pinecone/core-2020-05-10-deduplication huggingface dataset:
Comparison ofin Vitroandin VivoInfectivity of Different Clade B HIV-1 Envelope Chimeric Simian/Human Immunodeficiency Viruses inMacaca mulatta

AbstractThe use of HIV-1env/SIVmacchimeric viruses expressing divergent HIV-1 envelopes of clinical isolates, facilitates homologous and heterologous evaluation of various recombinant HIV-1 envelope vaccine candidates in lower primates. In this study we compare thein vitroandin vivoinfectivity, via intravenous (IV) and intravaginal (IVAG) routes of infection, of stocks of chimeric viruses expressingenvfrom four different clade B HIV-1 isolates. The TCID50/ml was 7.1 × 104, 1.0 × 104, 6.3 × 104, and 1.2 × 103for SHIVsf13, SHIVHan2, SHIVNM-3rn, and SHIVW6.1D, respectively, with a MID50/ml upon IV inoculation of 3.2 × 103, 3.2 × 104, 3.2 × 104, and 3.2 × 103, respectively. The same SHIVsf13stock was infectious after IVAG administration, requiring a 300-fold higher virus dose. Plasma antigenemia and cell-associated viremia were generally highest at weeks 2 or 4 after infection and decreased to subdetectable levels after 8–12 weeks. All infected animals tested developed anti-HIV-1 gp120 antibodies. Inoculated virus dose showed no (linear) quantitative correlation with cellular virus load, duration of viremia, plasma antigenemia, and anti-gp120 antibody titers. No significant changes in peripheral blood CD4 cell levels were observed and none of the animals has shown evidence of disease progression to date (i.e., 13 months postinfection). Fourin vivopassages of cell-associated SHIVW6.1Ddid not result in increased virulence. Vaccine development studies in macaques monkeys have become feasible with the use of various clade B HIV-1envSHIV chimeras
Chimeric HIV-1 Virus-like Particles Containing gp120 Epitopes as a Result of a Ribosomal Frameshift Elicit Gag- and SU-Specific Murine Cytotoxic T-Lymphocyte Activities

AbstractInsect cell expression of the HIV-1 Gag precursor protein by recombinant baculoviruses results in the assembly and budding of noninfectious virus-like particles (VLPs). The VLPs resemble immature virus in ultrastructural morphology and can be purified by conventional retroviral techniques. The virus-like appearance of the particles suggested that they could be used to package additional peptides. The retroviral frameshift mechanism was used to translate thepolgene products by expressing additional genetic information as chimeric Gag–Pol fusion proteins. Sequences encoding the carboxyl 65% of the HIV-1 surface glycoprotein (gp120, SU) were inserted into the Gag–Pol reading frame immediately downstream of the Gag stop codon. The assembly and budding of large quantities of Gag and chimeric Gag–SU VLPs were observed by standard transmission electron microscopy. The presence of gp120 epitopes in the Gag–SU VLPs was confirmed by immunoelectron microscopy and Western blot analysis using monoclonal anti-gp120 antibodies. Mice inoculated with the Gag–SU pseudovirions developed cytotoxic lymphocyte responses to both HIV-1 Gag and Env epitopes yet humoral immune responses only to Gag epitopes. The chimeric Gag–SU particles may have applications as vaccines or immunotherapeutic treatments for HIV-1 infection. In addition, the frameshift mechanism can be applied to the packaging of other viral or cellular proteins
Identification of a Novel Peptide Substrate of HSV-1 Protease Using Substrate Phage Display

AbstractThe method of substrate phage display was used to select a preferred substrate from three monovalent display libraries using the HSV-1 protease. The display libraries consisted of four random amino acids, six random amino acids, and a biased library containing four amino acids from the P side of the HSV-1 maturation site followed by four random amino acids. A series of consensus peptides was synthesized based upon the results from these screens and tested in peptide cleavage assays. An eight amino acid consensus peptide (LVLASSSF) derived from the phage results was cleaved as efficiently as a 20-mer maturation site peptide. The selected amino acid sequences also allowed the design of a four amino acid paranitroanilide substrate for continuous assay of HSV-1 protease. Similar to HCMV protease, these results define P4 to P1 as a minimal substrate recognition domain for the HSV-1 protease and suggest that P4 to P1 is the minimal substrate domain which all herpesvirus proteases recognize
The Role of Proximal and Distal Sequence Variations in the Presentation of an Immunodominant CTL Epitope Encoded by the Ecotropic AK7 MuLV

AbstractAnemv-14-derived, replication-competent ecotropic murine leukemia virus [MuLV], designated AK7, was previously cloned from the AKXL-5 recombinant inbred mouse strain and partially characterized. While genetically encoding for an envelope-derived immunodominant CTL epitope [KSPWFTTL] located in the transmembrane region of p15TM, this virus, unlike theemv-11-derived virus AKR623, fails to be efficiently recognized by AKR/Gross MuLV-specific cytotoxic T lymphocytes [CTL]. AK7 thus provides the opportunity to study the role of retroviral sequence variations that are located outside of the immunodominant epitope as a mechanism of escape from CTL-mediated immune survellience. In an attempt to identify which region[s] of the AK7 genome could account for its ability to evade efficient recognition by AKR/Gross MuLV-specific CTL, we have constructed recombinant murine retroviruses. The direct influence of a sequence variation twelve amino acids N-terminal to KSPWFTTL was explored with the use of chimeric viruses and determined not to significantly impair the presentation of KSPWFTTL to AKR/Gross MuLV-specific CTL. The long terminal repeat [LTR] derived from the AK7 virus, which possesses only one copy of the 99-base pair transcriptional enhancer in the U3 region, in contrast to AKR623 that possesses two copies of the tandem direct repeat enhancers, was also analyzed for its influence on the presentation of KSPWFTTL. Interestingly, our data indicate that the enhancer region derived from AK7 negatively influences the presentation of KSPWFTTL in the context of a recombinant AKR623 virus
Characterization of the Reverse Transcriptase of a Human Immunodeficiency Virus Type 1 Group O Isolate

AbstractThe catalytic properties and sensitivity to different inhibitors have been determined for the reverse transcriptase (RT) of group O human immunodeficiency virus type 1 (HIV-1). The RT-coding region was cloned from a new HIV-1 group O isolate from Spain, expressed inEscherichia coli,and purified by affinity chromatography. This new RT showed 79% amino acid sequence identity with the corresponding enzyme of group M subtype B strain BH10. The two enzymes showed very similar kinetics of RNA-dependent DNA polymerization using homopolymeric template-primers and RNase H specific activity. Inhibitor sensitivity to ddTTP and 3′-azido-2′,3′-dideoxythymidine triphosphate (AZTTP) was also similar for both enzymes. However, the two enzymes differed dramatically in their sensitivity to several inhibitors. While the RT of the BH10 isolate was sensitive to nevirapine and loviride (IC50ranged from 0.16 to 8.2 μM,depending on the substrates used), the enzyme of the Spanish HIV-1 group O isolate showed high-level resistance to those compounds (IC50> 200 μM). The amino acid sequence of the RT of group O HIV-1 contains three amino acids (Cys-181, Glu-179, and Gly-98), which are found in group M subtype B strains resistant to nonnucleoside RT inhibitors. The recombinant group O HIV-1 RT should be useful for studies aimed at discovering and designing drugs directed toward group O isolates of HIV-1
Expression of Influenza B Virus Hemagglutinin Containing Multibasic Residue Cleavage Sites

AbstractThe hemagglutinin (HA) protein of influenza B virus contains a single arginine residue at its cleavage site and the HA0precursor is not cleaved to the HA1and HA2subunits by tissue culture cell-associated proteases. To investigate if an HA protein could be obtained that could be cleaved by an endogenous cellular protease, the cDNA for HA of influenza B/MD/59 virus was subjected to site-specific mutagenesis. Three HA mutant proteins were constructed, through substitution or insertion of arginine residues, that have 4, 5, or 6 basic residues at their cleavage sites. Chemical cross-linking studies indicated that all three HA cleavage site mutants could oligomerize to a trimeric species, like WT HA. The three HA cleavage site mutant proteins were efficiently transported to the cell surface and bound erythrocytes in hemadsorption assays. The mutants were cleaved at a low level to HA1and HA2by an endogenous host cell protease and cleavage could be increased somewhat by addition of exogenous trypsin. The fusogenic activities of the HA cleavage site mutants were assessed in comparison to the WT HA protein by determining their syncytium formation ability and by using an R18 lipid-mixing assay and a NBD-taurine aqueous-content mixing assay. While the fusion activity of the WT HA protein was dependent on exogenous trypsin to activate HA, the three HA cleavage site mutant proteins were able to induce fusion in the absence of trypsin when assayed with the R18 lipid-mixing and NBD-taurine aqueous-content mixing assays, but were unable to induce syncytium formation in either the presence or absence of exogenous trypsin. Our results suggest that while the presence of a subtilisin-like protease cleavage sequence at the influenza B virus HA1/HA2boundary does enable some HA0molecules to be cleaved intracellularly, it alone is not sufficient for efficient cleavage
Characterization of a Specific Region in the Hepatitis B Virus Enhancer I for the Efficient Expression of X Gene in the Hepatic Cell

AbstractHepatitis B virus (HBV) enhancer I has been shown to consist of severalcis-acting sequences for the HBV gene expression efficiently in certain types of cells. Transcriptional regulation of HBV X gene mediated by enhancer I might be one of the mechanisms by which HBV obtains hepatotropism. By mutagenesis analysis of enhancer I function in the enhancer I/X gene promoter complex, we characterized a specific transcriptional regulatory region (designated as a LSR element, nt 989–1030) of enhancer I for the X gene promoter by means of the transient transfection technique using hepatic and nonhepatic cells. Based on the analysis of protein factors interacting with the LSR element, liver-enriched transcriptional factors, HNF3 and HNF4 or retinoid X receptor α (RXRα), are probably implicated in the activity of enhancer I for the efficient expression of X gene through their interaction with the LSR element in the hepatic cell. Furthermore, the isolated LSR element was demonstrated to function alone as a specificcis-acting element and to be able to activate transcription from the X gene promoter efficiently in the hepatic cell in an orientation-independent manner
Coat Protein Gene Replacement Results in Whitefly Transmission of an Insect Nontransmissible Geminivirus Isolate

AbstractGeminiviruses are transmitted by whiteflies, leafhoppers, or treehoppers. The whitefly speciesBemisia tabaci(Gennadius) is the most efficient vector of Subgroup III geminiviruses. An isolate of Abutilon mosaic virus (AbMV), a bipartite geminivirus, was not detectable in the DNA extract from insects by Southern blot analysis, nor was the isolate transmissible by the B-biotype ofB. tabaci,although the virus DNA was amplified (by PCR) from some insects. In contrast, Sida golden mosaic virus (SiGMV-Co), a closely related geminivirus, was acquired and transmitted byB. tabacito various host plants. The coat protein of AbMV was replaced with that of SiGMV-Co. The resulting chimeric AbMV was acquired and transmitted to various host plants byB. tabaci, indicating that the coat protein plays an essential role in the transmission process byB. tabaci
HsN3 Proteasomal Subunit as a Target for Human Immunodeficiency Virus Type 1 Nef Protein

AbstractHIV-1 Nef protein is important for pathogenicity, but its biochemical function remains obscure. To clarify its role, a yeast two-hybrid system (ths) screening was utilized to identify Nef cellular partners. Of 79 yeast clones harboring cDNAs for putative Nef binding proteins, 27 (34%) contained the coding region for HsN3 proteasomal subunit. HsN3 behaved asbona fideNef partner in ths control crosses. Nef–HsN3 interaction was confirmed byin vitrobinding experiments. In particular, recombinant Nef was able to capture the HsN3 subunit from a natural proteasome preparation. In Nef, the interacting region was mapped within aa 34–143, which span the structured portion of the protein, including the SH3-binding domain. In HsN3, Nef-binding portion was restricted to aa 73–249, and the tract 219–249—reminiscent of SH3 domain N-terminal 3/5ths—was shown to be essential, though not sufficient. Attempts to purify a Nef–HsN3 complex from transfected COS7 cells were unsuccessful. However, Nef was found to markedly downregulate intracellular levels of both a coexpressed HsN3 and the endogenous simian homologue. These results suggest that Nef, by binding to a subunit, might alter proteasome function in infected cells
Characterization of the Transcription Map and Rev Activity of a Highly Cytopathic Feline Immunodeficiency Virus

AbstractA highly cytopathic feline immunodeficiency virus, FIV-Oma, was previously isolated from a nondomestic cat. In this report, we describe experiments to characterize its transcription map and examine its Rev activity. The temporal progression of viral gene expression is similar to that of HIV-1. The splicing pattern of viral transcripts was determined by sequence analysis of RT-PCR-amplified viral cDNAs.In vitrotranscription and translation of two putativerevcDNAs revealed that they encode at least one 22-kDa protein. The Rev-responsive element (RRE) of FIV-Oma, identified by computer-assisted RNA secondary structure analysis, was inserted into the intron of an HIV-1-derived reporter plasmid and used in a transient transfection assay for Rev activity. Cotransfection of the RRE construct with the tworevcDNA clones significantly increased the expression of the reporter gene linked to the RRE, indicating that both transcripts encode an active Rev protein. The Rev activity of FIV-Oma is 5 to 8 times higher than that of a domestic cat FIV isolate, FIV-PPR. Our experiments also demonstrate the heterologous interaction of FIV-PPR Rev with the FIV-Oma RRE, even though the RREs of the two viruses have very little nucleotide sequence identity
The Enigma of pX: A Host-Dependentcis-Acting Element with Variable Effects on Tombusvirus RNA Accumulation

AbstractTomato bushy stunt virus (TBSV) is a small isometric virus that contains a single-stranded RNA genome with five major genes. In this study, we have analyzed the importance of an additional small sixth open reading frame (ORF) of 207 nucleotides, designated pX, which resides at the 3′ end of the genome. Bioassays showed that deletions or additions of nucleotides at the 5′ end of the pX gene that were designed to disrupt the ORF, or site-specific inactivation of its start codon, all gave rise to TBSV mutants which were unable to accumulate to detectable levels in cucumber orNicotiana benthamianaprotoplasts. Although these results suggested a role for the putative pX protein, introduction of a premature stop codon in the pX gene had no strong negative effect. However, a comparable mutation that affected the same nucleotides without changing the predicted amino acid sequence greatly reduced RNA accumulation. Therefore, we hypothesize thatcis-acting RNA sequences within the pX gene, rather than the predicted protein influence genome accumulation. The requirement of thecis-acting pX ORF sequences appears to be host-dependent because comparisons revealed that subtle pX gene mutations that prohibited accumulation of TBSV RNA in cucumber orN. benthamiana,failed to interfere substantially with replication inChenopodium quinoaprotoplasts or plants. Irrespective of the host, thecis-acting pX gene sequences were dispensable on replicase-deficient RNAs that require helper TBSV for replicationin trans.In addition, the pX gene was not essential forin vitrotranslation of replicase proteins from genomic RNA. These results suggest that neither translation nor polymerase activity of the replicase proteins require pX gene sequences. However, it is possible that very early in the replication cycle of genomic RNAin vivo,the pX genecis-acting element is essential for some other unidentified function which involves interaction with one or more host components whose composition varies slightly between different plants
Transcriptional Analysis of Walleye Dermal Sarcoma Virus (WDSV)

AbstractWalleye dermal sarcoma virus (WDSV) is a complex retrovirus associated with dermal sarcomas of walleye that develop and regress on a seasonal basis. WDSV contains, in addition togag, pol,andenv,three open reading frames (ORFs) designated ORF A, ORF B, and ORF C. The polymerase chain reaction technique was used to amplify and clone cDNAs representing subgenomic viral mRNAs isolated from developing (fall) and regressing (spring) tumors. Nine different singly or multiply spliced viral transcripts were identified and all were found to utilize a common 5′ leader sequence. This leader sequence is spliced to thepol/envjunction or downstream ofenvto generate singly spliced transcripts. Multiply spliced transcripts contain the 5′ leader, the pol/env junction, and sequences derived from the 3′ end of the genome. One multiply spliced transcript was isolated with the potential to encode the full-length ORF A protein. In addition, WDSV produced mRNAs that utilize alternative splice acceptor sites which would allow synthesis of five variant forms of the ORF A protein. In contrast, the ORF B protein is postulated to arise from a singly spliced transcript with the potential to encode the entire open reading frame. Spliced subgenomic transcripts representing ORF C mRNAs were not identified, suggesting that ORF C may be encoded from the full-length viral genomic transcript. We estimate that at least a 100-fold lower amount of the accessory/regulatory subgenomic transcripts exists in developing vs regressing tumors. These results demonstrate that WDSV undergoes an elaborate pattern of mRNA splicing similar to that of other complex retroviruses
Adenovirus Infection Inactivates the Translational Inhibitors 4E-BP1 and 4E-BP2

AbstractInfection with many viruses results in the selective shutoff of host protein synthesis. A common target for virus interference with host protein synthesis is the cap-binding protein complex, eIF4F. The large subunit of the complex, eIF4G, is cleaved upon picornavirus (except cardiovirus) infection. Infection with adenovirus and influenza virus causes dephosphorylation of the cap-binding subunit, eIF4E. Recently, it has been shown that infection with poliovirus or encephalomyocarditis virus activates 4E-BP1, which is a specific inhibitor of eIF4E. Here we show that early in adenovirus infection, 4E-BP1 and its related protein 4E-BP2 are phosphorylated and hence inactivated. This is not consistent with a role of 4E-BPs in adenovirus-induced shutoff, but could explain the increase in protein synthesis reported early in infection. Phosphorylation of 4E-BP1 and 4E-BP2 is consistent with earlier findings in adenovirus-infected cells on the activation of the protein kinase p70S6k, whose phosphorylation lies on the same pathway as 4E-BPs, by E1A. Findings similar to those described here were reported for 4E-BP1 by D. Feigenblum and R.J. Schneider (1996,Mol. Cell. Biol.16, 5450–5457)
Activation of the Envelope Proteins by a Metalloproteinase Enables Attachment and Entry of the Hepatitis B Virus into T-Lymphocyte

AbstractPreviously, we identified an HBV binding factor (HBV-BF), a 50-kDa serum glycoprotein which interacts with HBV envelope proteins and which is also located in the membrane of normal human hepatocyte (A. Budkowskaet al.(1993)J. Virol.67, 4316). Here we show that HBV-BF is a neutral metalloproteinase which shares substrate specificity and properties with a newly described family of membrane type matrix metalloproteinases. HBV-BF treatment of the HBV resulted in the cleavage of the N-terminal part of the middle HBV envelope protein at the pre-S2(136–141) amino acid sequence VRGLYF/L (containing a single arginine cleavage site). HBV-BF affected the reactivity of the large HBV protein with pre-S1-specific MAbs, probably inducing the conformational change of the pre-S1 domain. The HBV-BF-digested virus remained morphologically intact with unchanged S antigenic determinants. The structural modifications of the viral envelope proteins induced by HBV-BF enabled cell membrane attachment and viral entry into the T-lymphocyte. Both processes were blocked by the metalloproteinase inhibitor 1,10 phenanthroline. Thus, the host-dependent proteolytic activation of the envelope proteins seems to be essential for the HBV entry into the cell. HBV-BF under a membrane bound or a secreted form could be (one of) the molecule(s) responsible for the HBV proteolytic activation
Paramyxovirus Fusion (F) Protein and Hemagglutinin–Neuraminidase (HN) Protein Interactions: Intracellular Retention of F and HN Does Not Affect Transport of the Homotypic HN or F Protein

AbstractTo investigate a possible intracellular coassociation of the paramyxovirus simian virus 5 (SV5) and human parainfluenza virus type 3 (HPIV-3) fusion (F) and hemagglutinin–neuraminidase (HN) glycoproteins in a living cell, without resorting to chemical crosslinking and antibody coimmunoprecipitation, we tagged the cytoplasmic N-terminus of SV5 HN with a RRRRR motif and HPIV-3 HN with a RRR motif for endoplasmic reticulum (ER) retention. In addition, we tagged the cytoplasmic C-terminus of SV5 and HPIV-3 F with a KK motif. The RRR- or RRRRR-tagged HN molecules were coexpressed in mammalian cells together with the homologous wt F proteins, and the KK-tagged F molecules were coexpressed with the homologous wt HN proteins, and in each case the transport of the wt F or HN molecules was investigated. The data suggest that an association of F and HN of sufficient affinity to alter the transport of the reporter molecule does not occur intracellularly in the ER or the Golgi apparatus
Determinants of Substrate Specificity in the NS3 Serine Proteinase of the Hepatitis C Virus

AbstractProcessing of the nonstructural polyprotein of the hepatitis C virus (HCV) requires the serine-type proteinase located in the amino-terminal domain of NS3. To identify residues within NS3 determining substrate specificity, a mutation analysis was performed. Using sequence alignments and three-dimensional structure predictions, amino acids assumed to be important for specificity were replaced and the enzymes were tested in an intracellulartrans-processing assay for their effects on cleavage of an NS4B-5B substrate. For some of the substitutions at positions 133, 134, 135, 136, 138, 152, 155, 157, and 169, slightly reduced processing efficiencies were observed but in no case was the substrate specificity altered. In contrast, substitutions of the phenylalanine at position 154 resulted in a modified cleavage pattern, suggesting an important role for this residue in substrate specificity. To substantiate this assumption, a panel of NS4B-5B substrates carrying different P1 residues at the NS4B/5A site were tested for cleavage by these altered proteinases. We found that substitution of Phe-154 by alanine, by valine, and particularly by threonine generated enzymes with the following affinities for aliphatic P1 residues: C > L > I > V for 154 F → A, C = L > I > V for 154 F → V and L > C > I > V for 154 F → T. Neither leucine nor isoleucine nor valine was accepted by the parental NS3 proteinase, showing that Phe-154 is an important determinant for substrate specificity. Furthermore, we present evidence that Ala-157 plays an additional but minor role for this property