bonds, but can be processed into stable molecules in E. coli, if the homologies permit formation of circular molecules. This reaction provides a starting point for investigating the mechanism of viral concatemer formation and can be used to clone PCR-amplified DNA
Orthopoxvirus IL-18 Binding Proteins: Affinities and Antagonist Activities

AbstractThe affinities of purified recombinant human IL-18 binding protein (BP) and ectromelia and cowpox virus homologs for human and murine IL-18 were compared by plasmon resonance. The dissociation constants of human IL-18BP were similar for murine and human IL-18. By contrast, the dissociation constants of the viral proteins for murine IL-18 were 12- to 50-fold lower than that for human IL-18. The ectromelia and cowpox virus proteins were biologically active, as judged by their ability to inhibit induction of interferon-γ by murine and human IL-18. The relative affinities of the orthopoxvirus IL-18BPs are consistent with the rodent host range of the viruses
The Equine Herpesvirus 1 UL45 Homolog Encodes a Glycosylated Type II Transmembrane Protein and Is Involved in Virus Egress

AbstractExperiments to analyze the product of the equine herpesvirus type 1 (EHV-1) UL45 homolog were conducted. Using an antiserum generated against the carboxylterminal 114 amino acids of the EHV-1 UL45 protein, proteins of Mr 32,000, 40,000, and 43,000 were detected specifically in EHV-1-infected cells. Neither form of the protein was located in purified virions of EHV-1 wild-type strain RacL22 or the modified live vaccine strain RacH, but UL45 was demonstrated to be expressed as a late (γ−2) protein. Fractionation of infected cells and deglycosylation experiments demonstrated that the EHV-1 UL45 protein represents a type II membrane glycoprotein. Deletion of the UL45 gene in RacL22 and RacH (LΔ45 and HΔ45) showed that UL45 is nonessential for EHV-1 growth in vitro, but that deletion reduced the viruses' replication efficiency. A marked reduction of virus release was observed although no significant influence was noticed either on plaque size or on the syncytial phenotype of the EHV-1 strain RacH
1-Methylguanosine in Place of Y Base at Position 37 in Phenylalanine tRNA Is Responsible for Its Shiftiness in Retroviral Ribosomal Frameshifting

AbstractMany mammalian retroviruses express their protease and polymerase by ribosomal frameshifting. It was originally proposed that a specialized shifty tRNA promotes the frameshift event. We previously observed that phenylalanine tRNAPhe lacking the highly modified wybutoxosine (Y) base on the 3′ side of its anticodon stimulated frameshifting, demonstrating that this tRNA is shifty. We now report the shifty tRNAPhe contains 1-methylguanosine (m1G) in place of Y and that the m1G form from rabbit reticulocytes stimulates frameshifting more efficiently than its m1G-containing counterpart from mouse neuroblastoma cells. The latter tRNA contains unmodified C and G nucleosides at positions 32 and 34, respectively, while the former tRNA contains the analogous 2′-O-methylated nucleosides at these positions. The data suggest that not only does the loss of a highly modified base from the 3′ side of the anticodon render tRNAPhe shifty, but the modification status of the entire anticodon loop contributes to the degree of shiftiness. Possible biological consequences of these findings are discussed
The Transmembrane Domain of the Hepatitis C Virus E2 Glycoprotein Is Required for Correct Folding of the E1 Glycoprotein and Native Complex Formation

AbstractHepatitis C virus (HCV) encodes two glycoproteins, E1 and E2, that interact to form both native and aggregated complexes in tissue culture cells. In native complexes, E1 and E2 are associated by noncovalent interactions and such complexes are considered to constitute the authentic interactions between the proteins. By contrast, the proteins are linked by covalent, disulfide bonds in aggregated complexes. From studies with a mutant in which cysteine residues in E1 have been substituted with other amino acids, we show that E1 continues to associate with E2, although the migratory patterns of the proteins on gels are consistent with the formation of aggregated complexes. Therefore, such complexes can be stabilized by noncovalent as well as covalent interactions. To further examine the requirements for native complex formation, segments of foreign glycoproteins were linked to regions of E2. Our data provide direct evidence for the requirement of C-terminal sequences in E2 that contain the transmembrane domain to permit oxidation of E1 and assembly of a native complex. By contrast, native complexes and oxidized E1 are not found in the presence of chimeric proteins containing the E2 ectodomain. These data suggest that interaction of E1 with the E2 transmembrane domain is critical for native complex formation
Hepatitis C Virus Core Protein Inhibits Interleukin 12 and Nitric Oxide Production from Activated Macrophages

AbstractA characteristic feature of hepatitis C virus (HCV) infection is a high frequency of persistence and the progression to chronic liver diseases. Recent data suggest that prevalent T helper (Th) 2 immunity as well as weak HCV-specific T-cell response is associated with viral persistence. Here, we showed that the production of interleukin 12 (IL-12) and nitric oxide (NO) that is critical for the induction of Th1 and innate immunity, but not that of tumor necrosis factor α (TNF-α), was significantly suppressed in both HCV core-expressing macrophage cell lines and mouse peritoneal macrophages treated with recombinant core protein. In addition, IL-12 p40 promoter activity was repressed by the presence of HCV core in macrophages stimulated with lipopolysaccharride (LPS) following IFN-γ treatment, indicating that IL-12 production may be downregulated at the transcriptional level. We also found that proliferation of T cells and IFN-γ production in mixed lymphocyte reactions (MLR) with core-expressing cells were inhibited. Taken together, our results suggest that HCV core protein could play roles in suppressing the induction of Th1 immunity through inhibition of IL-12 and NO production
Sequential Immunization of Macaques with Two Differentially Attenuated Vaccines Induced Long-Term Virus-Specific Immune Responses and Conferred Protection against AIDS Caused by Heterologous Simian Human Immunodeficiency Virus (SHIV89.6P)

AbstractFour rhesus macaques were sequentially immunized with live vaccines ΔvpuΔnefSHIV-4 (vaccine-I) and Δvpu SHIVPPC (vaccine-II). The vaccine viruses did not replicate productively in the peripheral blood mononuclear cells (PBMCs) of the vaccinated animals. All four animals developed binding antibodies against both the vaccine-I and -II envelope glycoproteins but neutralizing antibodies only against vaccine-I. They developed vaccine virus-specific CTLs that also recognized homologous as well as heterologous pathogenic SHIVs. Thirty weeks after the last immunization, the vaccinated animals and three unvaccinated control animals were challenged iv with a highly virulent heterologous SHIV89.6P. As expected, the three unvaccinated control animals developed large numbers of infectious PBMCs, high plasma viremia, and precipitous loss of CD4+ T cells. Two controls did not develop any immune response and succumbed to AIDS in about 6 months. The third control animal developed neutralizing antibodies and had a more chronic disease course, but eventually succumbed to AIDS-related complications 81 weeks after inoculation. The four vaccinated animals became infected with challenge virus as indicated by the presence of challenge virus-specific DNA in the PBMCs and RNA in plasma. However, virus in these animals replicated approximately 200- to 60,000-fold less efficiently than in control animals and eventually, plasma viral RNA became undetectable in three of the four vaccinates. The animals maintained normal CD4+ T-cell levels throughout the observation period of 85 weeks after a transient drop at Week 3 postchallenge. They also maintained CTL responses throughout the observation period. These studies thus showed that the graded immunization schedule resulted in a safe and highly effective long-lasting immune response that was associated with protection against AIDS by highly pathogenic heterologous SHIV89.6P
Genetic and Biological Variation in Equine Infectious Anemia Virus Rev Correlates with Variable Stages of Clinical Disease in an Experimentally Infected Pony

AbstractGenetic and biological variation in the regulatory protein Rev of equine infectious anemia virus (EIAV) were examined throughout a clinically dynamic disease course of an experimentally infected pony. Following infection with the virulent EIAVWyo, the pony underwent a variable disease course, including an acute fever episode at 12 days postinfection (DPI), multiple recurrent fever episodes until 135 DPI, a prolonged subclinical period, and two late fever episodes. Viral RNA was isolated from the inoculum and sequential sera samples, and the rev exon 2/gp45 overlapping ORFs were amplified, cloned, and sequenced. Novel variants were found throughout infection, and genetic analyses indicated that both the Rev and gp45 ORFs were under selective pressure. The Rev variant predominant in the inoculum, R1, remained predominant during the early periods following infection (until 35 DPI); however, R1 was replaced by new predominant variants during the recurrent fever period (67–135 DPI). R1 reemerged as the predominant variant during the afebrile period, but a new predominant variant, R93, was associated with the late fever episodes. Rev variants predominant during recurrent febrile and late-febrile periods had significantly higher Rev-mediated nuclear export activity than the variants predominant during the acute and afebrile periods. Statistical correlation was found between Rev activity and different stages of clinical disease. Together, these results suggest that genetic and biological variation in rev may be a contributing factor in EIAV disease progression
Direct Measurement of CD4+ and CD8+ T-Cell Responses to CMV in HIV-1-Infected Subjects

AbstractData from murine models of chronic viral infection suggest that CD4+ T-cell responses to viral pathogens are important in sustaining the number and/or function of CD8+ cytotoxic T-cell (CTL) effectors. In this study, we used cytokine flow cytometry (CFC), staining with HLA-A*0201-peptide tetramers, and peptide stimulation with epitopic peptides to study functional CD4+ and CD8+ T-cell responses to cytomegalovirus (CMV) in human subjects coinfected with CMV and the human immunodeficiency virus, type 1 (HIV-1). We show that strong CD4+ and CD8+ T-cell responses to CMV antigens are sustained over time in HIV-1-infected individuals. Those who maintain a strong CD4+ T-cell response to CMV are also likely to maintain higher frequencies of CD8+ T cells capable of binding to HLA-A*0201-CMV pp65 (A2-pp65) tetramers as well as responses to pp65 peptide stimulation with effector cytokine production. These data support the hypothesis that declines in frequencies of CD4+ T-cell responses to CMV are associated with an inability to sustain high levels of CMV-specific CD8+ T-cell responses in HIV-1-infected subjects. These declines may precede the onset of CMV-associated end organ disease