pinecone/core-2020-05-10-deduplication huggingface dataset:
Role of Cellular Actin in the Gene Expression and Morphogenesis of Human Respiratory Syncytial Virus

AbstractCytoskeletal protein actin and nonactin cellular proteins were essential for human respiratory syncytial virus (RSV) gene expression.In vitro,specific antibodies against actin inhibited RSV transcription, whereas antibodies against other cytoskeletal proteins had little or no effect. Affinity purified cellular actin or bacterially expressed recombinant actin activated RSV transcription. However, optimal transcription required additional cellular protein(s) that appeared to function as accessory factor(s) for actin. In the absence of actin, these proteins did not activate viral transcription. Purified viral nucleocapsids contained actin, but no cytokeratin, tubulin, or vimentin. Cytochalasin D or DNaseI—agents that destabilize actin polymers—had little effect on RSV transcription. RSV infection itself seemed to alter the structure of the cellular actin filaments. Treatment of infected cells with cytochalasin D produced a more severe disruption of the filaments and drastically reduced the production of infectious virus particles but still had little effect on intracellular synthesis of viral macromolecules. Thus actin seems to serve a dual role in RSV life cycle: its monomeric form as well as polymeric form activate viral transcription, while only the microfilament form may take part in viral morphogenesis and/or budding
Loss of Retrovirus Production in JB/RH Melanoma Cells Transfected with H-2Kband TAP-1 Genes

AbstractJB/RH1 melanoma cells, as well as other melanomas of C57BL/6 mice (B16 and JB/MS), express a common melanoma-associated antigen (MAA) encoded by an ecotropic melanoma-associated retrovirus (MelARV). JB/RH1 cells do not express the H-2Kbmolecules due to down-regulation of the H-2Kband TAP-1 genes. When JB/RH1 cells were transfected with the H-2Kband cotransfected with the TAP-1 gene, it resulted in the appearance of H-2Kbmolecules and an increase in their immunogenicity, albeit they lost expression of retrovirus-encoded MAA recognized by MM2-9B6 mAb. Loss of MAA was found to result from a complete and stable elimination of ecotropic MelARV production in the H-2Kb/TAP-1-transfected JB/RH1 cells. Northern blot analysis showed no differences in ecotropic retroviral messages in MelARV-producing and -nonproducing melanoma cells, suggesting that loss of MelARV production was not due to down-regulation of MelARV transcription. Southern blot analysis revealed several rearrangements in the proviral DNA of H-2Kb-positive JB/RH1 melanoma cells. Sequence analysis of the ecotropic proviral DNA from these cells showed numerous nucleotide substitutions, some of which resulted in the appearance of a novel intraviralPstI restriction site and the loss of aHindIII restriction site in thepolregion. PCR amplification of the proviral DNAs indicates that an ecotropic provirus found in the H-2Kb-positive cells is novel and does not preexist in the parental H-2Kb-negative melanoma cells. Conversely, the ecotropic provirus of the parental JB/RH1 cells was not amplifable from the H-2Kb-positive cells. Our data indicate that stable loss of retroviral production in the H-2Kb/TAP-1-transfected melanoma cells is probably due to the induction of recombination between a productive ecotropic MelARV and a defective nonecotropic provirus leading to the generation of a defective ecotropic provirus and the loss of MelARV production and expression of the retrovirus-encoded MAA
Sequences 5′ of the Conserved tRNA-like Promoter Modulate the Initiation of Minus-Strand Synthesis by the Brome Mosaic Virus RNA-Dependent RNA Polymerase

AbstractEach of the brome mosaic virus (BMV) genomic RNAs contains a conserved tRNA-like structure that is sufficient to direct minus-strand RNA synthesisin vitro.The tRNA-like promoters, tB1 and tB3, direct approximately equal amounts of synthesisin vitro.However, 5′ sequences were found to affect the amount of minus-strand synthesis, suggesting that sequences beyond the tRNA-like structure are important in moderating minus-strand synthesis. Consistent with this, sequences upstream the tRNA-like structure are able to partially suppress mutations at or near the initiation site. This activity is observed in the 5′ sequences of both BMV and CMV (cucumber mosaic virus) templates. However, a chimeric RNA containing the CMV tRNA-like promoter fused to the 5′ sequences of BMV was not able to suppress mutations at the initiation site, suggesting that homologous 5′ and 3′ sequences are required to affect initiation. The ability to suppress mutations at the initiation site was correlated with a slight increase in the ability of the BMV RNA-dependent RNA polymerase to interact with the RNA
Herpes Simplex Virus 1 DNA Cleavage/Packaging: The UL28 Gene Encodes a Minor Component of B Capsids

AbstractAn antiserum directed against a bacterial fusion protein containing UL28 protein sequences specifically recognized an 86,000 apparent Mrprotein in immunoblots of wild-type capsids. This protein was not detected in immunoblots of capsids purified from cells infected with a UL28 deletion virus, indicating that the protein was a product of UL28. The 86,000 Mrprotein was also detected in capsids purified from cells infected with mutant viruses lacking the UL6, UL15, and UL25 genes, indicating that the UL28 protein can associate with capsids independently of successful DNA packaging and other minor capsid components. The UL6 protein, full-length UL15 protein, and UL25-encoded proteins were also detected in capsids purified from cells infected with the UL28 deletion virus. The UL28 and UL6 proteins remained associated with capsids treated with 1.0 M guanidine–HCl, indicating that, like the UL6 protein, the UL28 protein was an integral component of capsids. Amounts of UL28 protein were reduced in DNA-containing capsids and UL28 protein was not detected in virions, suggesting that some UL28 protein is lost during the cleavage-packaging reaction
Murine Leukemia Virus Recombinants That Use Phosphate Transporters for Cell Entry Induce Similar Spongiform Encephalomyelopathies in Newborn Mice

AbstractAmphotropic Moloney-murine leukemia virus recombinants (Mo-AmphoV) induce a severe spongiform encephalomyelopathy in newborn mice. We show here that a coisogenic recombinant with a 10A1-MuLV host range (Mo-10A1V) also induces a neurodegenerative disease, clinically characterized by mild tremor and ataxia. Spongiform lesions are most severe in the metencephalon and mesencephalon but extend into the prosencephalon and spinal cord. Significantly, the quality of histopathology was indistinguishable between Mo-AmphoV and Mo-10A1V, probably reflecting a final common pathogenic pathway. Common receptor use thus may be an important determinant in the pathogenicity of these viruses. These results have implications for the clinical use of retroviral pseudotypes that use phosphate transporters for cell entry
Induction of Fas Ligand Expression by an Acutely Lethal Simian Immunodeficiency Virus, SIVsmmPBj14

AbstractSimian immunodeficiency virus strain PBj14, SIVsmmPBj14, is unique among primate lentiviruses in its ability to trigger the proliferation of resting simian lymphocytes and to cause the rapid death of experimentally inoculated pigtailed macaques. Severe enteropathy, immune activation, and extensive apoptosis, particularly within gut-associated lymphoid tissue, characterize the acute disease syndrome associated with SIVsmmPBj14infection. In the present study, we examined whether the ability of this virus to cause widespread apoptosis might be linked to the up-regulation of Fas ligand (CD95L) expression in virally infected cells.In vitrostudies revealed that expression of the viral Nef protein, in the absence of any other viral gene product, was sufficient to up-regulate the transcriptional activity of the CD95L promoter and to cause cell surface expression of Fas ligand. This up-regulation was NFAT dependent (inhibited by cyclosporin A) and did not occur in cells that expressed a mutated derivative of the viral Nef protein, lacking a previously defined immunoreceptor tyrosine-based activation motif. These findings were corroborated by analysis of tissue sections from virally infected macaques. Immunohistochemical staining revealed that Fas ligand expression was efficiently up-regulated in the GALT of animals that had been experimentally infected with wild-type SIVsmmPBj14but not in animals that were infected with a nonacutely pathogenic viral mutant lacking the Nef ITAM. Taken together, these results suggest that the ability of SIVsmmPBj14to cause acutely lethal disease and to up-regulate FasL expression may be linked. Additional studies will be required to determine whether the induction of FasL expression is in itself important for acute disease pathogenesis
A Distal Element in the HPV-11 Upstream Regulatory Region Contributes to Promoter Repression in Basal Keratinocytes in Squamous Epithelium

AbstractIn benign squamous lesions and in organotypic epithelial cultures, the human papillomavirus (HPV) E6 and E7 genes are transcriptionally up-regulated in differentiated, spinous keratinocytes. We previously identified sequence elements in the enhancer-promoter regions of HPV types 18 and 11 important for this promoter regulation by using the bacterial LacZ reporter gene in stratified raft cultures of primary human keratinocytes (PHKs) or in submerged, proliferating cultures acutely transduced with recombinant retroviruses. Notably, mutations in the promoter-proximal Sp1, Oct1, and AP1 sites each significantly reduce reporter activity in differentiated cells, indicating that the bound factors are transcription transactivators. In the present study, we performed further mutagenesis on distal motifs in the HPV-11 regulatory region in PHKs in submerged and raft cultures. Mutations in an AP2-like site, three individual NF-1 sites, or five NF-1 sites collectively reduced promoter activity slightly in differentiated cells. A mutation in a putative glucocorticoid response element had no discernable effect in the presence or the absence of dexamethasone. However, mutations in a C/EBP binding site, especially the distal site, strikingly up-regulated reporter gene expression, particularly in basal and lower spinous cells, implicating bound protein as a transcription repressor. Collectively, these results demonstrate that the overall differentiation-dependent papillomaviral gene expression observedin vivoandin vitroinvolves promoter repression in the lower strata and activation in the upper, differentiated strata
Isolation of a Mutant That Changes Genomic Packaging Specificity in φ6

AbstractBacteriophage φ6 has a genome of three segments of double-stranded RNA enclosed in a polyhedral procapsid. Plus strand transcripts of the segments are packaged in a serially dependent fashion in which S can package alone, M depends on S, and L depends on S and M. We have isolated a mutant form of the virus in the carrier state that has lost segment S. This finding presented an apparent anomaly with respect to the packaging program. Sequencing of gene 1 of segment L in this virus showed a translational change of arginine to glycine at the 14th position. Procapsids prepared from cDNA containing this mutation show behavior inin vitropackaging that is consistent with the phenotype of the mutant virus. The procapsids are able to package segment S alone, but this RNA is present in reduced amounts when the other segments are present. Segments M and L package without dependence on segment S. The mutant virus appears to produce procapsids that are at the second stage of the packaging program
Coinfection of a Fungal Pathogen by Two Distinct Double-Stranded RNA Viruses

AbstractUnsegmented double-stranded (ds)RNA viruses belonging to the family Totiviridae persistently infect protozoa and fungi. In this study, two totiviruses were found to coinfect the filamentous fungusSphaeropsis sapinea, a well known pathogen of pines. Isometric, virus-like particles ∼35 nm in diameter were isolated from extracts of this fungus. The nucleotide sequences of the genomes of the twoS. sapineaRNA viruses named SsRV1 and SsRV2 were established. The linear genomes of 5163 and 5202 bp, respectively, are identically organized with two large, overlapping ORFs. The 5′ located ORF1 probably encodes the coat protein, whereas the gene product of ORF2 shows the typical features of RNA-dependent RNA polymerases. The absence of a pseudoknot and a slippery site at the overlapping region between ORF1 and ORF2, as well as the shortness of that region, leads us to suggest that the translation of ORF2 of both viruses is internally initiated. The mode of translation and the genomic organization are similar to those ofHelminthosporium victoriae190S virus (Hv190SV; Huang, S., and Ghabrial, S. A. (1996).Proc. Natl. Acad. Sci. USA93, 12541–12546). Hv190SV thus appears to be closely related to the SsRVs. Interestingly, based on amino acid sequence homology SsRV1 is more closely related to Hv190SV than to SsRV2
Rapid Subtyping of Dengue Viruses by Restriction Site-Specific (RSS)–PCR

AbstractDengue is a major public health problem worldwide. It is caused by four dengue virus serotypes, each further divided into distinct genetic subtypes. Strain typing is important for understanding the epidemiology and viral factors associated with disease transmission. However, most of the existing subtyping methods are expensive and technically unwieldy for timely, practical applications in developing countries. Here we describe a simple, rapid, PCR-based subtyping method, restriction site-specific (RSS)–PCR, which we used to analyze dengue virus serotypes 2 and 3. For each serotype, four primers targeted to sequences spanning polymorphic endonuclease restriction sites in the envelope gene were used to reverse transcribe and amplify viral RNA. These RT–PCR products generated distinct electrophoretic band patterns for different strains. Analysis of 73 dengue-2 strains and 54 dengue-3 strains representing a broad geographic distribution over several decades revealed that the RSS–PCR fingerprints reproducibly fell into 7 and 3 groups, respectively. These groups correlated well with previous phylogenetic classifications. This one-step assay should be widely accessible and allow more detailed epidemiologic investigations in dengue-endemic countries. This novel PCR approach to subtyping organisms based on restriction site polymorphisms should be applicable to other pathogens
Phenotype of HIV-1 Lacking a Functional Nuclear Localization Signal in Matrix Protein ofgagand Vpr Is Comparable to Wild-Type HIV-1 in Primary Macrophages

AbstractHuman immunodeficiency virus type 1 (HIV-1) is considered to infect nondividing cells because nuclear localization signals (NLS) in matrix (MA, p17Gag) and Vpr allow active nuclear transport of the preintegration complex. Previous studies demonstrated that HIV-1 reverse transcription is successful only in cells with proliferative potential, thus restricting HIV-1 replication to cycling cells. To sort out this apparent discrepancy we compared the phenotype of a chimeric HIV-1 variant lacking a functional Vpr and MA-NLS (R7.ΔVpr.ΔNLS), and previously described to lack replicative capacity in macrophages and growth-arrested cells, with a chimera lacking a functional Vpr (R7.ΔVpr). Both variants replicated efficiently in primary macrophages, with only minimal differences in the kinetics of reverse transcription, integration, or p24 production. In agreement with our previous observation, elongation of reverse transcription was restricted to the proliferating subpopulation of macrophages. Replication of R7.ΔVpr and R7.ΔVpr.ΔNLS could also be demonstrated in aphidicolin-treated macrophages, indicating efficient nuclear transport in G1/S phase-arrested cells. In conclusion, our results confirm the dependency of the process of HIV-1 reverse transcriptase on cell proliferation in primary macrophages and exclude an important role of MA-NLS and Vpr in macrophage infection
Functional Analysis of Vaccinia Virus B5R Protein: Role of the Cytoplasmic Tail

AbstractVaccinia extracellular enveloped virus (EEV) is important for cell-to-cell and long-range virus spread bothin vitroandin vivo. Six genes have been identified that encode protein constituents of the EEV outer membrane, and some of these proteins are critical for EEV formation. The B5R gene encodes an EEV-specific type I membrane protein, and deletion of this gene markedly decreases EEV formation and results in a small plaque phenotype. Data suggest that the transmembrane domain, cytoplasmic tail, or both contain the EEV localization signals that are required for targeting of the B5R protein to EEV and for EEV formation. Here, we report the construction of mutant vaccinia viruses in which the wild-type B5R gene was replaced with a mutated one that encodes a protein with the putative cytoplasmic tail deleted. The mutated protein showed normal intracellular distribution and was properly incorporated into EEV. Vaccinia viruses expressing the B5R protein lacking the cytoplasmic tail formed plaques that were similar in type and size to those formed by wild-type viruses and produced equivalent amounts of infectious EEV. These results indicate that the B5R cytoplasmic tail is not necessary for EEV formation and points to the transmembrane domain as the major determinant for targeting the B5R protein to the outer membrane of EEV and for supporting EEV formation
Identification of a Sequence Element Involved in AC2-Mediated Transactivation of the Pepper Huasteco Virus Coat Protein Gene

AbstractThe geminivirusAC2gene product transactivates the expression of the coat and movement protein (CPandBV1) genes, and this effect seems to be mediated by specific although hitherto unknowncis-acting elements. In this work we examined regions from theCPandBV1gene promoters of pepper huasteco virus (PHV) to define the sequence elements involved in regulation by AC2. Results from transient gene expression and transgenic plant assays suggest that a truncated 115-nt CP promoter is still responsive to the viral transactivator. This promoter contains three elements similar to a sequence motif termed conserved late element (CLE), which is found in the regulatory regions of many geminiviruses and that was previously suggested, on a theoretical basis, to be a potential functional target for AC2 (Argüello-Astorgaet al.(1994),Virology203, 90–100). To confirm these results, an oligonucleotide containing two CLE motifs was synthesized and characterized in gain-of-function experiments. Transient expression assays showed that this 29-nt sequence is able to confer AC2 responsiveness to heterologous promoters. A smaller oligonucleotide (16 nt) containing a single CLE also conferred this activity. In addition, when the CLE motifs were mutated in their original context (truncated 115-nt promoter), this modified promoter lost its ability to be transactivated by AC2. All these results support the involvement, at least in the case of PHV, of CLE sequences in the process of transactivation
Site-Directed Mutagenesis of the AcMNPVp143Gene: Effects on Baculovirus DNA Replication

AbstractAutographa californicamulticapsid nucleopolyhedrovirus (AcMNPV) encodes a 143-kDa protein (P143) required for viral DNA synthesis and involved in host range determination. The predicted amino acid sequence of P143 contains seven motifs (I, Ia, II–VI) shared with a superfamily of helicases involved in the unwinding of duplex nucleic acids; a putative DNA binding motif; a putative nuclear localization signal (NLS); and a demonstrated host range motif. In this study, the functional significance of these conserved P143 motifs was examined by site-specific mutation resulting in amino acid substitutions of conserved residues within each of them. Anin vivocomplementation replication assay was developed and each mutated P143 protein expressed from a transfected plasmid was tested for its ability to complement the replication-negative ts8 baculovirus mutant for the amplification of an origin-containing plasmid. Mutations in the helicase motifs I, Ia, and II and in a potential helix-turn-helix motif abolished the ability of P143 to complement the ts8 defect in DNA replication, suggesting that these conserved amino acid residues may be essential for the replication function of the protein. In contrast, mutation of conserved amino acid residues in the helicase motifs IV, V, and VI did not affect the ability of the P143 proteins to complement the replication defect of ts8. A mutation in motif III caused a reduction in the replication function of P143. Deletion of Gly552 in the host range region eliminated the replication function of P143. Mutations within a putative NLS had no effect on the ability of P143 to support DNA replication, suggesting that these residues are nonessential and that the putative P143 NLS sequence may not be responsible for the nuclear localization of the protein. The transient complementation system used in this study provides a simple method for functional analysis of essential baculovirus genes in infected cell cultures
Reactivation of Kaposi's Sarcoma-Associated Herpesvirus Infection from Latency by Expression of the ORF 50 Transactivator, a Homolog of the EBV R Protein

AbstractKaposi's sarcoma (KS)-associated herpesvirus (KSHV), or human herpesvirus 8, is a lymphotropic virus strongly linked to several AIDS-related neoplasms. The primary reservoir of infection consists of latently infected B lymphocytes and possibly other mononuclear cells. Viral reactivation from latency and spread from this lymphoid reservoir is presumably required for development of nonlymphoid tumors like KS. Here we show that deregulated expression of a single viral gene, ORF 50, which encodes a transactivator able to selectively upregulate delayed-early viral genes, suffices to disrupt latency and induce the lytic gene cascade in latently infected B cells. The identification of this gene opens the way to studies of the physiologic mechanisms controlling reactvation of KSHV from latency