bluuebunny/crossref_metadata_2025_split huggingface dataset:
Importance of Basic Residues in the Nucleocapsid Sequence for Retrovirus Gag Assembly and Complementation Rescue
Authors: Bowzard J. Bradford , Bennett Robert P. , Krishna Neel K. , Ernst Sandra M. , Rein Alan , Wills John W. 
Published: December 2019

ABSTRACT
The Gag proteins of Rous sarcoma virus (RSV) and human immunodeficiency virus (HIV) contain small interaction (I) domains within their nucleocapsid (NC) sequences. These overlap the zinc finger motifs and function to provide the proper density to viral particles. There are two zinc fingers and at least two I domains within these Gag proteins. To more thoroughly characterize the important sequence features and properties of I domains, we analyzed Gag proteins that contain one or no zinc finger motifs. Chimeric proteins containing the amino-terminal half of RSV Gag and various portions of the carboxy terminus of murine leukemia virus (MLV) (containing one zinc finger) Gag had only one I domain, whereas similar chimeras with human foamy virus (HFV) (containing no zinc fingers) Gag had at least two. Mutational analysis of the MLV NC sequence and inspection of I domain sequences within the zinc-fingerless C terminus of HFV Gag suggested that clusters of basic residues, but not the zinc finger motif residues themselves, are required for the formation of particles of proper density. In support of this, a simple string of strongly basic residues was found to be able to substitute for the RSV I domains. We also explored the possibility that differences in I domains (e.g., their number) account for differences in the ability of Gag proteins to be rescued into particles when they are unable to bind to membranes. Previously published experiments have shown that such membrane-binding mutants of RSV and HIV (two I domains) can be rescued but that those of MLV (one I domain) cannot. Complementation rescue experiments with RSV-MLV chimeras now map this difference to the NC sequence of MLV. Importantly, the same RSV-MLV chimeras could be rescued by complementation when the block to budding was after, rather than before, transport to the membrane. These results suggest that MLV Gag molecules begin to interact at a much later time after synthesis than those of RSV and HIV.
Persistent Baculovirus Infection Results from Deletion of the Apoptotic Suppressor Gene p35
Authors: Lee Jin-Ching , Chen Hong-Hwa , Chao Yu-Chan 
Published: December 2019

ABSTRACTInfection with the wild-type baculovirusAutographa californicamultiple nuclear polyhedrosis virus (AcMNPV) results in complete death ofSpodoptera frugiperda(Sf) cells. However, infection of Sf cells with AcMNPV carrying a mutation or deletion of the apoptotic suppressor genep35allowed the cloning of surviving Sf cells that harbored persistent viral genomes. Persistent infection established with the virus withp35mutated or deleted was blocked by stable transfection ofp35in the host genome or by insertion of the inhibitor of apoptosis (iap) gene into the viral genome. These artificially established persistently virus-infected cells became resistant to subsequent viral challenge, and some of the cell lines carried large quantities of viral DNA capable of early gene expression. Continuous release of viral progenies was evident in some of the persistently virus-infected cells, and transfection ofp35further stimulated viral activation of the persistent cells, including the reactivation of viruses in those cell lines without original continuous virus release. These results have demonstrated the successful establishment of persistent baculovirus infections under laboratory conditions and that their establishment may provide a novel continuous, nonlytic baculovirus expression system in the future.
Persistence of Herpes Simplex Virus Type 1 DNA in Chronic Conjunctival and Eyelid Lesions of Mice
Authors: Maggs David J. , Chang Ed , Nasisse Mark P. , Mitchell William J. 
Published: December 2019

ABSTRACTHerpes simplex virus type 1 (HSV-1) causes chronic blepharitis and conjunctivitis as well as keratitis in humans. The pathogenesis of these inflammatory ocular and dermal lesions is not well understood. We have examined the persistence of HSV-1 DNA and its relationship to inflammatory lesions in the conjunctiva and eyelid skin of mice which were inoculated with HSV-1 by the corneal route. Viral DNA was detected by in situ PCR in the conjunctiva and eyelid tissue of infected mice at 5, 11, 23, and 37 days postinfection (p.i.). This DNA was localized in the epithelial cells of the conjunctiva and hair follicles and in the epidermal cells of the eyelid skin. Viral proteins were not detected in the conjunctiva or the eyelid skin after 5 days p.i., even though histopathological lesions were found at 23 and 37 days p.i. in both tissues. The DNA-containing cells were adjacent to sites of inflammation in the chronic lesions in both the conjunctiva and the eyelid skin. A similar temporal and spatial relationship between HSV-1 DNA and inflammatory lesions has been previously reported for the cornea. Our data suggest that the lesions in the cornea, conjunctiva, and eyelid skin progress similarly. Further studies are required to determine whether the long-term presence of HSV-1 is involved in the mechanism by which these chronic inflammatory lesions develop. The presence of HSV-1 DNA in these extraocular tissues for extended periods may constitute persistent viral infection of nonneuronal cells.
The 5′ and 3′ TAR Elements of Human Immunodeficiency Virus Exert Effects at Several Points in the Virus Life Cycle
Authors: Das Atze T. , Klaver Bep , Berkhout Ben 
Published: December 2019

ABSTRACTThe human immunodeficiency virus type 1 RNA genome contains a terminal repeat (R) sequence that encodes the TAR hairpin motif, which has been implicated in Tat-mediated activation of transcription. More recently, a variety of other functions have been proposed for this structured RNA element. To determine the replicative roles of the 5′ and 3′ TAR hairpins, we analyzed multiple steps in the life cycle of wild-type and mutant viruses. A structure-destabilizing mutation was introduced in either the 5′, the 3′, or both TAR motifs of the proviral genome. As expected, opening of the 5′ TAR hairpin caused a transcription defect. Because the level of protein expression was not similarly reduced, the translation of this mRNA was improved. No effect of the 3′ hairpin on transcription and translation was measured. Mutations of the 5′ and 3′ hairpin structures reduced the efficiency of RNA packaging to similar extents, and RNA packaging was further reduced in the 5′ and 3′ TAR double mutant. Upon infection of cells with these virions, a reduced amount of reverse transcription products was synthesized by the TAR mutant. However, no net reverse transcription defect was observed after correction for the reduced level of virion RNA. This result was confirmed in in vitro reverse transcription assays. These data indicate that the 5′ and 3′ TAR motifs play important roles in several steps of the replication cycle, but these structures have no significant effect on the mechanism of reverse transcription.
Subunit Rotavirus Vaccine Administered Parenterally to Rabbits Induces Active Protective Immunity
Authors: Ciarlet Max , Crawford Sue E. , Barone Christopher , Bertolotti-Ciarlet Andrea , Ramig Robert F. , Estes Mary K. , Conner Margaret E. 
Published: December 2019

ABSTRACTVirus-like particles (VLPs) are being evaluated as a candidate rotavirus vaccine. The immunogenicity and protective efficacy of different formulations of VLPs administered parenterally to rabbits were tested. Two doses of VLPs (2/6-, G3 2/6/7-, or P[2], G3 2/4/6/7-VLPs) or SA11 simian rotavirus in Freund’s adjuvants, QS-21 (saponin adjuvant), or aluminum phosphate (AlP) were administered. Serological and mucosal immune responses were evaluated in all vaccinated and control rabbits before and after oral challenge with 10350% infective doses of live P[14], G3 ALA lapine rotavirus. All VLP- and SA11-vaccinated rabbits developed high levels of rotavirus-specific serum and intestinal immunoglobulin G (IgG) antibodies but not intestinal IgA antibodies. SA11 and 2/4/6/7-VLPs afforded similar but much higher mean levels of protection than 2/6/7- or 2/6-VLPs in QS-21. The presence of neutralizing antibodies to VP4 correlated (P< 0.001,r= 0.55; Pearson’s correlation coefficient) with enhanced protection rates, suggesting that these antibodies are important for protection. Although the inclusion of VP4 resulted in higher mean protection levels, high levels of protection (87 to 100%) from infection were observed in individual rabbits immunized with 2/6/7- or 2/6-VLPs in Freund’s adjuvants. Therefore, neither VP7 nor VP4 was absolutely required to achieve protection from infection in the rabbit model when Freund’s adjuvant was used. Our results show that VLPs are immunogenic when administered parenterally to rabbits and that Freund’s adjuvant is a better adjuvant than QS-21. The use of the rabbit model may help further our understanding of the critical rotavirus proteins needed to induce active protection. VLPs are a promising candidate for a parenterally administered subunit rotavirus vaccine.
The Bipartite Geminivirus Coat Protein Aids BR1 Function in Viral Movement by Affecting the Accumulation of Viral Single-Stranded DNA
Authors: Qin Shenwei , Ward Brian M. , Lazarowitz Sondra G. 
Published: December 2019

ABSTRACTThe movement of bipartite geminiviruses such as squash leaf curl virus (SqLCV) requires the cooperative interaction of two essential virus-encoded movement proteins, BR1 and BL1. While the viral coat protein AR1 is not essential for systemic infection, genetic studies demonstrate that its presence masks the defective phenotype of certain BR1 missense mutants, thus suggesting that coat protein does interact with the viral movement pathway. To further examine the mechanism of this interaction, we have constructed alanine-scanning mutants of AR1 and studied them for the ability to mask the infectivity defects of appropriate BR1 mutants, for the ability to target to the nucleus and to bind viral single-stranded DNA (ssDNA) and multimerize, and for effects on the accumulation of replicated viral ssDNA. We identified a specific region of AR1 required for masking of appropriate BR1 mutants and showed that this same region of AR1 was also important for ssDNA binding and the accumulation of viral replicated ssDNA. This region of AR1 also overlapped that involved in multimerization of the coat protein. We also found that the accumulation in protoplasts of single-stranded forms of a recombinant plasmid that included the SqLCV replication origin but was too large to be encapsidated was dependent on the presence of AR1 but did not appear to require encapsidation. These findings extend our model for SqLCV movement, demonstrating that coat protein affects viral movement through its ability to induce the accumulation of replicated viral ssDNA genomes. They further suggested that encapsidation was not required for the AR1-dependent accumulation of viral ssDNA.
Chemokine Coreceptor Usage by Diverse Primary Isolates of Human Immunodeficiency Virus Type 1
Authors: Zhang Linqi , He Tian , Huang Yaoxing , Chen Zhiwei , Guo Young , Wu Sam , Kunstman Kevin J. , Brown R. Clark , Phair John P. , Neumann Avidan U. , Ho David D. , Wolinsky Steven M. 
Published: December 2019

ABSTRACT
We tested chemokine receptor subset usage by diverse, well-characterized primary viruses isolated from peripheral blood by monitoring viral replication with CCR1, CCR2b, CCR3, CCR5, and CXCR4 U87MG.CD4 transformed cell lines and STRL33/BONZO/TYMSTR and GPR15/BOB HOS.CD4 transformed cell lines. Primary viruses were isolated from 79 men with confirmed human immunodeficiency virus type 1 (HIV-1) infection from the Chicago component of the Multicenter AIDS Cohort Study at interval time points. Thirty-five additional well-characterized primary viruses representing HIV-1 group M subtypes A, B, C, D, and E and group O and three primary simian immunodeficiency virus (SIV) isolates were also used for these studies. The restricted use of the CCR5 chemokine receptor for viral entry was associated with infection by a virus having a non-syncytium-inducing phenotype and correlated with a reduced rate of disease progression and a prolonged disease-free interval. Conversely, broadening chemokine receptor usage from CCR5 to both CCR5 and CXCR4 was associated with infection by a virus having a syncytium-inducing phenotype and correlated with a faster rate of CD4 T-cell decline and progression of disease. We also observed a greater tendency for infection with a virus having a syncytium-inducing phenotype in men heterozygous for the defectiveCCR5 Δ32 allele (25%) than in those men homozygous for the wild-type CCR5 allele (6%) (P = 0.03). The propensity for infection with a virus having a syncytium-inducing phenotype provides a partial explanation for the rapid disease progression among some men heterozygous for the defectiveCCR5 Δ32 allele. Furthermore, we did not identify any primary viruses that used CCR3 as an entry cofactor, despite this CC chemokine receptor being expressed on the cell surface at a level commensurate with or higher than that observed for primary peripheral blood mononuclear cells. Whereas isolates of primary viruses of SIV also used STRL33/BONZO/TYMSTR and GPR15/BOB, no primary isolates of HIV-1 used these particular chemokine receptor-like orphan molecules as entry cofactors, suggesting a limited contribution of these other chemokine receptors to viral evolution. Thus, despite the number of chemokine receptors implicated in viral entry, CCR5 and CXCR4 are likely to be the physiologically relevant chemokine receptors used as entry cofactors in vivo by diverse strains of primary viruses isolated from blood.
Level of ICAM-1 Surface Expression on Virus Producer Cells Influences both the Amount of Virion-Bound Host ICAM-1 and Human Immunodeficiency Virus Type 1 Infectivity
Authors: Paquette Jean-Sébastien , Fortin Jean-François , Blanchard Luc , Tremblay Michel J. 
Published: December 2019

ABSTRACTUsing virions harvested from 293T cells stably expressing either low or high levels of surface ICAM-1, we determined that the number of virus-embedded host ICAM-1 proteins is positively influenced by the expression level of ICAM-1 on virus producer cells. Moreover, the increase in virion-bound host cell membrane ICAM-1 led to a concomitant enhancement of virus infectivity when a T-cell-tropic strain of human immunodeficiency virus type 1 (HIV-1) was used. The phenomenon was also seen when primary human cells were infected with virions pseudotyped with the envelope protein from a macrophage-tropic HIV-1 isolate, thus ruling out any envelope-specific effect. We also observed that target cells treated with NKI-L16, an anti-LFA-1 antibody known to increase the affinity of LFA-1 for ICAM-1, were markedly more susceptible to infection with HIV-1 particles bearing on their surfaces large numbers of host-derived ICAM-1 proteins. Given that cellular activation of leukocytes is known to modify the conformational state of LFA-1 and induce ICAM-1 surface expression, it is tempting to speculate that activation of virus-infected cells will lead to the production of HIV-1 particles bearing more host ICAM-1 on their surfaces and that such progeny virions will preferentially infect and replicate more efficiently in activated cells which are prevalent in lymphoid organs.
Roles of the Human Immunodeficiency Virus Type 1 Nucleocapsid Protein in Annealing and Initiation versus Elongation in Reverse Transcription of Viral Negative-Strand Strong-Stop DNA
Authors: Rong Liwei , Liang Chen , Hsu Mayla , Kleiman Lawrence , Petitjean Patrice , de Rocquigny Hugues , Roques Bernard P. , Wainberg Mark A. 
Published: December 2019

ABSTRACTTo study the initiation of human immunodeficiency virus type 1 reverse transcription, we have used the viral nucleocapsid protein (NC7) to anneal tRNA3Lysprimer onto viral genomic RNA and have then eliminated NC7 from this primer-template complex by digestion with proteinase K and phenol-chloroform extraction of residual protein. Our data show that saturating concentrations of NC7 resulted in the formation of an active tRNA-template complex that yielded enhanced production of full-length negative-strand strong-stop DNA [(−)ssDNA] and that this complex remained active even after the elimination of NC7. While both of the two Zn finger motifs found within NC7 were essential for efficient elongation, NC protein that contained a point mutation in the first Zn finger or that was devoid of both Zn fingers yielded primer-template complexes that could still be initiated in 1-base-extension assays. In contrast, the use of heat annealing to produce primer-template complexes resulted in proportions of full-length (−)ssDNA lower than those seen with NC protein, and the addition of NC protein to such preformed primer-template complexes was able to reverse this defect only to a marginal extent.
RNA-Binding Activity of the E1B 55-Kilodalton Protein from Human Adenovirus Type 5
Authors: Horridge Jackie J. , Leppard Keith N. 
Published: December 2019

ABSTRACT
The human adenovirus 5 E1B 55-kDa protein is required for efficient nucleocytoplasmic transport of late viral mRNAs. This protein is shown to have RNA-binding activity which maps to a region of the protein with homology to a family of RNA-binding proteins and which has been shown previously to be essential for functionality of the protein in vivo.
Definition of Amino Acid Residues on the Epitope Responsible for Recognition by Influenza A Virus H1-Specific, H2-Specific, and H1- and H2-Cross-Reactive Murine Cytotoxic T-Lymphocyte Clones
Authors: Tamura Manabu , Kuwano Koichi , Kurane Ichiro , Ennis Francis A. 
Published: December 2019

ABSTRACTWe defined the epitopes recognized by three influenza A virus-specific,H-2Kd-restricted CD8+cytotoxic T-lymphocyte (CTL) clones: H1-specific clone A-12, H2-specific clone F-4, and H1- and H2-cross-reactive clone B7-B7. The A-12 and B7-B7 clones recognized the same peptide, which comprises amino acids 533 to 541 (IYSTVASSL) of A/PR/8 hemagglutinin (HA). The F-4 and B7-B7 clones both recognized the peptide which comprise amino acids 529 to 537 (IYATVAGSL) of A/Jap HA. Amino acids 533 to 541 of A/PR/8 HA are compatible with amino acids 529 to 537 of A/Jap HA. Amino acid S at positions 3 and 7 was responsible for recognition by H1-specific clone A-12, while amino acid G at position 7 was responsible for recognition by H2-specific clone F-4. Two conserved amino acids, T at position 4 and A at position 6, were responsible for recognition by H1-, and H2-cross-reactive clone B7-B7. These results indicate that a single nine-amino-acid region is recognized by HA-specific CTL clones of three different subtype specificities and that the amino acids responsible for the recognition by the CTL clones are different.
Guanylyltransferase Activity of the LEF-4 Subunit of Baculovirus RNA Polymerase
Authors: Guarino Linda A. , Jin Jianping , Dong Wen 
Published: December 2019

ABSTRACT

The baculovirus
Autographa californica
nuclear polyhedrosis virus encodes a DNA-dependent RNA polymerase that transcribes viral late genes. This polymerase is composed of four equimolar subunits, LEF-4, LEF-8, LEF-9, and p47. Here we present data indicating that the LEF-4 subunit of RNA polymerase is a guanylyltransferase. Incubation of RNA polymerase in the presence of divalent cation and radiolabeled GTP resulted in the formation of a covalent enzyme-guanylate complex that comigrated with the LEF-4 subunit. The label transfer assay showed an absolute requirement for divalent cation which could be satisfied by either manganese or magnesium. The reaction was specific for guanine nucleotides, and GTP was more effective than dGTP in the formation of enzyme-guanylate complex. To demonstrate that LEF-4 was the guanylyltransferase, the single subunit was overexpressed in baculovirus-infected cells. The overexpressed protein was primarily cytosolic, indicating that other proteins in the RNA polymerase complex were responsible for nuclear targeting of LEF-4. LEF-4 alone was able to covalently bind GMP, although less efficiently than viral RNA polymerase.
Multigene Tracking of Hepatitis C Virus Quasispecies after Liver Transplantation: Correlation of Genetic Diversification in the Envelope Region with Asymptomatic or Mild Disease Patterns
Authors: Sullivan Daniel G. , Wilson Jeffrey J. , Carithers Robert L. , Perkins James D. , Gretch David R. 
Published: December 2019

ABSTRACT

To investigate the role of hepatitis C virus (HCV) quasispecies mutation in the pathogenesis of HCV infection, we analyzed changes in the genetic diversity of HCV genomes in 22 patients before and after liver transplantation by using heteroduplex mobility assay (HMA) technology. All patients were infected with HCV genotype 1 and developed high-titer posttransplant viremia. Each patient was classified according to the severity of posttransplant hepatitis, as assessed by standard biochemical and histological criteria. HCV quasispecies were characterized by HMA analysis of eight separate subgenomic regions of HCV, which collectively comprise 44% of the entire genome. The glycoprotein genes E1 and E2, as well as the nonstructural protein genes NS2 and NS3, had the greatest genetic divergence after liver transplantation (the change in the heteroduplex mobility ratio [HMR] ranged from 2.5 to 7.0%). In contrast, genes encoding the core, NS4, and NS5b proteins had the least amount of genetic divergence after liver transplantation (range, 0.3 to 1.2%). The E1/E2 region showed the greatest change in genetic diversity after liver transplantation, and the change in HMRs was 2.5- to 3.3-fold greater in patients with asymptomatic or moderate disease than in those with severe disease. The E1-5′ region of HCV quasispecies isolated from patients in the asymptomatic group had a significantly greater degree of diversification after liver transplantation than the same regions of HCV quasispecies isolated from patients in the severe disease group (
P
= 0.05). While changes in the genetic diversity of some nonstructural genes were also greater in asymptomatic patients or in patients with mild disease than in patients with severe disease, the results were not significant. Data from this cohort demonstrate that greater rates of HCV quasispecies diversification are associated with mild or moderate liver disease activity in this immunosuppressed population.
Mutational Analysis of the Role of Nucleoside Triphosphatase P4 in the Assembly of the RNA Polymerase Complex of Bacteriophage φ6
Authors: Paatero Anja O. , Mindich Leonard , Bamford Dennis H. 
Published: December 2019

ABSTRACT

Bacteriophage φ6 is a complex enveloped double-stranded RNA virus with a segmented genome and replication strategy quite similar to that of the
Reoviridae
. An in vitro packaging and replication system using purified components is available. The positive-polarity genomic segments are translocated into a preformed polymerase complex (procapsid) particle. This particle is composed of four proteins: the shell-forming protein P1, the RNA polymerase P2, and two proteins active in packaging. Protein P7 is involved in stable packaging, and protein P4 is a homomultimeric potent nucleoside triphosphatase that provides the energy for the RNA translocation event. In this investigation, we used mutational analysis to study P4 multimerization and assembly. P4 is assembled onto a preformed particle containing proteins P2 and P7 in addition to P1. Only simultaneous production of P1 and P4 in the same cell leads to P4 assembly on P1 alone, whereas the P1 shell is incompetent for accepting P4 if produced separately. The C-terminal part of P4 is essential for particle assembly but not for multimerization or enzymatic activity. Altering the P4 nucleoside triphosphate binding site destroys the ability to form multimers.