pinecone/core-2020-05-10-deduplication huggingface dataset:
Herpes Simplex Virus 1 Late Gene Expression Is Preferentially Inhibited during Infection of the TAF250 Mutant ts13 Cell Line

AbstractA key component of the polymerase II transcription machinery is the transcription factor TFIID, a complex that contains the TATA-box binding protein and several (10–12) associated factors designated as TAFs (TBP-associated factors). ts13 cells, which contain a temperature-sensitive mutant in TAF250, the largest subunit of TFIID, exhibit promoter-specific defects in gene expression at the nonpermissive temperature, suggesting that individual TAFs are required for transcription of specific subsets of eukaryotic genes. Herpes simplex virus 1 (HSV-1) does not replicate in ts13 cells at the nonpermissive temperature, but the point at which the replicative process is blocked is not known. We used the TAF250 defect in ts13 cells to investigate the role of TAF250 in the expression of HSV-1 genes of each temporal class. At a low m.o.i., expression of most immediate-early mRNAs is reduced at the nonpermissive temperature, and consequently, there is little expression of early genes and no viral DNA replication. In contrast, at high m.o.i., expression of immediate-early genes is unaffected by the TAF250 defect and is not dependent on de novo viral protein synthesis. Early genes and early proteins are produced under these conditions, and viral DNA replication ensues, albeit at somewhat reduced levels. In contrast, late gene expression and late protein synthesis are severely restricted, even in the presence of appreciable viral DNA replication. Thus the lack of late protein synthesis is responsible for the inability of HSV-1 to replicate in ts13 cells at the nonpermissive temperature. Further, it appears that late viral gene expression may be preferentially inhibited by the TAF250 mutation in ts13 cells
The Vaccinia Virus A36R Protein Is a Type Ib Membrane Protein Present on Intracellular but Not Extracellular Enveloped Virus Particles

AbstractVaccinia virus gene A36R encodes a 45-kDa protein that is conserved in orthopoxviruses. A virus lacking the A36R protein formed a small plaque, was unable to induce the polymerization of actin tails, and was avirulent in vivo. Here we present a further characterization of the A36R protein by in vitro transcription and translation and analysis of infected cells by confocal microscopy and immunoelectron microscopy of cryosections using a monoclonal antibody raised against the C-terminal domain of the A36R protein. Translation of the A36R mRNA in vitro produced a protein of the same size whether or not the translation reaction was performed in the presence of canine pancreatic microsomes. However, the polypeptide synthesized in the presence of microsomes was associated integrally with the membrane and was sensitive to digestion by exogenous protease without permeabilization of the membrane with detergent, indicating that the majority of the protein is exposed on the outside of the vesicle. Consistent with this, immunofluorescent analysis of virus-infected cells demonstrated that the C-terminal domain of A36R was not exposed on the cell surface but was detected once the cell membrane was permeabilized. Immunoelectron microscopy of cryosections of infected cells showed that the protein was absent from IMV particles but present on intracellular enveloped virus (IEV) particles, predominantly on the cytosolic face of the IEV outer membrane. Where cell-associated enveloped virus (CEV) particles were attached to the cell surface, the A36R protein was detected only on the cytosolic surface of the plasma membrane where the virus particle remained attached to the cell and not elsewhere on the plasma membrane or on the CEV particle. A36R and actin copurified with EEV particles due to the association of fragments of cellular membranes with the EEV particles. Therefore, A36R represents the first example of a virus-encoded protein that is present on IEV but not CEV particles
Multidrug Resistance Genotypes (Insertions in the β3–β4 Finger Subdomain and MDR Mutations) of HIV-1 Reverse Transcriptase from Extensively Treated Patients: Incidence and Association with Other Resistance Mutations

AbstractMultiple nucleoside resistance involves specific mutational patterns of the HIV-1 pol gene that are independent of the classic mutations conferring resistance to individual dideoxynucleosides. These include a cluster of five mutations in the reverse-transcriptase (RT) coding region (A62V, V75I, F77L, F116Y, and Q151M) generally referred to as multidrug resistance (MDR) mutations, and insertions of one or several amino acid residues between codons 67 and 70 of RT, a flexible region joining two antiparrallel β sheets (β3–β4 insertions). The objectives of this study were (i) to determine the prevalence of multidrug resistance genotypes (MDR mutations and β3–β4 insertions) in a cohort of 632 patients who were extensively pretreated with anti-HIV drugs and not responding to their current antiretroviral therapy, and (ii) to analyze the association of multidrug resistance genotypes with other resistance mutations in the RT and protease genes. Among viruses sequenced from these patients, 15 (2.4%) of them contained an insertion and 2 (0.3%) contained a deletion in the β3–β4 finger subdomain of RT. In 9 cases, the insertion was associated with a D67S, G, or E mutation. In addition, we identified 13 (2.1%) viruses harboring specific MDR mutations (mainly Q151M and/or A62V, V75I, F116Y). Interestingly, the A62V mutation was found in 6 of the 15 strains with an insertion, whereas the other MDR mutations were not observed in insertion mutant strains. Especially high levels of resistance to zidovudine were observed for viruses with a β3–β4 insertion in the background of A62V, L210W, and T215Y. Otherwise, MDR mutations and β3–β4 insertions were found in association with the classic mutations conferring resistance to zidovudine, lamivudine, nonnucleoside RT inhibitors, and protease inhibitors, according to treatment history. Finally, we observed a genome with a deletion of codon 70 associated with a Q151M MDR mutation. These data suggest that the emergence of HIV-1 multidrug resistance, which may occur in various genetic contexts, poses a challenging problem in formulating treatment strategies
Sequences Flanking the cAMP Responsive Core of the HTLV-I Tax Response Elements Influence CREB Protease Sensitivity

AbstractThe human T cell leukemia virus type I (HTLV-I) Tax protein activates transcription from the viral long terminal repeat and select cellular promoters by interacting with cellular DNA-binding proteins. The HTLV-I promoter contains three copies of a Tax-responsive element (TRE-1), each of which possesses a core cAMP response element (CRE). The cAMP response element-binding protein, CREB, binds TRE-1 and mediates Tax association with, and transactivation of, the viral promoter. These activities depend on DNA sequences that flank the core CRE. Although CREs are found in a variety of cellular promoters, cellular CREs vary in sequence from TRE-1, especially in the flanking regions, and are generally not Tax responsive. The molecular basis for differential Tax responsiveness of viral and cellular CREs has not been determined. Here we demonstrate that the conformation of CREB is influenced by the nucleotide sequence of its DNA-binding element. CREB showed altered sensitivity to V8, chymotrypsin, and trypsin proteases when bound to the HTLV-I TRE-1 element as compared to the rat somatostatin CRE element. The phosphorylation state of CREB did not influence its protease sensitivity on either element. Sequences flanking the core CRE-binding site in each element were found to specify protease sensitivity. Since the TRE-1-flanking sequences also modulate Tax association with CREB, and Tax transactivation of CREB-dependent LTR transcription, these results suggest that CREB conformation may determine the ability of Tax to bind CREB
Crystallization of Newcastle Disease Virus Hemagglutinin-Neuraminidase Glycoprotein

AbstractThe hemagglutinin-neuraminidase (HN) glycoprotein of Newcastle disease virus was isolated by cleaving HN (cHN) from reconstituted virosome with chymotrypsin. N-terminal sequence analysis of the purified cHN showed that chymotrypsin cleavage had occurred at amino acid 123, freeing the C-terminal 454 amino acids. The purified cHN retained its neuraminidase and receptor binding activities and reacted with specific monoclonal antibodies, showing that the isolated cHN was biologically and antigenically functional. The crystals of the cHN were obtained in acetate buffer (pH 4.6) containing polyethylene glycol 3350 and ammonium sulfate and belong to the orthorhombic space group P212121 with unit cell dimension of approximately a = 72 Å, b = 78 Å, and c = 198 Å. Crystals of cHN grown in the presence of sialic acid (Neu5Ac) were grown in HEPES buffer (pH 6.2) containing polyethylene glycol 3350 and belong to the hexagonal space groups P61 or P65 with unit cell dimensions of a = b = 137.5 Å and c = 116.6Å. The orthorhombic crystals produced in this study diffract X rays to at least 2.0-Å resolution, thereby setting the stage for the solution of the three-dimensional structure of the HN glycoprotein of a paramyxovirus
Mutational Analysis of the Bovine Respiratory Syncytial Virus Nucleocapsid Protein Using a Minigenome System: Mutations That Affect Encapsidation, RNA Synthesis, and Interaction with the Phosphoprotein

AbstractThe nucleocapsid (N) protein of bovine respiratory syncytial virus (BRSV) is a multifunctional protein that plays a central role in transcription and replication of viral genomic RNA. To investigate the domains and specific residues involved in different N activities, we generated a total of 27 deletion and 12 point mutants of the N protein. These mutants were characterized using an intracellular BRSV-CAT minigenome replication system for the ability to (1) direct minigenome RNA synthesis, (2) direct minigenome encapsidation, and (3) form a complex with the phosphoprotein (P). The mutations tested were defective in synthesis of RNA from the BRSV-CAT minigenome template with the exception of the following: a deletion involving the first N-terminal amino acid and mutations involving conservative substitution at the second amino acid and at certain internal cysteine residues. Micrococcal nuclease enzyme protection assays showed that mutations involving amino acids 1–364 of the 391-amino-acid N protein prevented minigenome encapsidation. Thus the BRSV N protein has a C-terminal, 27-amino-acid tail that is not required for encapsidation. Interestingly, two of the mutations that ablated encapsidation did not greatly affect RNA synthesis; the mutant involving deletion of the N-terminal amino acid and the mutant involving a substitution at position 2. This finding indicates that the formation of a nucleocapsid sufficient to protect the RNA from nuclease is not required for template function. Coimmunoprecipitation of N and P using N- or P-specific antiserum revealed two regions of the N protein that are important for association with the P protein: a central portion of 244–290 amino acids and a C-terminal portion of 338–364 amino acids
Interaction of the Papillomavirus E2 Protein with Mitotic Chromosomes

AbstractThe bovine papillomavirus E2 transactivator protein is a multifunctional protein that activates viral transcription, cooperates in initiation of viral DNA replication, and is required for long-term episomal maintenance of viral genomes. We have shown previously that the E2 transactivator protein and bovine papillomavirus type 1 genomes are associated with mitotic chromosomes and have proposed that E2 links the genomes to cellular chromosomes to ensure segregation to daughter nuclei. In this study, we show that E2 is associated with cellular chromosomes at all stages of mitosis. We also further map the regions of E2 that are required for this association. The transactivation domain of E2 is necessary and sufficient to mediate the interaction with mitotic chromosomes; the DNA binding domain, and the flexible hinge region that separates the two domains, is not required. Furthermore, mutation of previously identified phosphorylation sites (serine residues 235, 298, and 301) has no effect on the ability of the E2 protein to bind mitotic chromosomes
HTLV-1 Tax Oncoprotein Binds to DNA Topoisomerase I and Inhibits Its Catalytic Activity

AbstractHTLV-1 Tax oncoprotein exerts pleiotropic effects on cellular regulatory systems, such as transcription and the cell cycle, through the interaction with various cellular factors. During our search for additional cellular targets of Tax using a yeast two-hybrid screening system, we isolated a cDNA encoding human DNA topoisomerase I. Tax was demonstrated to bind to topoisomerase I in vitro, and the Tax-topoisomerase I complex was also detected in HTLV-1-infected T-cells by immunoprecipitation. Furthermore, Tax inhibited the catalytic activity of topoisomerase I as measured by relaxation of supercoiled DNA, although complete inhibition was not observed under the conditions used. The binding of topoisomerase I to DNA was inhibited by the addition of the wild type of Tax but not by a mutant of Tax that cannot bind to topoisomerase I. Consistent with these observations, expression of Tax induced an in vivo reduction of the covalent association of topoisomerase I with chromosomal DNA, which accumulates in the presence of camptothecin. These results suggest that Tax has a novel potential to affect various cellular processes such as transcription and maintenance of genomic stability, in which DNA topoisomerase I is involved
Fusion Protein of the Paramyxovirus SV5: Destabilizing and Stabilizing Mutants of Fusion Activation

AbstractThe fusion (F) protein of the paramyxovirus SV5 strain W3A causes syncytium formation without coexpression of the SV5 hemagglutinin-neuraminidase (HN) glycoprotein, whereas the F protein of the SV5 strain WR requires coexpression of HN for fusion activity. SV5 strains W3A and WR differ by three amino acid residues at positions 22, 443, and 516. The W3A F protein residues P22, S443, and V516 were changed to amino acids found in the WR F protein (L22, P443, and A516, respectively). Three single-mutants, three double-mutants, and the triple-mutant were constructed, expressed, and assayed for fusion using three different assays. Mutant P22L did not cause fusion under physiological conditions, but fusion was activated at elevated temperatures. Compared with the W3A F protein, mutant S443P enhanced the fusion kinetics with a faster rate and greater extent, and had a lower activation temperature. Mutant V516A had little effect on F protein-mediated fusion. The double-mutant P22L,S443P was capable of causing fusion, suggesting that the two mutations have opposing effects on fusion activation. The WR F protein requires coexpression of HN to cause fusion at 37°C, and does not cause fusion at 37°C when coexpressed with influenza virus hemagglutinin (HA); however, at elevated temperatures coexpression of WR F protein with HA resulted in fusion activation. In the crystal structure of the core trimer of the SV5 F protein (Baker, K. A., Dutch, R. E., Lamb, R.A., and Jardetzky, T. S. (1999). Mol. Cell 3, 309–319), S443 is the last residue (with interpretable electron density) in an extended chain region and the temperature factor for S443 is high, suggesting conformational flexibility at this point. Thus, the presence of prolines at residues 22 and 443 may destabilize the F protein and thereby decrease the energy required to trigger the presumptive conformational change to the fusion-active state
Hepatitis B Virus Assembly Is Sensitive to Changes in the Cytosolic S Loop of the Envelope Proteins

AbstractAmong the three related L, M, and S envelope proteins of the hepatitis B virus (HBV), the L and S polypeptides are required for virion production. Whereas the pivotal function of the pre-S region of L in nucleocapsid envelopment has been established, the contribution of its S domain and the S protein is less clear. In this study, we evaluated the role of the cytosolic S loop, common to L and S, in HBV assembly by performing mutagenesis experiments. To distinguish between the effect of the mutations on either envelope or virion formation, we investigated the ability of the mutants to assemble into secretable subviral empty envelopes and to replace the wild-type proteins in virion maturation, respectively. Virion production was found to be blocked by each of the secretion-competent deletion and substitution mutants SΔ35–39, SΔ40–46, SΔ50–56, and Sς56–59, while an insertion within the loop is tolerated. Surprisingly, single mutations of the arginines terminating the loop had an opposite effect: while a conservative exchange of Arg-73 still allowed virion formation, the same mutation of Arg-79 did not. The critical sequences and/or structural requirements of the cytosolic S loop involved in nucleocapsid envelopment primarily act in the S background. These findings can be related to a model for a synergistical function of both L and S proteins in HBV morphogenesis
Characterization of a Novel Human Immunodeficiency Virus Type 1 Neutralizable Epitope within the Immunodominant Region of gp41

AbstractPreviously, we generated human monoclonal antibodies using peripheral blood mononuclear cells from an asymptomatic human immunodeficiency virus type 1 (HIV-1)-seropositive donor. One of these monoclonal antibodies (designated clone 3, CL3) recognized 10 amino acids (GCSGKLICTT) within the immunodominant region (cluster I) of the transmembrane envelope glycoprotein gp41 and neutralized infection of target cells with different laboratory isolates. Because the epitope recognized by CL3 has two cysteine residues that could potentially produce a disulfide loop in gp41, we analyzed binding of our monoclonal antibody to the cyclic and linear motif of the peptide sequence IWGCSGKLICTTAVP (residues 600–614). The CL3 antibody did not bind to the synthetic cyclic peptide but did recognize the linear form. Two polyclonal rabbit sera against both the linear and cyclic peptides were then generated. Both antisera bound to viral glycoproteins gp41 and gp160, but neither sera neutralized HIV-1 laboratory isolates. Using a set of alanine-substituted IWGCSGKLICTTAV peptides, we analyzed binding of polyclonal antisera and CL3. The profile of binding of polyclonal antisera to these peptides was different from that of CL3 to the same peptides. This suggests that CL3 recognized a unique neutralizable core epitope, which was not immunogenic in either the cyclic or the linear IWGCSGKLICTTAVP peptides used as immunogens in the rabbits
Helix-Destabilizing Properties of the Baculovirus Single-Stranded DNA-Binding Protein (LEF-3)

AbstractThe helix-destabilizing properties of a single-stranded DNA-binding protein, LEF-3, of Bombyx mori nucleopolyhedrovirus (BmNPV) were studied. Partial duplexes of DNA containing single-stranded (ss) tails of different sizes and orientations were used as substrates for assay of the unwinding ability of LEF-3. Upon noncooperative binding to ssDNA, LEF-3 was capable of unwinding the duplexes with 5′ ss tails. However, it did not cause melting of the duplexes containing 3′ ss tails, even at oversaturation of ssDNA adjacent to the duplexes. Upon cooperative binding to long ss tails, LEF-3 also produced the polar melting effect; it unwound the duplexes with long 5′ ss tails, but not those with long 3′ ss tails. These data suggest that LEF-3 has a preferential direction for entry into duplex DNA, namely 5′ to 3′ with respect to the bound DNA strand. In agreement with its polarity, LEF-3 efficiently melted the primer-template complexes which serve as substrates for DNA polymerases. However, the formation of a complex with viral DNA polymerase before addition of LEF-3 protected the primer-templates from the destabilization effect of LEF-3. Although the destabilization effect of LEF-3 was highly sensitive to monovalent and divalent salts, the protein was capable of melting DNA duplexes in a polar manner at physiological conditions, i.e., 30°C in 0.15 M NaCl. Therefore, the polar destabilization effect of LEF-3 seems to be physiologically important and may be connected, in particular, with the polar action of viral helicase holoenzyme during baculovirus replication
Viral Recombinant Vaccines to the E6 and E7 Antigens of HPV-16

AbstractMost cancerous lesions of the uterine cervix are linked to persistent infections with human papillomaviruses (HPV), most notably HPV-16 or -18. Vaccine-induced immune responses to the HPV early antigens E6 and E7, which contribute to cell transformation and are thus expressed in these cervical cancers, could potentially eradicate malignant cells. We generated recombinant vaccines based on E1-deleted adenovirus human strain 5 or on vaccinia virus strain Copenhagen expressing either the E6 or E7 oncoproteins of HPV-16. The different vaccines were compared in two experimental mouse tumor models employing Balb/c or C57Bl/6 mice. Data presented here demonstrate that depending on the model either CD4+ or CD8+ T cells provide protection to tumor cell challenge, resulting in striking differences in the efficacy of the four vaccines under investigation
Minor Capsid Protein of Human Genital Papillomaviruses Contains Subdominant, Cross-Neutralizing Epitopes

AbstractVaccination with virus-like particles (VLP), comprising both L1 and L2 of human papillomavirus (HPV) genital types 6, 16, and 18, induces predominantly type-specific neutralizing antibodies. L2 polypeptide vaccines protect animals against experimental challenge with homologous papillomavirus and cross-reactive epitopes are present in HPV L2. To assess L2-specific cross-neutralization of HPV genotypes, sheep were immunized with purified, bacterially expressed HPV6, 16, or 18 L2. In addition to neutralizing the homologous HPV type in vitro, antisera to each HPV L2 also cross-neutralized both heterologous HPV types. This suggests that unlike VLP-based prophylactic HPV vaccines, an L2 polypeptide vaccine may provide broad-spectrum protection
The Herpes Simplex Virus 1 Us11 Protein Cooperates with Suboptimal Amounts of Human Immunodeficiency Virus Type 1 (HIV-1) Rev Protein to Rescue HIV-1 Production

AbstractThe human immunodeficiency virus type 1 (HIV-1) RNA-binding Rev protein governs the expression of structural and enzymatic viral proteins at a posttranscriptional level. Binding of Rev to the stem-loop IIB (SLIIB) sequence of the Rev-response element (RRE) within unspliced and singly spliced viral mRNAs and to the nuclear export signal-binding receptor, hCRM1 (or exportin 1), is required for the export of these transcripts to the cytoplasm. We have previously shown that herpes simplex virus type 1 (HSV-1) RNA-binding Us11 protein is able to bind the RRE and substitute for Rev in inducing the expression of HIV-1 envelope glycoproteins. We show here that Us11 cannot substitute for Rev in rescuing a rev-deleted HIV-1 provirus. However, HIV-1 production is observed when Us11 is expressed with suboptimal amounts of Rev. An in vivo RNA–protein binding assay indicates that Us11 is unable to directly interact with the SLIIB RNA but can bind Rev assembled on that stem–loop structure. This association of US11 with Rev, which was confirmed by in vivo coimmunoprecipitation and GST-pulldown assays, therefore underlies a biological Us11–Rev cooperation. Furthermore this cooperation was shown to remain susceptible to the effect of leptomycin B, which blocks the binding of hCRM1 to the nuclear export signal of Rev. These observations performed with intron-containing constructs provide evidence that HSV-1 Us11 protein is not directly involved in the cytoplasmic accumulation of viral mRNAs but may be rather acting as an auxiliary protein, thus allowing this retroviral protein to fulfill the nuclear export of these transcripts and to rescue HIV-1 production