pinecone/core-2020-05-10-deduplication huggingface dataset:
Retroviral Recombination Is Nonrandom and Sequence Dependent

AbstractSequence variation plays a significant role in the pathogenesis and persistence of retroviral infections and is a major obstacle in the development of vaccines as well as therapies against lethal diseases caused by retroviruses. Recombination is one means by which sequence variation is generated. However, the basic molecular mechanisms of recombination are not adequately understood. In the present study, a spleen necrosis virus (SNV) recombination system was used to ask whether a known hot spot for mutation was also a hot spot for retroviral recombination. The system consisted of a pair of SNV vectors expressing two drug-resistance genes, constructed so that recombinants could be selected by a double resistant phenotype. Restriction enzyme site differences engineered into the vectors were used to map the location of recombination sites within relatively small intervals (55 to 420 bp). The vectors were modified to create two pairs that differed only by the presence of runs of identical nucleotides. The runs of identical nucleotides had been shown previously to be hot spots for frameshift mutations during SNV reverse transcription. Each vector pair was introduced into DSDh helper cells by infection. Viruses were harvested from doubly infected DSDh helper cells and used to infect D-17 target cells. Proviral sequences from 228 cell clones were analyzed by polymerase chain reaction and restriction enzyme digestion. Significant differences in the patterns of recombination were found between the two pairs of vectors. In particular, the frequency of recombination was higher than expected in the interval immediately following the runs. For both pairs of vectors, the overall pattern of recombination was nonrandom and one region was refractory toward recombination
Recombinant Swinepox Virus Expressing β-Galactosidase: Investigation of Viral Host Range and Gene Expression Levels in Cell Culture

AbstractSwinepox virus (SPV) has been proposed as a potential vector for generating recombinant vaccines for swine. However, little is known about important aspects of SPV biology, such as the functionality of SPV promoters or the host range of SPV. Using a transient expression assay, well-characterized vaccinia virus promoters were shown to be active in cells infected with SPV. A recombinant SPV expressing β-galactosidase (β-gal) was constructed and characterized. TheE. coli LacZgene was placed under the control of a strong vaccinia synthetic early/late promoter and was inserted by homologous recombination in a noncoding region of the SPV genome. The recombinant SPV expressing β-gal was used to characterize the host range of the virus by measuring protein expression and virus production in different cell lines. In general, SPV expressed more protein and grew more efficiently than vaccinia virus in porcine cell lines. Surprisingly, the recombinant SPV was able to infect and replicate in several cell lines of nonswine origin. The virus directed regulated early and late gene expression of β-gal in those cells and formed blue plaques in cell monolayers in the presence of X-gal. Upon infection with the recombinant SPV, there was a significant level of viral replication, and the virus can be serially passaged in some nonswine cell lines. The data presented suggest that despite the strict host tropism of SPV, the virus exhibits a relatively broad host range in cell culture
Structure–Function Analysis of the Herpes Simplex Virus Type 1 UL12 Gene: Correlation of Deoxyribonuclease Activityin Vitrowith Replication Function

AbstractAlthough the product of the UL12 gene of herpes simplex virus type 1 (HSV-1) has been shown to possess both exonuclease and endonuclease activitiesin vitro,and deletion of most of the gene within the viral genome results in inefficient production and maturation of infectious virions, the function of the deoxyribonuclease (DNase) activity per se in virus replication remains unclear. In order to correlate thein vitroandin vivoactivities of the protein encoded by UL12, mutant proteins were tested for nuclease activityin vitroby a novel hypersensitivity cleavage assay and for their ability to complement the replication of a DNase null mutant, AN-1. Rabbit reticulocyte lysates programmed with wild-type UL12 RNA cleaved at the same sites cleaved by purified HSV-1 DNase, but distinct from those cleaved by DNase I or micrococcal nuclease. All mutants which lacked DNase activityin vitroalso failed to complement the replication of AN-1 in nonpermissive cells. Likewise, all mutants which contained HSV-1 DNase activity, as detected by the hypersensitivity cleavage assay, were capable of complementing the replication of the DNase null mutant, though to varying extents. Of particular note was the d1-126 mutant protein, which, despite having the same specific activity as the wild-type enzymein vitro,complemented the replication of AN-1 significantly less than the wild-type protein. The results suggest that DNase activity per se is required for efficient replication of HSV-1in vivo.However, residues, including the N-terminal 126 amino acids, which are dispensable for enzymatic activityin vitromay facilitate the accessibility or activity of the proteinin vivo
Reactivities of Human Sera with Human Herpesvirus-8-Infected BCBL-1 Cells and Identification of HHV-8-Specific Proteins and Glycoproteins and the Encoding cDNAs

AbstractThe reactivates of human sera with uninduced and phorbol ester (TPA)-induced human herpesvirus-8 (HHV-8)-infected BCBL-1 cells were examined by immunofluorescence assay (IFA) and by radioimmunoprecipitation reactions (RIP). The seroprevalence of HHV-8 infections is low in the United States general population and only low levels of HHV-8 antibodies were detected in the seropositive sera. In contrast, high levels of antibodies against HHV-8 lytic and latent antigens were detected by IFA in the sera from HIV+Kaposi's sarcoma (KS)-positive individuals. These sera recognized several proteins and glycoproteins from BCBL-1 cells in RIP reactions. Two types of antibody responses were detected in the sera from HIV+KS−homosexual men. In majority of the sera with and without detectable HHV-8 DNA in the peripheral blood mononuclear cells (PBMC), significantly low levels of HHV-8 antibodies were detected by IFA. These sera recognized only a subset of HHV-8 proteins and glycoproteins in RIP reactions. In contrast, in a subgroup of sera from HIV+KS−homosexual men, higher levels of IFA antibodies against HHV-8 lytic and latent antigens were detected. These sera also recognized several viral proteins and glycoproteins in RIP reactions. These results suggest that antibody response profiles to HHV-8 infection vary significantly and serologic assays to detect antibody responses to a panel of both lytic and latent antibodies may be required for maximum sensitivity. Screening of a cDNA library from TPA-induced BCBL-1 cells with an HIV+KS+serum identified cDNAs encoding 12 HHV-8 proteins. Further characterization of these HHV-8 proteins would define the HHV-8 antigens useful for seroepidemiological studies and in discriminating lytic, latent, past, and/or reactivation infections
Two Regions of Simian Virus 40 Large T-Antigen Independently Extend the Life Span of Primary C57BL/6 Mouse Embryo Fibroblasts and Cooperate in Immortalization

AbstractExpression of the SV40 large T-antigen allows primary cells to escape senescence and thereby become immortalized. Immortalization occurs in two steps, extension of life span and acquisition of unlimited cell division potential. By following the increase in expression of a senescence-associated marker with increased cell passage, we show that C57Bl/6 mouse embryo fibroblast (B6MEF) cultures senesce by passage 4. Thus, the development of colonies from cultures transfected with T-antigen expressing constructs indicates extension of life span. Two T-antigen regions independently extended the life span of B6MEF. Expression of either a T-antigen consisting of amino acids 1–147 (T1-147) or a T-antigen consisting of amino acids 251–708 (T251-708) resulted in colony development. However, the colonies expressing these truncated T-antigens could not be expanded into cell lines efficiently. In contrast, coexpression of T1-147 and T251-708 produced colonies that could be expanded into cell lines as efficiently as could colonies expressing full-length T-antigen. Thus, the two regions of T-antigen contain analogous activities that are sufficient to extend cell life span; they cooperate to immortalize primary B6MEF; and they actin trans,indicating that the functions involved are independent
Mutation in a 17D-204 Vaccine Substrain-Specific Envelope Protein Epitope Alters the Pathogenesis of Yellow Fever Virus in Mice

AbstractThe heterogeneous nature of the yellow fever (YF) 17D-204 vaccine virus population was exploited in this study to isolate virus variants able to escape neutralization by the 17D-204 vaccine-specific MAb 864. The conformational change on the virus surface that resulted in the loss of the MAb 864-defined epitope was effected in each variant by a single amino acid mutation in the envelope (E) protein at either position E-305 or E-325. Interestingly, both positions were mutated during attenuation of the 17D-204 vaccine substrain from the wildtype Asibi strain. The mutations in several of the variants represented reversion to the wildtype Asibi virus sequence consistent with loss of a 17D-204 substrain-specific epitope. The majority of the variant viruses were shown to have altered mouse neurovirulence phenotypes, ranging from complete avirulence through to increased virulence. The avirulent variants are the first flavivirus MAb-neutralization-resistant variants to be attenuated for neurovirulence in the adult mouse model. Overall, the results indicate that the E protein epitope recognized by MAb 864 defines a functionally important region that encodes major molecular determinants of YF virus pathogenesisin vivo
Identification of a 24-kDa Polypeptide Processed from the Coronavirus Infectious Bronchitis Virus 1a Polyprotein by the 3C-like Proteinase and Determination of Its Cleavage Sites

AbstractWe report here the identification of a 24-kDa polypeptide in IBV-infected Vero cells by immunoprecipitation with a region-specific antiserum raised in rabbits against the IBV sequence encoded between nucleotides 10928 and 11493. Coexpression, deletion, and mutagenesis studies have demonstrated that this protein is encoded by ORF 1a from nucleotide 10915 to 11544 and is released from the 1a polyprotein by the 3C-like proteinase-mediated proteolysis. A previously predicted Q-S (Q3462S3463) dipeptide bond encoded by the IBV sequence from nucleotide 10912 to 10917 is identified as the N-terminal cleavage site, and a Q-N (Q3672N3673) dipeptide bond encoded by the IBV sequence between nucleotides 11542 and 11547 is identified as the C-terminal cleavage site of the 24-kDa polypeptide
Characterizations of Coronaviruscis-Acting RNA Elements and the Transcription Step Affecting Its Transcription Efficiency

AbstractSeven to eight species of viral subgenomic mRNAs are produced in coronavirus-infected cells. These mRNAs are produced in different quantities, and their molar ratios remain constant during viral replication. We studied RNA elements that affect coronavirus transcription efficiency by characterizing a series of cloned coronavirus mouse hepatitis virus (MHV) defective interfering (DI) RNAs containing an inserted intergenic sequence, from which subgenomic DI RNA is transcribed in MHV-infected cells. Certain combinations of upstream and downstream flanking sequences of the intergenic sequence suppressed subgenomic DI RNA transcription, yet changing one of the flanking sequences to a different sequence eliminated transcription suppression. The suppressive effect of certain combinations of flanking sequences, but not all combinations, could be counteracted by altering the intergenic sequence. Thus, the combination of intergenic sequence and flanking sequence affected transcription efficiency. We also characterized another set of DI RNAs designed to clarify which transcription step determines the relative molar ratios of coronavirus mRNAs. Our study indicated that if subgenomic mRNAs were exclusively synthesized from negative-strand genomic RNA, then the relative molar ratios of coronavirus mRNAs were most likely determined after synthesis of the genomic-sized template RNA. If negative-strand subgenomic RNAs were templates for subgenomic mRNAs, then the relative molar ratios of coronavirus mRNAs probably were determined after synthesis of the genomic-sized template RNA used for subgenomic-sized RNA transcription but prior to the completion of the synthesis of subgenomic-sized RNAs containing the leader sequence. The relative molar ratios of coronavirus mRNAs, therefore, seem to have been established prior to a putative replicon-type amplification of subgenomic mRNAs
Stimulation of Local Immunity and Protection in Mice by Intramuscular Immunization with Triple- or Double-Layered Rotavirus Particles and QS-21

AbstractBased on studies in animal models, parenteral immunization has become recognized as a potential vaccination strategy against rotavirus. Using an adult mouse model, the effects of the saponin adjuvant QS-21 on protection against murine rotavirus (strain EDIM) infection was determined following two intramuscular (i.m.) immunizations with purified EDIM particles including triple-layered (tl) infectious particles, tl particles inactivated with psoralen/UV, and double-layered (dl) inactivated particles. All three particles stimulated large serum rotavirus IgG responses and small amounts of serum rotavirus IgA, but undetectable stool rotavirus IgA. Inclusion of QS-21 during immunization increased the serum responses approximately 2- to 10-fold and also stimulated low levels of stool rotavirus IgA. Protection based on reduced shedding of rotavirus following EDIM challenge was significant (P< 0.001) with each immunized group and was enhanced (P< 0.001) by inclusion of QS-21 during immunization. Mice immunized with either live or inactivated tl particles and QS-21 were almost fully protected. Furthermore, animals inoculated with dl particles and the adjuvant shed significantly (P= .02) less virus following challenge than mice immunized with inactivated tl particles even though the latter induced measurable titers of neutralizing antibody to EDIM. These results demonstrate significant protection against rotavirus following i.m. immunization with both dl and tl EDIM particles which is consistently enhanced with QS-21
Stoichiometry of Reovirus Structural Proteins in Virus, ISVP, and Core Particles

AbstractAll eight reovirus structural proteins were resolved in a new tris, glycine, and urea (TGU) electrophoretic gel system. The specific identities of proteins were determined immunologically, biochemically, and genetically. Structural proteins of reovirus type 1 Lang had different mobilities in the TGU gel than did type 3 Dearing proteins. Intertypic reassortant viruses that contained various combinations of parental genes were used to identify each of the viral protein bands. Type 1 Lang virions were metabolically-labelled with either3H-amino acids or35S-methionine/cysteine and gradient purified. Aliquots of purified virions were treated to generate infectious subviral particles (ISVPs) and core particles. Radiolabelled virus, ISVP, and core proteins were resolved in the TGU gel and protein band intensities were used to determine copy numbers of each structural protein. These studies confirmed the copy numbers and locations of most reovirus proteins. However, important new findings include the discovery that virions contain approximately 120 copies of major core protein ς2 and 20 copies of the polymerase cofactor protein μ2, and ISVP particles contain about 24 copies of μ1C that has not been processed to the δ peptide. These data are used to generate a new model of the arrangement of structural proteins within the reovirus particle
Inhibition of Friend Virus Replication by a Compound That Reacts with the Nucleocapsid Zinc Finger: Anti-Retroviral Effect Demonstratedin Vivo

AbstractThe zinc finger structure that is found in the nucleocapsid protein of nearly all retroviruses has been proposed as a target for antiviral therapy. Since compounds that chemically attack the cysteines of the finger have been shown to inactivate both human immunodeficiency virus type 1 (HIV-1) and murine leukemia virus (MuLV)in vitro,14 of these compounds were tested in an MuLV-induced Friend disease model to assess their ability to inhibit retroviral replicationin vivo.Of the 14 compounds tested, only Aldrithiol-2 clearly exhibited anti-retroviral activity as measured indirectly by the delay of Friend disease onset (P< 0.05). These results were confirmed by quantitative competitive polymerase chain reaction studies which monitored viral spread by measuring the level of viral DNA in the peripheral blood mononuclear cells of treated mice. Comparison of treated mice with untreated mice revealed that Aldrithiol-2 produced a greater than 2-log reduction in virus levels. These results functionally demonstrate that a zinc finger-attacking compound can inhibit viral replicationin vivo.Since only 1 of the 14 compounds studied was effective, this study also shows the importance ofin vivotesting of these types of antiviral compounds in an animal model. Given the strict conservation of the metal-coordinating cysteine structure within HIV-1 and MuLV zinc fingers, our results support the proposal that anti-retroviral drugs which target the nucleocapsid zinc finger may be clinically useful against HIV-1
The Short Sendai Virus Leader Region Controls Induction of Programmed Cell Death

AbstractThe replication of nonsegmented minus-strand RNA genomes, like that of Sendai paramyxovirus (SeV), are controlled by the short leader regions present at each end of the linear genomes and antigenomes; the left and right promoters (PLand PR), respectively. Wild-type SeV is highly cytopathic in cell culture, because it induces programmed cell death (PCD). We have found that a recombinant SeV (rSeVGP42), in which the first 42 nt ofle+sequences at PLwere replaced with the equivalent sequences of PR, and which produces infectious virus in amounts comparable to wild type, does not kill cells. Further, the increasing replacement of the terminalle+sequences at PLwithle−sequences led to a decreasing fraction of infected cells being apoptotic. This property (PCD−), moreover, is dominant in cells co-infected with SeVwtand rSeVGP42, and the mutant virus therefore appears to have gained a function which prevents PCD induced by SeVwt. Even though this virus has not been selected for naturally, it excludes SeVwtduring co-infections of cultured cells or embryonated chicken eggs. The noncytopathic nature of cells infected or co-infected with rSeVGP42leads automatically to stable, persistent infections. The mutation in rSeVGP42is not in the protein coding regions of the viral genome, but in the 55-nt-long leader region which controls antigenome synthesis from genome templates. The SeV leader regions, which are expressed as short RNAs, thus appear to control the induction of PCD
Identification and Characterization of Virus Assembly Intermediate Complexes in HIV-1-Infected CD4+T Cells

AbstractFor type-C and lentiviruses, including human immunodeficiency virus type 1 (HIV-1), the pathway of virus assembly remains poorly defined, and the assembly and budding of capsids are believed to occur simultaneously at the plasma membrane of the infected cell. We have now identified two putative HIV-1 assembly intermediate complexes in infected CD4+T cells. The first of these intermediates, a detergent-resistant complex (DRC), was identified as a large oligomer that had a density of 1.10–1.13 g/ml and was primarily composed of Pr55Gagand Pr160Gag-Polprecursors. The other putative intermediate was a detergent-sensitive complex (DSC) with a density of 1.15–1.17 g/ml, which apparently represented the products of extensive proteolytic processing of both the Pr55Gagand Pr160Gag-Polprecursors. Both complexes could be distinguished from released mature virions as well as immature viral particles. Surprisingly, the formation of DRC was not dependent upon the myristylation at the N-terminus of the Gag proteins, a signal required for plasma membrane targeting and virus production. However, the myristic acid modification was essential for the formation of DSC. These data suggest that interactions between individual Gag molecules and between Gag and Gag-Pol precursors may occur before their targeting to the plasma membrane during HIV-1 assembly. However, formation of the late virus assembly complex and productive processing of Pr55Gagand Pr160Gag-Polprecursors apparently do not occur until these precursors are targeted to the plasma membrane
Actin Binding and Nucleation byAutographa californicaM Nucleopolyhedrovirus

AbstractThe budded form ofAutographa californicaM nucleopolyhedrovirus enters permissive cells via adsorptive endocytosis. Shortly after nucleocapsid penetration into the cytoplasm, thick actin cables form, which frequently project toward the nucleus. These actin cables are transient structures, formed in association with viral nucleocapsids prior to viral gene expression and concomitant with nucleocapsid transport to the nucleus. In this paper we report that nucleocapsids are capable of nucleating actin polymerizationin vitroin a concentration-dependent manner. Two viral-encoded capsid proteins, p39 and p78/83, were found to bind actin directly and therefore could be involved in the observed acceleration of actin polymerization. When nucleocapsids were added to actin in the presence of cytochalasin D, actin polymerization was reduced to levels below those obtained with actin and cytochalasin D alone, suggesting that the nucleocapsids bound to the pointed ends of actin filaments. Finally, treatment of infected cells with the myosin inhibitor 2,3-butanedione monoxime delayed nucleocapsid transport to the nucleus. We postulate that upon entering the cytoplasm, AcMNPV nucleocapsids induce the polymerization of actin cables, which, in conjunction with a myosin-like motor, facilitate their transport to and/or into the nucleus
Structural Homology of the Central Conserved Region of the Attachment Protein G of Respiratory Syncytial Virus with the Fourth Subdomain of 55-kDa Tumor Necrosis Factor Receptor

AbstractThe attachment protein G of respiratory syncytial virus (RSV) has a modular architecture. The ectodomain of the protein comprises a small folded conserved region which is bounded by two mucin-like regions. In this study, a sequence and structural homology is described between this central conserved region of RSV-G and the fourth subdomain of the 55-kDa tumor necrosis factor receptor (TNFr). The three-dimensional structures of RSV-G and human TNFr were previously determined with NMR spectroscopy and X-ray crystallography, respectively. The C-terminal part of both subdomains fold into a cystine noose connected by two cystine bridges with the same spacing between cysteine residues and the same topology. Although a general structural similarity is observed, there are differences in secondary structure and other structural features. Molecular Dynamics calculations show that the BRSV-G NMR structure of the cystine noose is stable and that the TNFr crystal structure of the cystine noose drifts towards the BRSV-G NMR structure in the simulated solution environment. By homology modelling a model was built for the unresolved N-terminal part of the central conserved region of RSV-G. The functions for both protein domains are not known but the structural similarity of both protein domains suggests a similar function. Although the homology suggests that the cystine noose of RSV-G may interfere with the antiviral and apoptotic effect of TNF, the biological activity remains to be proven