pinecone/core-2020-05-10-deduplication huggingface dataset:
Integrin α2β1 Mediates the Cell Attachment of the Rotavirus Neuraminidase-Resistant Variant nar3

AbstractIt was previously reported that integrins α2β1, α4β1, and αXβ2 are involved in rotavirus cell infection. In this work we studied the role of integrin subunits α2, α4, and β2 on the attachment of rotaviruses RRV and nar3 to MA104 cells. Integrin α2β1 was found to serve as the binding receptor for the neuraminidase-resistant virus nar3, whereas the neuraminidase-sensitive strain RRV interacted with this integrin at a postattachment step. It was shown that nar3 binds α2β1 through the DGE integrin-recognition motif located in the virus surface protein VP5. Integrin subunits α4 and β2 do not seem to be involved in the initial cell binding of either virus
Recombinant Bovine Adenovirus Type 3 Expressing Bovine Viral Diarrhea Virus Glycoprotein E2 Induces an Immune Response in Cotton Rats

AbstractRecombinant bovine adenovirus is being developed as a live vector for animal vaccination and for human gene therapy. In this study, two replication-competent bovine adenovirus 3 (BAV-3) recombinants (BAV331 and BAV338) expressing bovine viral diarrhea virus (BVDV) glycoprotein E2 in the early region 3 (E3) of BAV-3 were constructed. Recombinant BAV331 contains chemically synthesized E2 gene (nucleotides modified to remove internal cryptic splice sites) under the control of BAV-3 E3/major late promoter (MLP), while recombinant BAV338 contains original E2 gene under the control of human cytomegalovirus immediate early promoter. Since E2, a class I membrane glycoprotein, does not contain its own signal peptide sequence at the 5′ end, the bovine herpesvirus 1 (BHV-1) glycoprotein D signal sequence was fused in frame to the E2 open reading frame (ORF) for proper processing of the E2 glycoprotein in both the recombinant viruses. Recombinant E2 protein expressed by BAV331 and BAV338 recombinant viruses was recognized by E2-specific monoclonal antibodies as a 53-kDa protein, which also formed dimer with an apparent molecular weight of 94 kDa. Insertion of an E2-expression cassette in the E3 region did not effect the replication of recombinant BAV-3s. Intranasal immunization of cotton rats with these recombinant viruses generated E2-specific IgA and IgG responses at the mucosal surfaces and in the serum. In summary, these results show that the pestivirus glycoprotein can be expressed efficiently by BAV-3. In addition, mucosal immunization with replication-competent recombinant bovine adenovirus 3 can induce a specific immune response against the expressed antigen
Ser2194 Is a Highly Conserved Major Phosphorylation Site of the Hepatitis C Virus Nonstructural Protein NS5A

AbstractPhosphorylation of the nonstructural NS5A protein is highly conserved among hepatitis C virus (HCV) genotypes. However, the precise site or sites of phosphorylation of NS5A have not been determined, and the functional significance of phosphorylation remains unknown. Here, we showed by two-dimensional phosphopeptide mapping that a protein kinase or kinases present in yeast, insect, and mammalian cells phosphorylated a highly purified HCV genotype 1b NS5A from insect cells on identical serine residues. We identified a major phosphopeptide (corresponding to amino acids 2193–2212 of the HCV 1b polyprotein) by using negative-ion electrospray ionization–microcapillary high performance liquid chromatography–mass spectrometry. The elution time of the phosphopeptide determined by negative-ion electrospray ionization–mass spectrometry corresponded with the elution time of the majority of 32P-label that was incorporated into the phosphopeptide by an in vitro kinase reaction. Subsequent analysis of the peak fraction by automated positive-ion electrospray ionization–tandem mass spectrometry revealed that Ser2194 was the major phosphorylated residue on the phosphopeptide GpSPPSLASSSASQLSAPSLK. Substitution for Ser2194 with Ala resulted in the concomitant disappearance of major in vivo phosphorylated peptides. Ser2194 and surrounding amino acids are highly conserved in all HCV genotypes, suggesting NS5A phosphorylation at Ser2194 may be an important mechanism for modulating NS5A biological functions
Differential Budding Efficiencies of Human T-Cell Leukemia Virus Type I (HTLV-I) Gag and Gag-Pro Polyproteins from Insect and Mammalian Cells

AbstractIn this study, we examined the ability of human T-cell leukemia virus type I (HTLV-I) Gag and Gag-Pro to assemble immature virus-like particles (VLPs) and bud from insect and mammalian cells. Transmission electron microscopy of insect cells infected with a recombinant baculovirus carrying the entire gag gene revealed that Pr53Gag is targeted to the plasma membrane, where it extensively accumulates and forms electron-dense evaginations. However, no particles could be detected either inside the cells or in the culture supernatants. With the Gag-Pro-expressing construct, we observed HTLV-I-specific cytoplasmic proteolysis of the Gag precursor, but again no particle released in the culture supernatants. Transmission electron microscopic analysis of insect cells expressing Gag-Pro polyprotein revealed large vacuoles in the cytoplasm and no budding particles at the plasma membrane. In contrast, human immunodeficiency virus type 1 Gag polyprotein expressed in insect cells is able to release VLPs. These data showed that unlike other retroviruses, Pr53Gag is unable to be released as immature VLPs from insect cells. To determine whether the block in particle budding and release is due to an intrinsic property of Pr53Gag or the absence of essential cellular factors in insect cells, we expressed Gag and Gag-Pro polyproteins in human 293 cells. The results indicate that Pr53Gag and p24 capsid are released within particles into the culture supernatants of human 293 cells. We found that the myristylation of the N-terminal glycine residue is essential for Gag release. Altogether, these results strongly suggest that the proper assembly of HTLV-I particles is dependent on mammalian host cell factors
Equine Herpesvirus 1 (EHV-1) Glycoprotein M: Effect of Deletions of Transmembrane Domains

AbstractEquine herpesvirus 1 (EHV-1) recombinants that carry either a deletion of glycoprotein M (gM) or express mutant forms of gM were constructed. The recombinants were derived from strain Kentucky A (KyA), which also lacks genes encoding gE and gI. Plaques on RK13 cells induced by the gM-negative KyA were reduced in size by 80%, but plaque sizes were restored to wild-type levels on gM-expressing cells. Electron microscopic studies revealed a massive defect in virus release after the deletion of gM in the gE- and gI-negative KyA, which was caused by a block in secondary envelopment of virions at Golgi vesicles. Recombinant KyA expressing mutant gM with deletions of predicted transmembrane domains was generated and characterized. It was shown that mutant gM was expressed and formed dimeric and oligomeric structures. However, subcellular localization of mutant gM proteins differed from that of wild-type gM. Mutant glycoproteins were not transported to the Golgi network and consequently were not incorporated into the envelope of extracellular virions. Also, a small plaque phenotype of mutant viruses that was indistinguishable from that of the gM-negative KyA was observed. Plaque sizes of mutant viruses were restored to wild-type levels by plating onto RK13 cells constitutively expressing full-length EHV-1 gM, indicating that mutant proteins did not exert a transdominant negative effect on wild-type gM
Herpesvirus Saimiri Pathogenicity Enhanced by Thymidine Kinase of Herpes Simplex Virus

AbstractHerpesvirus saimiri can be used as an efficient gene expression vector for human T lymphocytes and thus may allow applications in experimental leukemia therapy. We constructed recombinant viruses for the functional expression of the thymidine kinase (TK) of herpes simplex virus type 1 (HSV) as a suicide gene. These viruses reliably allowed the targeted elimination of transduced nonpermissive human T cells in vitro after the administration of ganciclovir. To test the reliability of this function under the most stringent permissive conditions, in this study we analyzed the influence of the prodrugs ganciclovir and acyclovir in common marmosets on the acute leukemogenesis induced by either wild-type herpesvirus saimiri C488 or by a recombinant derivative expressing TK of HSV. Antiviral drug treatment did not influence the rapid development of acute disease. In contrast, the presence of the HSV tk gene resulted in a faster disease progression. In addition, HSV TK-expressing viruses showed faster replication than wild-type virus in culture at low serum concentrations. Thus, HSV TK accelerates the replication of herpesvirus saimiri and enhances its pathogenicity. This should be generally considered when HSV TK is applied as a transgene in replication-competent DNA virus vectors for gene therapy
Latency-Associated Sense Transcripts Are Expressed during in Vitro Human Cytomegalovirus Productive Infection

AbstractPublished studies have identified novel sense transcripts in latent human cytomegalovirus (HCMV) infection. These cytomegalovirus latency-associated transcripts (CLTs) have start sites upstream from the MIE gene productive start site (PSS). We evaluated the expression of the sense CLTs in an in vitro HCMV productive infection. Transcripts with initiation sites upstream from the PSS were detected in infected human fibroblasts using reverse transcription–polymerase chain reaction and 5′ rapid amplification of cDNA ends (RACE) analysis. DNA sequencing of 5′ RACE PCR products confirmed that the start sites were consistent with sense CLT expression. Furthermore, ribonuclease protection assay analysis showed that transcripts initiating at the latent start site-1 were most abundant at late times postinfection after transcription from the PSS had decreased. In addition, transcription consistent with sense CLT expression was identified in HCMV-infected dTHP-1 cells, monocyte-derived macrophages, and endothelial cells, as well as in clinical isolate-infected human fibroblasts. These findings clearly demonstrate the expression of sense CLTs during in vitro HCMV infection
Human T-Cell Leukemia Virus Type 1 Tax Protein Activates Transcription through AP-1 Site by Inducing DNA Binding Activity in T Cells

AbstractHuman T-cell leukemia virus type 1 (HTLV-1) Tax protein induces the expression of various family members of the transcription factor AP-1, such as c-Jun, JunD, c-Fos, and Fra-1, at the level of RNA expression in T cells. We examined the activity of Tax in transcription through AP-1-binding sites (AP-1 site) in T cells. Transient transfection studies showed that Tax activated the expression of a luciferase gene regulated by two copies of an AP-1 site in the human Jurkat T-cell line. Tax activates the expression of viral and cellular genes through two different enhancers: a cAMP-responsive (CRE)-like element and a κB element. Two Tax mutants differentially activated expression of these two elements. Tax703 preferentially activated the κB element but not the CRE-like one, whereas TaxM22 showed the reverse. In addition, Tax703 and Tax, but not TaxM22, converted cell growth of a mouse T-cell line from being interleukin (IL)-2-dependent to being IL-2-independent. Unlike the wild-type Tax, Tax703 and TaxM22 only weakly activated the AP-1 site in the T-cell line. Thus, Tax seems to activate the AP-1 site via mechanisms distinct from those of κB or CRE-like elements, and the activation of the AP-1 site is dispensable for IL-2-independent growth of CTLL-2. Electrophoretic mobility shift assays showed that Tax induced strong binding activity to an AP-1 site in CTLL-2, whereas Tax703 did not, indicating that the induction of binding activity to the AP-1 site is essential for the transcriptional activation by Tax. The binding complex induced by Tax in CTLL-2 contained JunD and Fra-2. Other AP-1 proteins were undetectable. Activation of transcription through the AP-1 site in Jurkat cells by JunD and/or Fra-2 was weak. c-Jun, JunB, and c-Fos activation was greater, although the level was still less than that with Tax. Thus, the induction of AP-1 mRNA by Tax may not be sufficient for a complete activation of AP-1 site by Tax. Our results suggest that Tax activates the transcription of cellular genes with AP-1 sites by inducing the DNA-binding activity of AP-1 proteins in T cells, a mechanism distinct from those of CRE-like and κB elements
Temporal Loss of Nef-Epitope CTL Recognition following Macaque Lipopeptide Immunization and SIV Challenge

AbstractTo address the subtle interactions between antiviral cytotoxic T-cell (CTL) immune responses and the evolution of viral quasispecies variants in vivo, we performed a longitudinal study in a simian immunodeficiency virus (SIV)-infected rhesus macaque that had a long experimental SIV infection before developing simian AIDS. Before being infected with SIV, this animal was immunized with a mixture of seven lipopeptides derived from SIV Nef and Gag proteins and showed a bispecific antiviral CTL response directed toward Nef 169–178 and 211–225 peptides. After SIV infection, CTL activity against the Nef 169–178 epitope was no longer detectable, as assessed from peripheral blood mononuclear cells stimulated by autologous SIV. CTL activity against the 211–225 epitope was lost after 3 months, and an additional CTL response to the amino acids 112–119 Nef epitope emerged. Analysis of the Nef proviral sequence revealed the presence of immune escape variants first in the 211–225 epitope and much later in the 112–119 epitope. In contrast, epitope 169–178 showed only two mutations among all viral sequencing performed. We conclude that in this macaque, bispecific CTL exerted a strong selective pressure and escape virus mutants finally emerged. We identified CTL recognizing a conserved Nef epitope 112–119 (SYKLAIDM), essential for viral replication, which could be associated with a prolonged AIDS-free period. These results stress the importance of the induction of broader multispecific CTLs directed against highly conserved and functional T-cell epitopes by vaccination, with the aim of keeping HIV infection in check
Complete Nucleotide Sequence of the Chiba Virus Genome and Functional Expression of the 3C-Like Protease in Escherichia coli

AbstractWe cloned the genome RNA of the Chiba virus (ChV; Hu/NLV/Chiba 407/1987/JP) and determined its complete nucleotide sequence. The genome is predicted to be a positive-sense, single-stranded RNA of 7697 bases, excluding a poly(A) tract. Comparison of the nucleotide and amino acid sequences with those of other members of the species Norwalk virus (NV) revealed that ChV belongs to genogroup I NV. The ChV genome contains three open reading frames (ORFs). A large 5′-terminal ORF (ORF1) encodes a polyprotein with 1785 amino acids that are likely processed into functional proteins, including RNA helicase, VPg, protease, and RNA-dependent RNA polymerase. ORF2 encodes the capsid protein with 544 amino acids, and a small 3′-terminal ORF (ORF3) encodes a basic protein with 208 amino acids. The amino acid sequences of five cleavage sites in ORF1 are highly conserved compared with those of other members of NV. When expressed in Escherichia coli, the glutathione-S-transferase (GST) fusion protein of the ChV protease connected via a short peptide containing a human rhinovirus 3C protease cleavage site was cleaved into GST and the protease; however, this cleavage did not occur when the Cys mutation was introduced into the putative active site of the protease. Moreover, the ChV protease recognized and cleaved the predicted proteolytic sites between VPg and protease and between protease and RNA polymerase. Therefore, the ChV protease expressed in E. coli retained an enzymatic activity and a substrate specificity similar to that of the human rhinovirus 3C protease
Neutralization of Human Papillomavirus (HPV) Pseudovirions: A Novel and Efficient Approach to Detect and Characterize HPV Neutralizing Antibodies

AbstractThe development of vaccines against human papillomaviruses (HPVs) has long been hampered by the inability to grow HPVs in tissue culture and the lack of an efficient neutralization assay. To date, less than 10% of more than 100 different HPV types can be grown in athymic and “SCID” mouse xenograft systems or raft culture systems. Recently, the in vitro generation of HPV pseudovirions and their use in neutralization assays were demonstrated. The major shortcomings of the current approaches to HPV neutralization are the lack of HPV virions for most types for the xenograft methods and the time-consuming and inefficient generation of infective pseudovirions for the latter methods, which precludes their use in large-scale HPV clinical trials or epidemiological studies. We describe here a novel and efficient approach to generating pseudovirions in which HPV virus-like particles (VLPs) are coupled to the β-lactamase gene as a reporter. We show that it is not necessary to encapsidate the reporter gene constructs into the pseudovirions. Using sera from human volunteers immunized with HPV-11 VLPs expressed in yeast, we demonstrate that our novel neutralization assay compares favorably with the athymic mouse neutralization assay. Furthermore, our assay was used to define neutralizing monoclonal antibodies to HPV-6, which were previously unknown
Role of M Protein Aggregation in Defective Assembly of Temperature-Sensitive M Protein Mutants of Vesicular Stomatitis Virus

AbstractThe goal of these experiments was to determine the steps in virus assembly that are defective at the nonpermissive temperature in temperature-sensitive (ts) matrix (M) protein mutants of vesicular stomatitis virus. It has been proposed that mutations in M protein either reduce the binding affinity for nucleocapsids or lead to aggregation, reducing the amount of M protein available for virus assembly. Cytosolic or membrane-derived M proteins from wild-type VSV and two ts M protein mutant viruses, tsM301 and tsO23, as well as a revertant of tsO23 virus, O23R1, were analyzed for binding to nucleocapsid–M protein (NCM) complexes and for M protein aggregation. The experiments presented here showed that ts M proteins synthesized at the nonpermissive temperature were capable of binding to nucleocapsids and that aggregation of ts M proteins did not reduce the amount of soluble M protein below the amount required for assembly of the O23R1 virus. Instead, the most pronounced defect in ts M proteins was in the ability of membrane-derived M proteins to be solubilized in the presence of the detergent Triton X-100. It is proposed that this detergent-insoluble form of M protein interferes with a step necessary to initiate assembly of NCM complexes. A similar detergent, Triton X-114, caused aggregation of membrane-derived wild-type M protein, disproving an earlier proposal that membrane-derived M protein behaves like an integral membrane protein in the presence of Triton X-114. Aggregation of wild-type M protein in the presence of Triton X-100 could be induced by incubation at 37°C with a high-molecular-weight fraction isolated from uninfected cells by sucrose gradient centrifugation. These results implicate host components in inducing M protein aggregation
Tobravirus 2b Protein Acts in Trans to Facilitate Transmission by Nematodes

AbstractAnalysis of RNA2 of TRV PaY4 showed it to be recombinant, carrying 3′-terminal sequences derived from RNA1. Virus produced using an infectious cDNA clone of PaY4 RNA2 was nematode transmissible, demonstrating that natural TRV recombinant isolates are not necessarily defective. Mutations introduced into PaY4 RNA2 showed that the 2b gene, but not the 2c gene, is required for transmission by both Paratrichodorus pachydermus and P. anemones nematodes. Experiments examined whether infection of plants with two different virus clones would impact upon nematode transmission of either virus. Simultaneous inoculation with TRV clones expressing green or red fluorescent proteins revealed that mixing of the two virus populations did not occur, although, in roots, adjacent cells were found containing green- or red-tagged viruses. Subsequently, in similar experiments it was found that a TRV PaY4 2b mutant was transmitted when combined with wild-type TRV PaY4. Also, transmission of a 2b mutant of an in vitro TRV/PEBV recombinant virus (TRV-C1) occurred after coinfection with wild-type virus. Thus, the tobravirus 2b transmission protein is trans-acting. Although TRV PaY4 and TRV PpK20 are both transmitted by P. pachydermus, a 2b mutant of TRV PaY4 was not transmitted when coinoculated to plants with TRV PpK20
Herpes Simplex Virus Type 1 Latency in the Murine Nervous System Is Associated with Oxidative Damage to Neurons

AbstractThe pathological consequences of herpes simplex virus type 1 (HSV-1) latency in the nervous system are not well understood. To determine whether acute and latent HSV-1 infections of the nervous system are associated with oxidative damage, mice were inoculated with HSV-1 by the corneal route, and the extent of viral infection and oxidative damage in trigeminal ganglia and brain was assessed at 7, 90, and 220 days after inoculation. Abundant HSV-1 protein expression in the nervous system was observed in neurons and non-neuronal cells at 7 days after inoculation, consistent with viral replication and spread through the trigeminal and olfactory systems. Acute HSV-1 infection was associated with focal, neuronal and non-neuronal 4-hydroxy-2-nonenal- and 8-hydroxyguanosine-specific immunoreactivity, indicating oxidative damage. Rare HSV-1 antigen-positive cells were observed at 90 and 220 days after inoculation; however, widespread HSV-1 latency-associated transcript expression was detected, consistent with latent HSV-1 infection in the nervous system. HSV-1 latency was detected predominantly in the trigeminal ganglia, brainstem, olfactory bulbs, and temporal cortex. Latent HSV-1 infection was associated with focal chronic inflammation and consistently detectable evidence of oxidative damage involving primarily neurons. These results indicate that both acute and latent HSV-1 infections in the murine nervous system are associated with oxidative damage
Change in Receptor-Binding Specificity of Recent Human Influenza A Viruses (H3N2): A Single Amino Acid Change in Hemagglutinin Altered Its Recognition of Sialyloligosaccharides

AbstractHuman H3N2 influenza A viruses were known to preferentially bind to sialic acid (SA) in α2,6Gal linkage on red blood cells (RBC). However, H3N2 viruses isolated in MDCK cells after 1992 did not agglutinate chicken RBC (CRBC). Experiments with point-mutated hemagglutinin (HA) of A/Aichi/51/92, one of these viruses, revealed that an amino acid change from Glu to Asp at position 190 (E190D) was responsible for the loss of ability to bind to CRBC. A/Aichi/51/92 did not agglutinate CRBC treated with α2,3-sialidase, suggesting that SAα2,3Gal on CRBC might not inhibit the binding of the virus to SAα2,6Gal on CRBC. However, the virus agglutinated derivatized CRBC resialylated with SAα2,6Galβ1,4GlcNAc. These findings suggested that the E190D change might have rendered the HA able to distinguish sialyloligosaccharides on the derivatized CRBC containing the SAα2,6Galβ1,4GlcNAc sequence from those on the native CRBC