pinecone/core-2020-05-10-deduplication huggingface dataset:
Human Adenoviruses of Subgenera B, C, and E with Various Tropisms Differ in Both Binding to and Replication in the Epithelial A549 and 293 Cells

AbstractAdenoviruses of six subgenera, namely, adenovirus 31 (Ad31) (subgenus A), Ad3, Ad7, Ad11p, Ad11a, and Ad35 (subgenus B), Ad5v and Ad5p (subgenus C), Ad37 (subgenus D), Ad4 (subgenus E), and Ad41 (subgenus F), were studied. The relative binding properties of different adenoviruses to 293 (human kidney embryonic cells) and A549 (human lung carcinoma cells) cells were compared by flow cytometry. All analyzed adenoviruses bound to cells in a dose-dependent manner. The binding capacity showed that Ad11p, Ad35 (subgenus B:2) with kidney tropism, and Ad4 (subgenus E), which can cause adenopharyngoconjunctivitis, bound strongly to both A549 and 293 cells. The other members of subgenus B and Ad37 of subgenus D manifested an intermediate binding capacity. The analyzed adenoviruses of subgenera A, C, and F manifested a low affinity. Adenoviruses of subgenera B:2 and E manifested high binding affinity to preparations of cell membranes from the epithelial cell lines. Reciprocal competition experiments using Ad11p and Ad4 demonstrated that the two viruses did not block each other. Antibodies against αvβ3 and αvβ5 reduced the binding of Ad5v virions and slightly impaired the binding of Ad4 but did not affect Ad11p binding to the A549 cell surface. Recombinant fiber proteins of Ad11p and Ad35 reciprocally blocked the binding of both viruses to the epithelial cells but they could not block Ad4. The hexon protein expression of Ad11p and Ad4 was 100 times more efficient than that of the Ad5 vector (pFG140), whereas the infectivity of Ad11p and Ad4 was 40- to 200-fold that of the commonly used Ad5v vector. Taken together, our findings demonstrate that Ad11p and Ad4 bind different receptor molecules and that the fibers of these two viruses provide the predominant high degree of binding, which obviously is a requirement for subsequent internalization and efficacious expression
Reorganization of Nuclear Domain 10 Induced by Papillomavirus Capsid Protein L2

AbstractNuclear domains (ND) 10 are associated with proteins implicated in transcriptional regulation, growth suppression, and apoptosis. We now show that the minor capsid protein L2 of human papillomavirus (HPV) type 33 induces a reorganization of ND10-associated proteins. Whereas the promyelocytic leukemia protein, the major structural component of ND10, was unaffected by L2, Sp100 was released from ND10 upon L2 expression. The total cellular amount of Sp100, but not of Sp100 mRNA, decreased significantly, suggesting degradation of Sp100. Proteasome inhibitors induced the dispersal of Sp100 and inhibited the nuclear translocation of L2. In contrast to Sp100, Daxx was recruited to ND10 by L2 expression. Coimmunoprecipitation demonstrated interaction of the two proteins. L2-induced reorganization of ND10 was observed both in cell culture and in natural HPV lesions. The differential change in protein composition observed provides further evidence to suggest that the ND10-associated proteins are an important interface of viral life cycle and host cell
DNA Binding of L1 Is Required for Human Papillomavirus Morphogenesis in Vivo

AbstractThe role of putative DNA-binding domains of human papillomavirus (HPV) capsid proteins for DNA encapsidation in vivo is still unknown. We have now analyzed mutants of the major capsid protein L1 of HPV type 33, which are defective for DNA binding, for their ability to encapsidate DNA using an in vivo packaging approach. Since the DNA-binding domain and the nuclear localization signal (NLS) of L1 overlap, both a carboxy-terminal deletion mutant (L1-1/470) and a substitution mutant (L1-1/477M9) were analyzed. L1-1/477M9 has the classical NLS replaced by a noncanonical NLS taken from the human hnRNP protein A1. The mutant proteins were defective for DNA binding in contrast to wild-type (wt) L1 proteins of several HPV types. L1-1/470 localized to the cytoplasm when expressed in human cell lines, whereas L1-1/477M9 was found mainly in the nucleus. When coexpressed with wt L2, mutant L1 proteins colocalized with L2 in nuclear domains 10 (ND10) similar to wt L1 and assembled into virus-like particles incorporating L2. Wt L1 and L2 but not mutant L1 proteins and wt L2 efficiently encapsidated DNA and transferred it into cells, suggesting that physical interaction of L1 with DNA is essential for DNA inclusion into VLPs. We also compared the infectivity of histone-free DNA-containing pseudovirions generated in vitro with the infectivity of pseudovirions generated in vivo, which most likely harbor DNA in the form of chromatin. Pseudovirions generated in vivo were, on a per genome basis, four to five times more infectious than pseudovirions generated in vitro, even though no structural or biochemical difference was obvious. This indicates that chromatin may enhance the infectivity of pseudovirions
Hydrophobic Residue Contributions to Sequence-Specific DNA Binding by the Bovine Papillomavirus Helicase E1

AbstractPreviously, mutational analyses of the DNA binding domain of the bovine papillomavirus E1 protein (E1DBD) identified several hydrophobic residues that are critical for DNA binding activity (M. West, D. Flanery, K. Woytek, D. Rangasamy, and V. G. Wilson, 2001, J. Virol. 75, 11948–11960). Hydrophobic interactions of nonpolar amino acid side chains can contribute to the function of DNA binding proteins through both conformational effects and direct interaction with nucleotides. To further investigate the role of hydrophobic residues in E1DBD function, a more extensive site-directed mutational analysis of hydrophobic amino acids was conducted. Alanine substitutions were made at residues V196, F197, F217, F, 237, V246, L249, and F276, and the mutants were tested for DNA binding activity in vitro and in vivo. The E1 F237A and F276A mutants were completely defective for site-specific DNA binding, while the other mutants retained partial to full wild-type binding activity. Consistent with their DNA binding defect, the F237A and F276A mutants were severely impaired for the ability to support transient in vivo replication of an origin plasmid. Combined with our previous study, five critical hydrophobic residues have been identified: F175, V193, F237, V246, and F276. These five residues localize to two internal clusters in the E1DBD structure designated hydrophobic clusters A (HCA; includes F175, V193, and F276) and B (HCB; includes F237 and V246). Amino acid side chains from residues in HCA and HCB have little surface accessibility and it is unlikely that they are involved in direct contact with DNA. HCA is distal to the DNA binding surface and presumably contributes to global conformational organization of the E1DBD. HCB is positioned beneath the DNA contact surface and we propose that it serves as an anchor or platform device to stabilize the DNA-binding element. A comparable hydrophobic cluster is present in the corresponding position in the T antigen DBD and likely serves a similar function
Pathogenic and Nef-Interrupted Simian–Human Immunodeficiency Viruses Traffic to the Macaque CNS and Cause Astrocytosis Early after Inoculation

AbstractSeveral studies have shown that deletion of the nef gene of simian immunodeficiency virus (SIV) and simian–human immunodeficiency virus (SHIV) results in attenuated viruses. However, studies have not critically examined trafficking of attenuated viruses to the central nervous system (CNS) at early stages after inoculation. In this study, we investigated the colocalization of pathogenic and vpu-negative, nef-interrupted SHIVs at early stages following inoculation. The first virus, designated SHIV50OLNV, was isolated from the lymph node of a pig-tailed macaque which developed severe CD4+ T cell loss and neurological disease. The second virus was a molecularly cloned virus in which the vpu gene was deleted and the gene for the enhanced green fluorescent protein from the jellyfish Aequoria victora had been inserted in-frame within the nef gene of the pathogenic SHIVKU-1bMC33 (designated SHIVKU-1bEGFP). Three pig-tailed macaques were inoculated intravenously with equivalent amounts of two viruses, two macaques were inoculated with SHIVKU-1bEGFP, and two macaques were inoculated with SHIV50OLNV. The peripheral blood mononuclear cells (PBMCs) were isolated from bleeds obtained 3, 7, 10, and 14 days postinoculation and monitored for syncytia-inducing virus and for fluorescent cells. Virus was detected in the PBMCs as early as 3 days postinoculation and was present throughout the course of this short-term study. At 14 days postinoculation, the macaques were sacrificed and examined for virus in lymphoid tissues and different regions of the CNS following necropsy. Our results revealed the presence of both viruses in lymphoid and CNS tissues, although SHIV50OLNV was present to a much greater extent. Histological examination revealed that one macaque displayed signs of meningitis and all three macaques developed massive cortical astrocyte activation as demonstrated by immunostaining for glial fibrillary acidic protein, but only limited microglial activation. In the two macaques inoculated with SHIV50OLNV, astrocyte activation similar to that in the macaques inoculated with both viruses was observed while no astrocyte activation was observed in macaques inoculated with SHIVKU-1bEGFP. Thus, this study demonstrates that SHIVs with an intact nef(SHIV50OLNV) as well as those lacking a vpu gene and with a nonfunctional nef gene (SHIVKU-1bEGFP) are capable of invading the CNS and that pathogenic SHIVs are capable of causing reactive astrocytosis early after inoculation
Interaction between Nef and Phosphatidylinositol-3-Kinase Leads to Activation of p21-Activated Kinase and Increased Production of HIV

AbstractThe negative factor (Nef) is one of six accessory proteins from primate lentiviruses (HIV-1, HIV-2, and SIV). It leads to high levels of viremia and the progression to AIDS in monkeys and humans. In this study, we demonstrated that Nef from HIV-1 binds to the regulatory subunit (p85) of phosphatidylinositol-3-kinase (PI3K). This interaction depended on the C-terminus of p85 and Nef. Moreover, PI3K was required to activate the Nef-associated p21-activated kinase (PAK). Finally, inhibition of PI3K blocked the activation of PAK and decreased the production of viral particles to levels observed with the Nef-deleted provirus. We conclude that Nef assembles a multiprotein signaling complex which is required for the optimal replication of HIV-1
Decreased IL-2, IFN-γ, and IL-10 Production by Aged Mice During the Acute Phase of E55+ Retrovirus Infection

AbstractWe previously reported that aged mice demonstrated a 12-week delay in virus clearance compared to young mice after infection with E55+ murine leukemia retrovirus (E55+MuLV). The current study demonstrates that both the levels of IL-2, IFN-γ, and IL-10 and the number of cells producing IL-2 and IFN-γ were lower at 2 and 4 weeks postinfection (p.i.) in aged compared to young mice after virus-specific stimulation of spleen cells in vitro. In both age groups, IL-2 and IL-10 were produced by CD4+ T and B cells, respectively. IFN-γ was produced mainly by CD4+ T cells at 2 weeks p.i. and by CD4+ and CD8+ T cells at 4 weeks p.i. in young, but primarily by CD8+ T cells, in aged mice. Therefore, delayed virus clearance is associated with age-related decreases in type 1 and type 2 cytokines and a shift in the primary source of at least one cytokine
Inhibition of Host ER Glucosidase Activity Prevents Golgi Processing of Virion-Associated Bovine Viral Diarrhea Virus E2 Glycoproteins and Reduces Infectivity of Secreted Virions

AbstractRecently, it was shown that replication of bovine viral diarrhea virus (BVDV) is sensitive to inhibitors of host ER glucosidases. Consistent with these findings, we report that incubation of BVDV-infected MDBK cells with the glucosidase inhibitor n-butyl-deoxynojirimycin (nB-DNJ) reduced BVDV yields by 70- to 100-fold (n = 27), while having no effect on MDBK cell viability. However, the 70- to 100-fold reduction in infectious virus was associated with only a 2-fold reduction in genomic RNA synthesis and secretion of enveloped virus particles. Analysis of secreted virions showed that in the absence of glucosidase inhibitor, approximately 50% of the virion-associated BVDV E2 glycoprotein was resistant to endoglycosidase H (endo H) digestion, whereas intracellular E2 was completely sensitive to endo H digestion. In the presence of glucosidase inhibitor, virion-associated E2 and intracellular E2 were completely sensitive to endo H digestion. Taken together, these results suggest that BVDV is secreted through a Golgi-mediated pathway and that host ER glucosidase activity is required for production of infectious virions and Golgi processing of envelope E2 protein during virus egress
Hepatitis C Virus Core Protein Inhibits Apoptosis via Enhanced Bcl-xL Expression

AbstractPrevious studies indicated that hepatitis C virus core protein influences cellular apoptosis. However, the precise mechanisms of the effects are not fully understood. Therefore, in this study, we examined the mechanisms of the effects on cell apoptosis by core protein, using transiently transfected and magnetically collected core-producing HepG2 cells. First, to elucidate the target site of core protein in the apoptotic pathway, we examined the activation of caspases after anti-Fas antibody stimulation. Core protein inhibited the apoptotic cascade downstream from caspase 8 and upstream from caspase 3. Next, to clarify more direct mechanisms of this effect, mRNA levels of several bcl-2-related genes were examined. An RNase protection assay showed that the mRNA of bcl-xl increased in the core-producing cells. We showed that this increase was mediated by the enhancement of bcl-x promoter activity by core protein through an extracellular-regulated kinase pathway. These results suggest that core protein inhibits apoptosis at the mitochondria level through augmentation of Bcl-x expression, resulting in an inhibition of caspase 3 activation
Mouse Neuroinvasive Phenotype of West Nile Virus Strains Varies Depending upon Virus Genotype

AbstractDespite recent advances in the genetics of West Nile (WN) virus, relatively little is known about the molecular basis of virulence of this virus. In particular, although the genotype of the WN virus strain that was recently introduced into North America has been determined, there have been few experimental studies on the virulence phenotype of the virus. We compared genetic and neurovirulence properties of 19 strains of WN virus, including 2 from North America, and observed significant differences in their neuroinvasive phenotype in mice and hamsters that correlated with virus genotype. Virus isolated in North America was found to be highly neuroinvasive with a lack of age-related resistance to infection in mice normally associated with mosquito-borne flaviviruses
An Altered Cellular Response to Interferon and Up-Regulation of Interleukin-8 Induced by the Hepatitis C Viral Protein NS5A Uncovered by Microarray Analysis

AbstractThere is evidence for an inhibition of interferon-α antiviral activity by the hepatitis C viral protein, NS5A. To identify the mechanisms through which NS5A blocks interferon activity, we compared the gene expression profile of interferon-treated Huh7 cells, stably expressing NS5A with control, using microarrays. Following interferon treatment, 50 genes were up-regulated by at least twofold in control clones, whereas induction of 9 of the 50 genes was significantly reduced in NS5A-expressing clones. The strongest effect of NS5A on interferon response was observed for the OAS-p69 gene. Remarkably, Huh7 cells expressing NS5A showed an up-regulation of interleukin-8. Up-regulation of interleukin-8 was also observed upon transient expression of NS5A mutants isolated from patients responsive or resistant to interferon therapy. Addition of interleukin-8 to Huh7 cells inhibited the antiviral activity of interferon and, similarly to NS5A, reduced the induction by interferon-α of selective genes including OAS-p69. Our findings provide a mechanism for NS5A-mediated interferon resistance
Stabilization and Functional Impairment of the Tumor Suppressor p53 by the Human Papillomavirus Type 16 E7 Oncoprotein

AbstractThe p53 tumor suppressor is stabilized in cells expressing the human papillomavirus type 16 (HPV-16) E7 oncoprotein. In contrast, expression of the HPV-16 E6 protein inactivates p53 by targeting it for proteasomal degradation. Since p53 activation is associated with protein accumulation we investigated the biochemical mechanisms and biological consequences of p53 stabilization in HPV-16 E7-expressing cells. Transcriptional reporter assays, expression profiling studies using cDNA arrays, and immunoblot analyses of known p53 target genes suggest that p53 remains transcriptionally inert in E7-expressing cells. The stabilized p53 in E7-expressing cells is in a wild-type conformation and the same number of phospho-forms is present. Furthermore, E7 expression does not alter p53 localization or generally block nuclear export or proteasomal degradation of p53. Moreover, the stabilized p53 remains susceptible to mdm2-induced proteasome-mediated degradation, and exogenous transfected p53 is transcriptionally active in E7-expressing cells. Taken together, these results suggest that E7 can interfere with the normal turnover of p53 but that the resulting increase of p53 has no detectable transcriptional consequences on the p53 targets that we investigated
Primary Isolated Human Brain Microvascular Endothelial Cells Express Diverse HIV/SIV-Associated Chemokine Coreceptors and DC-SIGN and L-SIGN

AbstractChemokines have received increasing attention due to their inhibitory activities on human immunodeficiency virus type-1 (HIV-1) and simian immunodeficiency virus (SIV) replication and the potential for chemokine receptors to assist in HIV-1/SIV entry into permissive cells. Besides CD4, which is the major receptor for HIV-1 and SIV, a number of chemokine receptors including but not limited to APJ, CCR3, CXCR4, and CCR5 may be coreceptors for HIV-1/SIV, not only in peripheral blood and lymphoid tissues but also in the central nervous system (CNS). The present studies reveal the lack of CD4, but the significant expression of various chemokine receptors, APJ, CCR3, CXCR4, and CCR5, plus C-type lectins DC-SIGN and L-SIGN on isolated primary human brain microvascular endothelial cells (MVECs). As these MVECs do not express CD4, this suggests a CD4-independent HIV/SIV entry/infection of these cells, which are the major cells constituting the human blood–brain barrier. We also found that chemokines for cognate chemokine receptors individually were unable to block binding of HIV-1 to brain MVECs. These results reveal that in primary isolated brain MVECs viral attachment is mediated by a possible previously unknown receptor(s) or by cooperative activity of various receptors. Moreover, mRNA transcripts for DC-SIGN/L-SIGN, as well as DC-SIGN protein expression, suggest the capability of MVECs to attach viral particles on cell surfaces, even though polyclonal antisera for DC-SIGN did not affect viral binding to these cells. These data will assist in further understanding lentiviral entry into the CNS
Replication of the RNA Segments of a Bipartite Viral Genome Is Coordinated by a Transactivating Subgenomic RNA

AbstractThe insect nodavirus Flock house virus (FHV) has a small genome divided between two segments of positive-sense RNA, RNA1 and RNA2. RNA1 encodes the RNA-dependent RNA polymerase (RdRp) catalytic subunit and templates the synthesis of a subgenomic RNA (RNA3) that encodes two small nonstructural proteins. Replication of RNA2, which encodes a precursor to the viral capsid proteins, suppresses RNA3 synthesis. Here we report that RNA1 mutants deficient in RNA3 synthesis failed to support RNA2 replication. This effect was not caused by alterations in the RdRp catalytic subunit nor by a lack of the proteins encoded by RNA3. Furthermore, RNA3 supplied in trans from an exogenous source restored RNA2 replication. These data indicate that RNA3 transactivates the replication of RNA2, a novel property for a viral RNA. We propose that the RNA3 dependence of RNA2 replication serves to coordinate replication of the FHV genome segments
Autographa californica M Nucleopolyhedrovirus ProV-CATH is Activated during Infected Cell Death

AbstractV-CATH, a cathepsin L-like cysteine protease encoded by the baculovirus Autographa californica M nucleopolyhedrovirus, has been shown to play an essential role in host liquefaction. Similar to cellular cathepsin L, V-CATH is synthesized as an inactive proenzyme and is activated by cleavage of the propeptide. Previous studies indicated that removal of the propeptide was rapid, occurring as soon as the protein could be detected by Western blot, 22 h postinfection. We found, however, that these results reflected artifactual processing of the proenzyme. When the protease inhibitor E-64 was used to prevent this aberration, we found that proV-CATH accumulated in infected cells and activation did not begin until the onset of cell death, at approximately 80 h postinfection. Western blot analysis of fractions of live and dead cells isolated by fluorescence-activated cell sorting revealed that mature V-CATH was found only in dead cells. The regulation of activation of proV-CATH, therefore, was quite different from that of cellular cathepsins. Acridine orange staining revealed that lysosome integrity was lost in dead cells, an occurrence that could lead to the activation of proV-CATH by lysosomal proteases
Mechanisms of Protection Induced by Attenuated Simian Immunodeficiency Virus V. No Evidence for Lymphocyte-Regulated Cytokine Responses upon Rechallenge

AbstractTo determine whether attenuated simian immunodeficiency virus (SIV) vaccines confer protection against superinfection via secondary cellular immune responses, we searched for markers of immune activation following rechallenge. Productive infection with either attenuated SIVmacC8 or wild-type SIVmacJ5 resulted in a transient increase in T-lymphocyte CD25 and Mafa-DR expression. A pronounced increase in the frequency of FAS+ CD8+ lymphocytes was observed following SIVmacJ5 infection only. A transient increase in lymphocytes positive for intracellular IFN-γ and IL-4 was observed following primary infection with either virus. In contrast, lymphocytes positive for intracellular IL-2 were reduced. Following SIVmacJ5 challenge of SIVmacC8-infected vaccinees, no evidence of detectable superinfection was obtained. Rechallenge of vaccinees did not alter the frequency of activated peripheral T-lymphocytes, perturb cytokine profiles, or generate an anamnestic antibody response. These data do not support the hypothesis that protection conferred by live attenuated SIV is mediated by the induction of vigorous T-cell responses upon rechallenge
Target-Cell-Derived tRNA-like Primers for Reverse Transcription Support Retroviral Infection at Low Efficiency

AbstractReverse transcription of a retroviral genome takes place in the cytoplasm of an infected cell by a process primed by a producer-cell-derived tRNA annealed to an 18-nucleotide primer-binding site (PBS). By an assay involving primer complementation of PBS-mutated vectors we analyzed whether tRNA primers derived from the target cell can sustain reverse transcription during murine leukemia virus (MLV) infection. Transduction efficiencies were 4–5 orders of magnitude below those of comparable producer-cell complementations. However, successful usage of a target-cell-derived tRNA primer was proven by cases of correction of single mismatches between Akv-MLV vectors and complementary tRNA primers toward the primer sequence in the integrated vector. Thus, target-cell-derived tRNA-like primers are able to initiate first-strand cDNA synthesis and plus-strand transfer leading to a complete provirus, suggesting that endogenous tRNAs from the infected cell may also have access to the intracellular viral complex at that step of the replication cycle