pinecone/core-2020-05-10-deduplication huggingface dataset:
The Requirement of Varicella Zoster Virus Glycoprotein E (gE) for Viral Replication and Effects of Glycoprotein I on gE in Melanoma Cells

AbstractThe glycoprotein E (gE) of varicella zoster virus (VZV), encoded by ORF68, is the most abundant viral glycoprotein. In the current experiments, we demonstrated that ORF68 deletion was incompatible with recovery of infectious virus from VZV cosmids. Replacing ORF68 at a nonnative AvrII site in the genome restored infectivity. Further, we found that VZV gE could be expressed under the control of the Tet-On promoter in stably transfected melanoma cell lines (Met-gE cells) without evidence of toxicity. In these Met-gE cells, gE colocalized with γ-adaptin, a trans Golgi network marker, in perinuclear sites, but did not reach plasma membranes. In order to investigate how infection altered gE localization, we made a recombinant virus, vOka-MSPgE, with ORF68 from the VZV MSP strain. VZV MSP encodes a mutant gE protein (D150N) that lacks the mAb epitope, 3B3 (Santos et al., Virology 275, 306–317, 2000), whereas Met-gE protein binds mAb 3B3. Within 48 h after Met-gE cells were infected with vOka-MSPgE, the steady-state distribution of Met-gE protein extended beyond the perinuclear areas to other cytoplasmic sites and to plasma membranes. A second recombinant, vOka-MSPgE without gI (vOka-MSPgEΔgI), was constructed to investigate Met-gE protein distribution in the absence of gI. The redistribution of Met-gE protein which was observed by 48 h after vOka-MSPgE infection did not occur until 5 days (140 h) within vOka-MSPgEΔgI infected cells. After vOka-MSPgE infection of Met-gE cells, most Met-gE protein was in the final 94K mature form by 72 h. However, progression to predominance of mature gE was delayed in Met-gE cells infected with vOka-MSPgEΔgI. These observations confirm our hypothesis that VZV gE is essential, based upon the demonstration of restored infectivity after replacing ORF68 in a nonnative site in the genome, and provide further evidence of the role of gI in facilitating the maturation and intracellular distribution of this critical VZV glycoprotein
Analysis of Human Cytomegalovirus US3 Gene Products

AbstractSimilar to other herpesviruses, human cytomegalovirus remains in the infected host following resolution of the primary infection. The ability to persist in the host after primary infection is believed to be strongly influenced by the ability of HCMV to down-regulate immune recognition of infected cells. One of the genes contributing to immune evasion is the US3 gene. The US3 gene has been shown to retain major histocompatibility complex type I molecules in the endoplasmic reticulum. The US3 gene gives rise to three alternatively spliced RNAs which encode distinct but related proteins. Each of the alternatively spliced transcripts is present early in viral infection, suggesting that the encoded proteins play a role in the viral life cycle. We demonstrate that only the protein encoded by the unspliced US3 transcript is able to retain MHC class I heavy chains in the endoplasmic reticulum. The protein encoded by the singly spliced US3 transcript appears to be processed through the secretory pathway while the protein encoded by the doubly spliced transcript becomes localized to the Golgi apparatus. These experiments raise interesting questions about the functions of the smaller US3 proteins during viral infection in the host
HPV Episomal Copy Number Closely Correlates with Cell Size in Keratinocyte Monolayer Cultures

AbstractW12E keratinocytes maintaining episomal copies of HPV DNA were separated according to size by centrifugal elutriation. HPV DNA copy number was greatly increased in the largest, most differentiated cells of the population. The large cells with the highest HPV copy number also showed evidence of endoreduplication of host cell DNA. Other cell lines maintaining episomal copies of HPV18 and HPV31 were also tested with all lines showing similar results. The results demonstrate that increase in HPV DNA copy number correlates well with increased cell size, a fundamental marker of keratinocyte differentiation. The results also indicate that simple monolayer cultures may be useful for studying the relationship between differentiation, HPV DNA replication, and cell-cycle events
Cellular Protein Binds to Sequences near the 5′ Terminus of Potato virus X RNA That Are Important for Virus Replication

AbstractThe sequences in the 5′-nontranslated region (NTR) of Potato virus X (PVX) genomic RNA were previously reported to contain several regulatory elements that are required for genomic and subgenomic RNA accumulation. To investigate whether cellular proteins bind to these elements, we conducted electrophoretic mobility shift assays (EMSA) with protoplast protein extracts and RNA sequences from the PVX 5′-NTR. These analyses showed that the 5′ region of PVX positive-strand RNA formed complexes with cellular proteins. UV cross-linking studies of complexes formed with various deletions of the PVX RNA indicated that a 54-kDa cellular protein (p54) was bound to nt 1–46 at the 5′ terminus of PVX RNA. Site-directed mutations introduced within this 46-nt region further indicated that an ACCA sequence element located at nt 10–13 was important for optimal binding. In addition, mutations that decreased the affinity of the template RNA for the cellular factor decreased PVX plus-strand RNA accumulation in protoplasts. These studies suggest that the p54 may function in PVX RNA replication by binding to the 5′ terminus of the viral genomic RNA
A Nuclear Localization Signal and a Membrane Association Domain Contribute to the Cellular Localization of the Tobacco Mosaic Virus 126-kDa Replicase Protein

AbstractA transient expression system using onion epidermal cells was used to investigate domains of the Tobacco mosaic virus (TMV) 126-kDa replicase protein involved in cellular localization. Initially, a nuclear localization signal (NLS), identified within the amino-terminus of the 126-kDa protein, was investigated for its functionality using fusion constructs containing the green fluorescent protein (GFP). Fusion of the amino-terminal 70 amino acids of the 126-kDa protein, containing the NLS, to a β-glucuronidase-GFP open reading frame (ORF), directed the accumulation of fluorescence to the nucleus. In contrast, similar constructs lacking the NLS or containing a mutated NLS sequence failed to accumulate within the nucleus. Additional investigations using GFP fusion constructs containing the first 178 or 388 amino acids of the 126-kDa protein also displayed nuclear localization. However, fusion constructs encoding the first 781 amino acids or the entire 126-kDa ORF did not accumulate within the nucleus but instead associated with the endoplasmic reticulum (ER), forming spot-like inclusions. Thus, a dominant ER association domain exists between amino acids 388 and 781 of the 126-kDa protein. Interestingly, a full-length 126-kDa GFP fusion construct encoding a nonfunctional NLS mutation also localized to the ER but did not form inclusions. Furthermore, a TMV mutant containing the same nonfunctional NLS mutation failed to replicate in protoplasts. Together these findings suggest that both the NLS and the ER retention domain contribute to the functional localization of the 126-kDa protein
Transcription Mapping and Expression Patterns of Genes in the Major Immediate-Early Region of Kaposi's Sarcoma-Associated Herpesvirus

AbstractViral immediate-early (IE) genes are the first class of viral genes expressed during primary infection or reactivation from latency. They usually encode regulatory proteins that play crucial roles in viral life cycle. In a previous study, four regions in the KSHV genome were found to be actively transcribed in the immediate-early stage of viral reactivation in primary effusion lymphoma cells. Three immediate-early transcripts were characterized in these regions, as follows: mRNAs for ORF50 (KIE-1), ORF-45 (KIE-2), and ORF K4.2 (KIE-3) (F. X. Zhu, T. Cusano, and Y. Yuan, 1999, J. Virol. 73, 5556–5567). In the present study, we further analyzed the expression of genes in these IE regions in BC-1 and BCBL-1 cells. One of the immediate-early regions (KIE-1) that encompasses ORF50 and other genes was intensively studied to establish a detailed transcription map and expression patterns of genes in this region. This study led to identification of several novel IE transcripts in this region. They include a 2.6-kb mRNA which encodes ORF48/ORF29b, a family of transcripts that are complementary to ORF50 mRNA and a novel K8 IE mRNA of 1.5 kb. Together with the IE mRNA for ORF50 which was identified previously, four immediate-early genes have been mapped to KIE-1 region. Therefore, we would designate KIE-1 the major immediate-early region of KSHV. In addition, we showed that transcription of K8 gene is controlled by two promoters, yielding two transcripts, an immediate-early mRNA of 1.5 kb and a delayed-early mRNA of 1.3 kb
Lysine-Independent Ubiquitination of Epstein–Barr Virus LMP2A

AbstractLatent membrane protein 2A (LMP2A) of latent Epstein–Barr virus (EBV) specifically associates with HECT domain-containing Nedd4-family ubiquitin-protein ligases (E3s). Here we demonstrate that LMP2A is specifically ubiquitinated by the HECT domains of AIP4 and WWP2. Deletion and site-specific mutation of LMP2A indicates that LMP2A is ubiquitinated at its amino-terminus and is not ubiquitinated on lysine residues. LMP2A and LMP1, also encoded by EBV, are two of only four proteins that have been identified that are ubiquitinated at the amino-terminus, indicating that EBV may specifically target and utilize this host cell protein modification
Passive Transfer of Serum Antibodies Induced by BBG2Na, a Subunit Vaccine, in the Elderly Protects SCID Mouse Lungs Against Respiratory Syncytial Virus Challenge

AbstractRespiratory syncytial virus (RSV) is responsible for severe low respiratory tract infections in young infants and the elderly. To investigate whether BBG2Na, a recombinant subunit vaccine comprising aa 130–230 of the RSV G protein, induced protective Abs in subjects over 60 years during phase II clinical trial, pre- and postimmunization sera of individuals immunized with BBG2Na or placebo were transferred into SCID mice before RSV challenge. These sera dose-dependently reduced lung RSV titers. However at some points of serial dilutions, postimmunization sera of BBG2Na-immunized subjects only were significantly more efficient than the corresponding preimmunization sera, in agreement with the induction of an increased Ab response against multiple epitopes on RSV-A G protein. Thus, BBG2Na is immunogenic in the elderly and confers passive protection in mice after serum transfer. To our knowledge, this is the first description of protective Abs induced by a subunit vaccine in human
Contacts between Influenza Virus N9 Neuraminidase and Monoclonal Antibody NC10

AbstractWe are interested in studying how influenza virus escapes antibody inhibition. Based on the structure of the complex between N9 NA and monoclonal antibody NC10 Fab (R. L. Malby, W. R. Tulip, V. R. Harley, J. L. McKimm-Breschkin, W. G. Laver, R. G. Webster, and P. M. Colman, 1994, Structure 2, 733–746), we investigated the contribution made by individual amino acids to the stability of the complex. We made conservative changes in residues that are centrally located in the epitope and more drastic changes in peripheral contacts. The mutations made were N200L (removing an N-linked oligosaccharide), N329Q, N345Q, S370T, S372A, N400L, and K432M. Binding of each mutant to NC10 was quantitated by NA inhibition assays and ELISA. Except for N200L and N329Q, the mutants were inhibited by NC10 to the same extent as wild-type NA although with less affinity. The enzyme activity (Kcat) of N200L is 80% reduced, indicating a defect in folding or assembly; therefore, the loss in binding activity due to the missing sugar residue cannot be assessed. The Kd for N329Q is sixfold higher than for wild-type NA in the inhibition test, but the same as wild-type in ELISA, indicating a change in disposition of the antibody but no loss of affinity. The results show that the NC10 epitope can accommodate a change at any site and is not dominated by a few high-energy interactions as was found in the NC41 epitope. We propose that the difference lies in the contribution of buried water molecules to the NA-NC10 complex
Cloning and Genomic Characterization of Felis domesticus Papillomavirus Type 1

AbstractA novel papillomavirus was cloned from hyperkeratotic cutaneous lesions of a Persian domestic cat. The Felis domesticus papillomavirus (FdPV-1) genome counts 8300 bp and has a typical genome structure with an early region (E1, E2, E4, E6, E7), a late region (L1, L2), and a noncoding upstream regulatory region (URR or NCR1) between the end of L1 and the beginning of E6. The FdPV-1 also shows an unusual second noncoding region (NCR2) of 1.3 kb, situated between the end of E2 and the beginning of L2. This NCR2 is uniquely related to a similar region in the canine oral papillomavirus (COPV). Phylogenetic analysis places FdPV-1 together with COPV, the cottontail rabbit papillomavirus, human papillomavirus type 1 (HPV-1), and HPV-63 in the group of the benign cutaneous papillomaviruses. The position of FdPV-1 in the phylogenetic tree allows us to hypothesize that already in an early phase of the papillomavirus molecular evolution, a split occurred into viruses with a dual tropism primarily for cutaneous epithelia but also secondarily for mucosal surfaces, and viruses with a specific monotropism for mucosal surfaces. The close relationship between FdPV-1 and COPV, and between their Canidae and Felidae hosts, supports the hypothesis that papillomaviruses have speciated and coevolved together with their hosts throughout vertebrate evolution. A papillomavirus mutation rate of 0.73 to 0.96 × 10−8 nucleotide substitutions per base per year was calculated
New Targets for Antivirals: The Ribosomal A-Site and the Factors That Interact with It

AbstractMany viruses use programmed −1 ribosomal frameshifting to ensure the correct ratio of viral structural to enzymatic proteins. Alteration of frameshift efficiencies changes these ratios, in turn inhibiting viral particle assembly and virus propagation. Previous studies determined that anisomycin, a peptidyl transferase inhibitor, specifically inhibited −1 frameshifting and the ability of yeast cells to propagate the L-A and M1 dsRNA viruses (J. D. Dinman, M. J. Ruiz-Echevarria, K. Czaplinski, and S. W. Peltz, 1997, Proc. Natl. Acad. Sci. USA 94, 6606–6611). Here we show that preussin, a pyrollidine that is structurally similar to anisomycin (R. E. Schwartz, J. Liesch, O. Hensens, L. Zitano, S. Honeycutt, G. Garrity, R. A. Fromtling, J. Onishi, and R. Monaghan, 1988. J. Antibiot. (Tokyo) 41, 1774-−1779), also inhibits −1 programmed ribosomal frameshifting and virus propagation by acting at the same site or through the same mechanism as anisomycin. Since anisomycin is known to assert its effect at the ribosomal A-site, we undertook a pharmacogenetic analysis of mutants of trans-acting eukaryotic elongation factors (eEFs) that function at this region of the ribosome. Among mutants of eEF1A, a correlation is observed between resistance/susceptibility profiles to preussin and anisomycin, and these in turn correlate with programmed −1 ribosomal frameshifting efficiencies and killer virus phenotypes. Among mutants of eEF2, the extent of resistance to preussin correlates with resistance to sordarin, an eEF2 inhibitor. These results suggest that structural features associated with the ribosomal A-site and with the trans-acting factors that interact with it may present a new set of molecular targets for the rational design of antiviral compounds
Phylogenetic Analysis of the Complete Genome of 18 Norwalk-like Viruses

Abstract“Norwalk-like viruses” (NLV), a member of the family Caliciviridae, are the major causative agents of acute gastroenteritis and are genetically divided into two groups, genogroup I (GI) and genogroup II (GII). We have determined the complete nucleotide sequences of 10 new NLV strains. Using this information together with eight known NLV sequences, the criteria to further classify genotypes of NLV were investigated. Validation of the topological error based on the bootstrap value and the branch length (distance) allowed us to identify two potential subgenomic regions suitable for the genotyping. They were the putative 3D-like RNA-dependent RNA polymerase (polymerase) and the capsid N-terminal/Shell domains (capsid N/S domain). When the distance distribution analysis was performed, the polymerase-based classification did not separate the strains into internal clusters within the genogroup. Furthermore, a diversity plot analysis of the complete nucleotide sequences of WUG1, a NLV GI strain, and Saitama U1, a NLV GII strain, indicated that the genotype was different between the polymerase and capsid N/S domain, suggesting that these strains are the genetic recombinants. Therefore, polymerase is not suitable for genotyping. On the other hand, the clustering based on the capsid N/S domain successfully distinguished the NLV as well as the grouping based on the antigenicity, as determined by both antigen and antibody ELISAs with recombinant virus-like particles. As the nucleotide sequences of the primers for the capsid N/S domain are highly conserved among the NLV, the amplification of the unknown genotype can be easily performed. This method will facilitate global surveying as well as epidemiologic study on NLV
Genome Analysis of an Inducible Prophage and Prophage Remnants Integrated in the Streptococcus pyogenes Strain SF370

AbstractThe mitomycin C inducible prophage SF370.1 from the highly pathogenic M1 serotype Streptococcus pyogenes isolate SF370 showed a 41-kb-long genome whose genetic organization resembled that of SF11-like pac-site Siphoviridae. Its closest relative was prophage NIH1.1 from an M3 serotype S. pyogenes strain, followed by S. pneumoniae phage MM1 and Lactobacillus phage phig1e, Listeria phage A118, and Bacillus phage SPP1 in a gradient of relatedness. Sequence similarity with the previously described prophages SF370.2 and SF370.3 from the same polylysogenic SF370 strain were mainly limited to the tail fiber genes. As in these two other prophages, SF370.1 encoded likely lysogenic conversion genes between the phage lysin and the right attachment site. The genes encoded the pyrogenic exotoxin C of S. pyogenes and a protein sharing sequence similarity with both DNases and mitogenic factors. The screening of the SF370 genome revealed further prophage-like elements. A 13-kb-long phage remnant SF370.4 encoded lysogeny and DNA replication genes. A closely related prophage remnant was identified in S. pyogenes strain Manfredo at a corresponding genome position. The two prophages differed by internal indels and gene replacements. Four phage-like integrases were detected; three were still accompanied by likely repressor genes. All prophage elements were integrated into coding sequences. The phage sequences complemented the coding sequences in all cases. The DNA repair genes mutL and mutS were separated by the prophage remnant SF370.4; prophage SF370.1 and S. pneumoniae phage MM1 integrated into homologous chromosomal locations. The prophage sequences were interpreted with a hypothesis that predicts elements of cooperation and an arms race between phage and host genomes
Transcription Analysis of Streptococcus thermophilus Phages in the Lysogenic State

AbstractThe transcription of prophage genes was studied in two lysogenic Streptococcus thermophilus cells by Northern blot and primer-extension experiments. In the lysogen containing the cos-site phage Sfi21 only two gene regions of the prophage were transcribed. Within the lysogeny module an 1.6-kb-long mRNA started at the promoter of the phage repressor gene and covered also the next two genes, including a superinfection exclusion (sie) gene. A second, quantitatively more prominent 1-kb-long transcript was initiated at the promoter of the sie gene. Another prophage transcript of 1.6-kb length covered a group of genes without database matches that were located between the lysin gene and the right attachment site. The rest of the prophage genome was transcriptionally silent. A very similar transcription pattern was observed for a S. thermophilus lysogen containing the pac-site phage O1205 as a prophage. Prophages from pathogenic streptococci encode virulence genes downstream of the lysin gene. We speculate that temperate phages from lactic streptococci also encode nonessential phage genes (“lysogenic conversion genes”) in this region that increase the ecological fitness of the lysogen to further their own evolutionary success. A comparative genome analysis revealed that many temperate phages from low GC content Gram-positive bacteria encode a variable number of genes in that region and none was linked to known phage-related function. Prophages from pathogenic streptococci encode toxin genes in this region. In accordance with theoretical predictions on prophage–host genome interactions a prophage remnant was detected in S. thermophilus that had lost most of the prophage DNA while transcribed prophage genes were spared from the deletion process
Chitin Synthesis in Chlorovirus CVK2-Infected Chlorella Cells

AbstractHyaluronan synthesis in chlorovirus PBCV-1-infected Chlorella cells was previously reported (DeAngelis et al., 1997). In contrast, we report here on the detection, characterization, and expression of a gene for chitin synthase (chs) encoded by chlorovirus CVK2 isolated in Kyoto, Japan. The CVK2 chs gene encoding an open reading frame of 516 aa was expressed as early as 10 min postinfection (p.i.), peaked at 20–40 min p.i., and disappeared at 120–180 min p.i. The chitin polysaccharide began to accumulate as chitinase-sensitive, hair-like fibers on the outside of the virus-infected Chlorella cell wall by 30 min p.i. All chloroviruses without the gene for hyaluronan synthase (has) alternatively contained the chs gene, suggesting the importance of polysaccharide production in the course of virus infection. A few chloroviruses possessed both the chs and has genes and produced chitin and hyaluronan simultaneously. Polysaccharide accumulation on the algal surface may protect virus-infected algae from uptake by other organisms, such as protozoa. Since CVK2 was reported to encode two chitinases and one chitosanase, CVK2 is a very peculiar virus that encodes enzymes required for both the synthesis and the degradation of chitin materials
Ornithine Decarboxylase Encoded by Chlorella Virus PBCV-1

AbstractSequence analysis of the 330-kb genome of chlorella virus PBCV-1 revealed an open reading frame, A207R, which encodes a protein with 37–41% amino acid identity to ornithine decarboxylase (ODC) from many eukaryotic organisms. The a207r gene was cloned and the protein was expressed as a His-A207R fusion protein in Escherichia coli. The recombinant protein catalyzes pyridoxal 5′-phosphate-dependent decarboxylation of ornithine to putrescine, the first step in the polyamine biosynthetic pathway. The enzyme has a pH optimum of 9.0 and a temperature optimum of 42°C, and it requires dithiothreitol for maximal activity. The enzyme has a Km for ornithine of 0.78 mM and a specific activity of 100 μmol/min/mg protein. PBCV-1 ODC is quite sensitive to the competitive inhibitor L-arginine and the irreversible inhibitor difluoromethylarginine but it is less sensitive to the irreversible inhibitor difluoromethylornithine. The a207r gene is expressed both early and late in PBCV-1 infection and is highly conserved among the chlorella viruses. The 42-kDa PBCV-1 ODC (372 amino acids) is the smallest ODC in the databases and, to our knowledge, is the first virus-encoded ODC