pinecone/core-2020-05-10-deduplication huggingface dataset:
Construction and Biological Characterization of Infectious Molecular Clones of HIV-1 Subtypes B and E (CRF01_AE) Generated by the Polymerase Chain Reaction

AbstractWe previously described the use of extended polymerase chain reaction (PCR) to amplify contiguous 9.2-kilobase (kb) single-long terminal repeat (LTR) proviral sequences from HIV-1 genetic subtypes A through G. We now extend these findings by describing a novel vector system to recover infectious molecular clones from long PCR amplicons. Directional ligation of 9.2-kb proviral amplicons into a recovery vector reconstitutes missing LTR sequences, providing candidate molecular clones for infectivity screening. We show that a previously characterized infectious molecular clone of HIV-1 retains its biological properties upon recovery with this strategy. Three additional infectious molecular clones generated, from primary isolates of subtype B (HIV-1WR27) and circulating recombinant form 01_AE (subtype E) (HIV-1CM235) by subtype-specific LTR reconstitution, displayed biological properties reflecting their cognate parental isolates. This represents the first report of infectious molecular clones from circulating recombinant form 01_AE (subtype E)
In Vivo Retroviral Integration: Fidelity to Size of the Host DNA Duplication Might Be Reduced When Integration Occurs near Sequences Homologous to LTR Ends

AbstractIntegrated retroviral DNAs are flanked by a short duplication of target DNA whose size is virus specific and invariable. We have sequenced the junctions between an ALSV (Avian Leukemia and Sarcoma Viruses)-based vector and quail DNA from five individual proviruses. Three proviruses were flanked by the expected 6-bp duplication of host DNA, whereas the two others were flanked by a 5-bp duplication. Nucleotide sequencing of the native integration sites of these two proviruses showed that these integrations had occurred at the immediate vicinity of either a CA or a TG dinucleotide, revealing striking microhomologies between the integration sites and viral LTR ends. These results suggest that size duplication of the target DNA might be influenced by nucleotidic sequence at the site of integration
Inhibition of Murine AIDS (MAIDS) Development in C57BL/6J Mice by Tyrphostin AG-1387

AbstractWe previously showed that certain tyrphostin derivatives, known as protein tyrosine kinase inhibitors, also act as topoisomerase I-specific antagonists and inhibit Moloney murine leukemia virus replication in vitro in acutely and chronically infected cells. However, an accurate portrayal of retroviral-induced disease cannot rely exclusively on extrapolations from in vitro data. Therefore, experiments with animal models are essential for evaluating the efficacy of a specific drug in vivo. In this study, we examined the effect of tyrphostin AG-1387 on murine AIDS (MAIDS) development in C57BL/6J mice injected with the LP-BM5 virus mixture. A single dose of tyrphostin, administered together with or 24 h post virus inoculation, decreased the development of MAIDS symptoms as measured by spleen and lymph node weight, the T-cell response to concanavalin A (con A), and spleen architecture. Furthermore, weekly treatment with tyrphostins totally abolished MAIDS symptoms and prevented the viral infection of the spleen cells as measured by the absence of viral RNA and the restoration of T-cell function in these spleens. These results implicate that prolonged treatment with tyrphostins is needed for the prevention of MAIDS development in infected mice and suggest that it may be applied as a legitimate remedy for the treatment of retroviral-induced diseases
Persistent Elevated Expression of Cytokine Transcripts in Ganglia Latently Infected with Herpes Simplex Virus in the Absence of Ganglionic Replication or Reactivation

AbstractInfection of mouse trigeminal ganglia by herpes simplex virus induces cytokine expression that persists long after infectious virus or viral antigens become undetectable. To examine mechanisms underlying this phenomenon, we used a thymidine kinase mutant, dlsptk, which fails to replicate in ganglia and does not reactivate upon ganglionic explant. Using quantitative reverse transcriptase–polymerase chain reaction assays, we found that levels of interferon-γ and tumor necrosis factor-α transcripts in dlsptk-infected ganglia were lower than those in wild type-infected ganglia, but were significantly (eight- to 10-fold) higher than those in mock-infected ganglia from Day 3 to Day 100 postinfection. We also studied latency-associated transcript (LAT) negative mutants that exhibit increased expression of productive cycle transcripts in ganglia. Ganglia infected with these mutants contained levels of cytokine transcripts similar to those in wild type-infected ganglia; any increases in viral antigen expression mediated by the LAT deletion were not accompanied by increased cytokine expression. Thus, neither viral replication, the ability to reactivate, nor LAT expression in ganglia is required for persistent elevated cytokine expression. The results provide indirect evidence that low-level expression of viral productive cycle genes in neurons can provide signals that elicit cytokine expression
Insertion of Cellular NEDD8 Coding Sequences in a Pestivirus

AbstractFor the cytopathogenic (cp) bovine viral diarrhea virus (BVDV) strain CP 821, a duplication of the genomic region encoding part of NS2, NS3, NS4A, and part of NS4B together with a nonviral insertion was detected. Further analyses including molecular cloning and sequencing of the putative cellular recombination partner showed that the insertion in CP 821 originated from a bovine mRNA encoding the cellular protein NEDD8, which is 58% identical to ubiquitin. To our knowledge the genome of CP 821 represents the first viral RNA with a NEDD8 coding insertion. Remarkably, the insertion site differs from that described for insertions of ubiquitin. The NEDD8 sequence allows an additional cleavage of the viral polyprotein, whereby an NS3 with an unusual N-terminus is generated. Furthermore, the CP 821-specific genomic alterations were introduced into an infectious noncytopathogenic (noncp) BVDV cDNA clone. After transfection of bovine cells with the respective RNA, a cp virus was recovered. This showed that the NEDD8 coding insertion together with the duplicated viral sequences represents the genetic basis for cytopathogenicity of CP 821. In addition to the recovered cp virus, noncp BVDV rapidly evolved after transfection. This is the first time that a change from the cp to the noncp phenotype was demonstrated in the course of replication in tissue culture cells
Differential Expression of TGF-β, IL-2, and Other Cytokines in the CNS of Theiler's Murine Encephalomyelitis Virus-Infected Susceptible and Resistant Strains of Mice

AbstractIntracranial inoculation of susceptible SJL mice with Theiler's murine encephalomyelitis virus (TMEV) results in biphasic disease consisting of early acute disease, followed by late chronic demyelinating disease, associated with mononuclear infiltrates and demyelinating lesions. In contrast, resistant C57BL/6 (B6) mice develop only early acute disease. We employed cytokine-specific RT-PCR to determine the expression of cytokine transcripts in the CNS of TMEV-infected SJL and B6 mice. During early acute disease, we have found a strong proinflammatory (Th1) cytokine response in the CNS of both TMEV-infected SJL and B6 mice, demonstrated by the expression of transcripts for IFN-γ, IL-1, IL-6, IL-12p40, and TNF-α. At 8 days postinfection (p.i.), TGF-β1 and TNF-α transcripts were present at significantly higher levels (P < 0.01) in the CNS of SJL susceptible mice in comparison to those found in the CNS of B6 mice. Immunohistochemical staining revealed that TGF-β protein was expressed in leptomeningeal mononuclear inflammatory cell infiltrates in the brain of SJL mice but not in B6 mice, at 8 days p.i. TGF-β may be responsible for the failure of SJL mice to develop an effective anti-TMEV CTL response. During late chronic demyelinating disease, high levels of proinflammatory Th1 cytokines were found in the CNS of SJL mice, but not B6 mice. Significantly higher levels (P < 0.01) of anti-inflammatory cytokine transcripts (IL-4, IL-5, and IL-10 (Th2 cytokines) and TGF-β) were found in the spinal cord of TMEV-infected SJL mice with chronic demyelinating disease than in the spinal cord of B6 mice during the same time period (39 or 60 days p.i.). These anti-inflammatory cytokines may contribute to the downregulation of the proinflammatory response in SJL mice. High levels of IL-2 transcripts and protein appeared transiently in the spinal cord of TMEV-infected SJL mice before the onset of demyelinating disease and coincided with an influx of new T cells into the CNS and/or expansion of remaining T cells that have not been eliminated after viral clearance
A C-Terminal Deletion Mutant of Pokeweed Antiviral Protein Inhibits Programmed +1 Ribosomal Frameshifting and Ty1 Retrotransposition without Depurinating the Sarcin/Ricin Loop of rRNA

AbstractPokeweed antiviral protein (PAP) is a ribosome-inactivating protein characterized by its ability to depurinate the sarcin/ricin (S/R) loop of the large rRNA of prokaryotic and eukaryotic ribosomes. Here, a series of PAP mutants were used to examine the relationship between depurination of the S/R loop and inhibition of +1 programmed ribosomal frameshifting (PRF) and to define PAP sequences critical for inhibition of +1 PRF and Ty1 retrotransposition in the yeast Saccharomyces cerevisiae. Using three different classes of mutants we present evidence that strong binding of a C-terminal PAP mutant (PAPc) to ribosomes is sufficient to inhibit +1 PRF and Ty1 retrotransposition in the absence of S/R loop depurination. PAPc did not affect the totivirus ScV-L-A and HIV-1-directed −1 PRF efficiencies or the ability of cells to maintain the M1-dependent killer phenotype, demonstrating the specificity of the effect of PAPc on +1 PRF
Ubiquitination of HIV-1 and MuLV Gag

AbstractOur previous biochemical studies of HIV-1 and MuLV virions isolated and identified mature Gag products, HIV-1 p6Gag and MuLV p12Gag, that were conjugated to a single ubiquitin. To study the importance of the monoubiquitination of Gag, a series of lysine to arginine mutants were constructed that eliminated ubiquitination at one or both of the lysines in HIV-1NL4-3 p6Gag and both lysines in Moloney MuLV p12Gag. HPLC and immunoblot analysis of the HIV-1 mutants demonstrated that either of the lysines in p6Gag, K27 or K33, could be monoubiquitinated. However, infectivity assays showed that monoubiquitination of HIV-1 p6Gag or MuLV p12Gag is not required for viral replication in vitro. Pulse-chase radiolabeling of HIV-1-producing cells revealed that monoubiquitination of p6Gag does not affect the short-term release of virus from the cell, the maturation of Pr55Gag, or the sensitivity of these processes to proteasome inhibitors. Experiments with protease-deficient HIV-1 showed that Pr55Gag can be monoubiquitinated, suggesting that p6Gag is first modified as a domain within Gag. Examination of the proteins inside an HIV-1 mutant found that free ubiquitin was incorporated into the virions in the absence of the lysines in p6Gag, showing that the ubiquitin inside the virus is not initially brought in as a p6Gag conjugate. Although our results establish that monoubiquitination of p6Gag and p12Gag is not required for viral replication in vitro, this modification may be a by-product of interactions between Gag and cellular proteins during assembly and budding
Simian Foamy Virus Infections in a Baboon Breeding Colony

AbstractThe prevalence, transmission, and variation of simian foamy viruses (SFVs) in baboons was investigated. Over 95% of adult baboons in the breeding colony as well as recently imported adult animals had high titers of anti-SFV serum IgG. Maternal antibody was detectable in infants' serum up to 6 months of age. Approximately 30% of infants in breeding harems experienced SFV infections by 1 year of age. Shedding of SFV in oral secretions was common, with 13% of samples from normal adult animals and 35% from immunosuppressed animals containing infectious SFV. SFV was isolated from three baboon subspecies (olive, yellow, and chacma baboons) and sequences from both the pol and the LTR regions of the provirus were amplified by PCR and sequenced. Phylogenetic analysis indicated that all baboon isolates formed a single lineage distinct from SFVs of other African monkey species. Within the baboon SFV lineage, two distinct clades were apparent, which consisted of isolates from yellow and olive baboons and isolates from chacma baboons. Competition ELISAs indicated that, while SFV isolates of these two groups were very closely related, antigenic differences do exist between them. SFV isolates from a drill and a mandrill were distinct from baboon SFV isolates, both genetically and antigenically
Establishment of Latent HIV-1 Infection of Resting CD4+ T Lymphocytes Does Not Require Inactivation of Vpr

AbstractThe introduction of highly active antiretroviral therapy (HAART) for the treatment of HIV-1 infection has allowed dramatic reductions in plasma virus levels to below the limit of detection in many patients. However, latently infected CD4+ memory T lymphocytes persist as an important reservoir for the virus in the presence of this aggressive therapy and represent a major barrier to HIV-1 eradication with HAART. The mechanism through which the latent compartment is formed has not yet been established. It may involve actively proliferating CD4+ T-cell intermediates that are infected with HIV-1 and revert back to a resting state, carrying integrated provirus at some low frequency. The HIV-1 accessory protein Vpr, which mediates G2 cell cycle arrest in host cells, may interfere with the formation of the latently infected T cells by preventing them from exiting the cell cycle to return to a resting state. To investigate the role of the Vpr in the formation of latently infected memory T cells, we cloned and characterized vpr genes from viruses in the latent reservoir. Both sequence analysis and functional assays demonstrated that the vpr gene products of the viruses isolated from the latent pool did not differ significantly from those of a functional Vpr (NL4-3). These results indicate that the generation of resting G0 memory T lymphocytes that carry latent HIV-1 provirus occurs despite the G2 arrest function of the vpr gene product
Induction of Long-Term Protective Effects against Heterologous Challenge in SIVhu-Infected Macaques

AbstractA group of three rhesus macaques were inoculated with SIV isolated from a human (SIVhu) accidentally exposed and infected with SIVsm. Extensive sequence analyses of SIVhu obtained from the human and macaques following infection indicated the presence of truncated nef. Not only did nef fail to repair itself in vivo postinfection (p.i.), but instead, further mutations added additional stop codons with increasing time p.i. Infection of these animals was associated with minimal acute viral replication, followed by undetectable plasma viral loads and only intermittent PCR detection up to 5 years p.i. The three SIVhu infected and three control monkeys were then challenged with the heterologous highly pathogenic SHIV89.6p. All three controls became infected and showed rapid declines in peripheral CD4+ lymphocytes, disease, and death at 10 and 32 weeks p.i., respectively. In contrast, all three animals previously infected with SIVhu are healthy and exhibit stable CD4+ lymphocyte levels and undetectable plasma viral loads at >20 months post-SHIV89.6p challenge. Only transient, low levels of SHIV replication were noted in these animals. Whereas responses to SIVgag/pol were noted, no evidence for SIV/SHIV envelope cross-reactivity was detected by antibody or CTL analyses, suggesting that the protective immune mechanisms to the heterologous challenge isolate were most likely not directed to envelope but rather to other viral determinants
Co-Replication of a Reovirus and a Polydnavirus in the Ichneumonid Parasitoid Hyposoter exiguae

AbstractA recently established colony of the ichneumonid parasitoid, Hyposoter exiguae, was found to carry both a reovirus (HeRV) and a polydnavirus (HePDV). Morphogenesis of these viruses was observed in all cells comprising the ovarian calyx epithelium, apparently without detrimental effect to the parasitoid. While polydnavirus replication in H. exiguae was restricted to the calyx region, HeRV was detected in ovarioles, oviducts, midguts, malpighian tubules, and accessory glands associated with the male reproductive system. In addition, HeRV was able to infect the fat body of parasitized host larvae and to establish a persistent infection in vitro. Electron microscopy revealed that both viruses were released into the calyx fluid compartment exclusively by budding, a phenomenon rarely observed among the Reoviridae; HeRV envelopes thus obtained, however, appeared to be subsequently shed within the oviducts. HeRV particles were concentrated to near homogeneity by differential centrifugation; mature virions consisted of seven to eight structural polypeptides and 10 dsRNA genome segments. Prominent spikes were observed at the vertices of icosahedral core particles. Most, but not all, individuals comprising the H. exiguae colony appeared to be infected with HeRV, suggesting a commensal relationship between wasp and virus; however, while this association is of obvious benefit to the virus, it seems unlikely that any advantage accrues to the parasitoid which carries it
Predominance of Canine Parvovirus (CPV) in Unvaccinated Cat Populations and Emergence of New Antigenic Types of CPVs in Cats

AbstractSerological, sequence, and in vitro host range analyses of feline parvovirus (FPV) isolates in Vietnam and Taiwan revealed that more than 80% of the isolates were of the canine parvovirus (CPV) type, rather than feline panleukopenia virus (FPLV). Although parvovirus isolates from three Vietnamese leopard cats were genetically related to CPV type 2a or 2b, they had a natural mutation of VP2 residue 300 Gly to an Asp, resulting in remarkable changes in their antigenic properties. These results indicated the possibility that CPV-2a/2b-type viruses can spread in cats more efficiently than conventional FPLV under natural conditions and that CPV-2a/2b viruses are further evolving in cats
Involvement of Minor Structural Proteins in Recombination of Polyoma Virus DNA

AbstractWe have previously observed that a polyoma-mouse chimeric DNA molecule (RmI) in which the murine DNA insert is flanked by directly repeated viral sequences is effectively converted into unit-length polyoma DNA upon transfection of permissive mouse cells. This intramolecular recombination event appears to be dependent on VmP1, a protein encoded by RmI which includes the 328 N-terminal amino acids of polyoma VP1, and nine amino acids of murine origin carrying the C-terminus of the protein. We report here that introducing mutations into the VP2/VP3 coding sequence reduces the ability of RmI to generate polyoma DNA, even though the same mutations seem to exert little or no effect on the ability of polyoma DNA to either replicate or accumulate inside transfected cells. A mutation affecting VP2 alone being as effective as one that affects both VP2 and VP3, VP2 appears to be playing a critical role in recombination
Assembly and Release of Human Immunodeficiency Virus Type 1 Gag Proteins Containing Tandem Repeats of the Matrix Protein Coding Sequences in the Matrix Domain

AbstractWe have constructed human immunodeficiency virus (HIV) gag mutants by increasing the matrix protein (MA) sequences via tandemly repeated duplication of the central 107-MA codons. Instead of a total of 132 amino acid residues for the wild-type MA, the resultant mutants designated as MA2, MA3, and MA4 contained a total of 242, 352, and 462 codons in the MA domains, respectively. Analysis indicated that the addition of 110 or 220 amino acid residues to the MA did not significantly affect the assembly, release, and processing of particles; however, particle production was markedly reduced when another copy of 110 residues was added to the MA. Subcellular fractionation analysis suggested that the MA tandem repeat mutations enhanced the Gag membrane affinity, in a manner which correlated with the copy number of MA sequences. The effects of enhanced membrane affinity were substantially reduced when sequences downstream of the capsid (CA) domain were deleted. Sucrose density gradient fractionation analysis showed that particles produced by the large insertion mutants possessed wild-type (wt) HIV particle density. Truncation of sequences downstream of the nucleocapsid (NC) domains of the mutants did not influence the budding of particles. In contrast, particle budding was severely impaired when sequences downstream of the CA domain were truncated. Particle densities for the large Gag proteins, which were truncated at the C-terminus of CA, were about 1.12–1.14 g/ml lower than that for wt. Our results suggest that the HIV MA domain could adopt insertions of large protein sequences, and strongly support the proposal that the NC and p2 domains play a crucial role in the process of correct Gag protein packing
Vaccinia Virus E10R Protein Is Associated with the Membranes of Intracellular Mature Virions and has a Role in Morphogenesis

AbstractThis study provides the initial biochemical, microscopic, and genetic characterization of the product of the vaccinia virus E10R gene, which belongs to the ERV1/ALR family of eukaryotic proteins, is conserved in all poxviruses and has homologs in other cytoplasmic DNA viruses. DNA encoding a short epitope tag was appended to the C-terminus of the 95-amino-acid open-reading frame without affecting virus reproduction. The E10R protein was synthesized after DNA replication and was associated with purified intracellular mature virions (IMV), from which it could be extracted with a nonionic detergent. Antibody to the tag decorated the surface of IMV, consistent with the anchorage of the E10R protein to the membrane via its hydrophobic N-terminus. Immunoelectron microscopy revealed that the E10R protein was associated with crescent membranes, immature virions, IMV, and extracellular particles. To investigate the role of E10R in the virus life cycle, we constructed an inducer-dependent null mutant. In the absence of inducer, the formation of infectious virus was severely inhibited and electron microscopy revealed an assembly block with accumulation of crescent membranes and immature virions. Cysteines 43 and 46, comprising a putative redox motif common to all poxvirus E10R homologs, were essential for complementation of the mutant virus by transfected E10R DNA
Gene Transfer Systems Derived from Visna Virus: Analysis of Virus Production and Infectivity

AbstractEfficient transfer of therapeutic genes into nondividing human cells can be accomplished by inserting the genes into lentiviruses and infecting the cells with the modified viruses. The most developed lentivirus gene transfer systems are based on HIV-1, but because of the widespread HIV epidemic, the use of HIV-based vectors for gene therapy may be associated with a safety risk. In an attempt to find another lentivirus which can transduce human cells and might be safer than HIV-1, we generated gene transfer constructs based on the sheep lentivirus Visna. Molecular analysis of the constructs in a transient production system indicated that Visna produced as many mature virus particles as did HIV-1. Moreover, the virus particles incorporated a heterologous surface protein marker-gene-containing vector RNAs as efficiently as did HIV-1. However, the Visna virus transduced target cells poorly because of defects in reverse transcription and integration of the vector. Further modifications must be made to the Visna gene transfer system if the system is to be used in clinical gene therapy applications