pinecone/core-2020-05-10-deduplication huggingface dataset:
Cross-Clade-Specific Cytotoxic T Lymphocytes in HIV-1-Infected Children

AbstractWe studied cytotoxic T lymphocyte (CTL) cross-reactivity between human immunodeficiency virus type 1 (HIV-1) subtypes within a group of infants infected with either HIV-1 B or non-B clade. Fifteen children were infected with a clade B virus. Nine were infected with non-B virus, including two clade A, four clade D, two clade F, and one clade G. CTL activities fromin vitroactivated peripheral blood mononuclear cells were tested against autologous cell line infected with recombinant vaccinia viruses encoding for Env, Gag, Pol, or Nef proteins from a clade A or B isolate. HIV-1-specific CTL elicited from infection with clade B virus could lyse targets expressing clade A proteins, and vice versa. In infants with positive CTL responses, cross-clade recognition was predominant and was detected within 88% of the Pol, 83% of the Nef, 67% of the Gag, and 55% of the Env responders. Longitudinal studies showed that CTL cross-reactivity to both B and A targets was stable for several years. Elicitation of CTL reactivities capable of elimination of virus-infected cells is an important goal for the development of an efficient AIDS vaccine. The significant cross-reactivity of CTL shown in this study supports the concept that vaccines developed using a single-clade immunogen may be applicable to induce broadly reactive T cell responses
The Role of Nucleocapsid of HIV-1 in Virus Assembly

AbstractThe role of the nucleocapsid protein of HIV-1 Gag in virus assembly was investigated using Gag truncation mutants, a nucleocapsid deletion mutant, and point mutations in the nucleocapsid region of Gag, in transfected COS cells, and in stable T-cell lines. Consistent with previous investigations, a truncation containing only the matrix and capsid regions of Gag was unable to assemble efficiently into particles; also, the pelletable material released was lighter than the density of wild-type HIV-1. A deletion mutant lacking p7 nucleocapsid but containing the C-terminal p6 protein was also inefficient in particle release and released lighter particles, while a truncation containing only the first zinc finger of p7 could assemble more efficiently into virions. These results clearly show that p7 is indispensable for virus assembly and release. Some point mutations in the N-terminal basic domain and in the basic linker region between the two zinc fingers, which had been previously shown to have reduced RNA bindingin vitro[Schmalzbauer, E., Strack, B., Dannull, J., Guehmann, S., and Moelling, K. (1996).J. Virol.70: 771–777], were shown to reduce virus assembly dramatically when expressed in full-length viral clones. A fusion protein consisting of matrix and capsid fused to a heterologous viral protein known to have nonspecific RNA binding activity [Ribas, J. C., Fujimura, T., and Wickner, R. B. (1994)J. Biol. Chem.269: 28420–28428] released pelletable material slightly more efficiently than matrix and capsid alone, and these particles had density higher than matrix and capsid alone. These results demonstrate the essential role of HIV-1 nucleocapsid in the virus assembly process and show that the positively charged N terminus of p7 is critical for this role
Amino Acids 16–275 of Minute Virus of Mice NS1 Include a Domain That Specifically Binds (ACCA)2–3-Containing DNA

AbstractGST-NS1 purified fromEscherichia coliand insect cells binds double-strand DNA in an (ACCA)2–3-dependent fashion under similar ionic conditions, independent of the presence of anti-NS1 antisera or exogenously supplied ATP and interacts with single-strand DNA and RNA in a sequence-independent manner. An amino-terminal domain (amino acids 1–275) of NS1 [GST-NS1(1–275)], representing 41% of the full-length NS1 molecule, includes a domain that binds double-strand DNA in a sequence-specific manner at levels comparable to full-length GST-NS1, as well as single-strand DNA and RNA in a sequence-independent manner. The deletion of 15 additional amino-terminal amino acids yielded a molecule [GST-NS1(1–275)] that maintained (ACCA)2–3-specific double-strand DNA binding; however, this molecule was more sensitive to increasing ionic conditions than full-length GST-NS1 and GST-NS1(1–275)and could not be demonstrated to bind single-strand nucleic acids. A quantitative filter binding assay showed thatE. coli- and baculovirus-expressed GST-NS1 andE. coliGST-NS1(1–275)specifically bound double-strand DNA with similar equilibrium kinetics [as measured by their apparent equilibrium DNA binding constants (KD)], whereas GST-NS1(16–275)bound 4- to 8-fold less well
Internal Ribosomal Entry Site Scanning of the Poliovirus Polyprotein: Implications for Proteolytic Processing

AbstractBased on previous studies of dicistronic polioviruses carrying two internal ribosomal entry sites (IRESes), we performed a novel experiment of IRES scanning through a polypeptide by inserting sequentially the IRES of encephalomyocarditis virus into the open reading frame (ORF) of the poliovirus polyprotein at selected 3Cpro-specific Q*G cleavage sites. No cytopathic effects were observed after transfection of HeLa cells with any of the dicistronic constructs, and no virus was recovered.In vitrotranslation of the dicistronic RNA transcripts in HeLa cell-free extracts revealed that multiple defects in the processing of the P2–P3 domain of the polyprotein is the primary reason for the lethal phenotypes. Surprisingly, the interruption of 3Cpro-catalyzed cleavages downstream of 2C interfered with the 2Apro-catalyzed, primary cleavage between P1 and P2. In contrast, insertion of a foreign coding sequence (V3 loop of human immunodeficiency virus type 1 gp120) into the ORF of the polyprotein at the 2C–3A junction yielded a viable virus that appeared to be genetically stable over several passages. The results of these experiments, which are generally applicable to analyses of viral polyproteins or multidomain polypeptides, suggest that processing of the P2–P3 domain by 3C–3CDprois rapid and accurate only in the context of the unperturbed P2-P3 precursor; this is consistent with cleavages occurringin cis.Moreover, an intact 2C–3A precursor is not required for viral proliferation
Evolutionary Inferences of Novel Simian T Lymphotropic Virus Type 1 from Wild-Caught Chacma (Papio ursinus) and Olive Baboons (Papio anubis)

AbstractA serological survey of 22 wild-caught South African (Transvaal) chacma baboons (Papio ursinus) and eight olive baboons (Papio anubis) from Kenya indicates that 13P. ursinusand oneP. anubishave antibodies reacting with human T cell leukemia/lymphoma virus type 1 (HTLV-1) antigens, whereas threeP. ursinushad a indeterminate reactivity on Western blot analysis. With six primer sets specific to either HTLV-1–Simian T-cell leukemia virus type 1 (STLV-1) or HTLV-2 and encompassing long terminal repeat (LTR),gag,pol,env, andtaxsequences, polymerase chain reaction was performed on genomic DNA from peripheral blood mononuclear cells of 18 animals, and the presence of HTLV-1–STLV-1-related viruses was determined in 13 seropositive and three seroindeterminate animals but not in the two HTLV seronegative individuals. Proviral DNA sequences fromenv(522 bp),pol(120 bp), and complete (755 bp) or partial (514 bp) LTR were determined for three STLV-1-infectedP. ursinusand oneP. anubis.Comparative and phylogenetic analyses revealed thatP. anubis(Pan-486) sequence clusters with one (Pan-1621) of two previously describedP. anubisSTLV-1. Likewise,P. ursinusviruses (Pur-529, Pur-539, and Pur-543) form a distinct group, different from all known HTLV-1 but closely affiliated with two STLV-1 strains from South African vervets (Cercopithecus aethiops pygerythrus). This study, reporting the first STLV-1 sequences from wild-caughtP. ursinusandP. anubis,corroborates the hypothesis of cross-species transmissions of STLV-1 in the wild. Further, phylogenetic analyses indicate that the known HTLV-1 strains do not share a common origin with nonhuman primates STLV in South Africa
Relationship between Anti-Tax Antibody Responses and Cocultivatable Virus in HTLV-I-Infected Rabbits

AbstractThe presence of anti-Tax antibody responses in human T cell leukemia virus type I (HTLV-I)-infected individuals has been correlated with increased proviral load, increased risk of transmitting infection, and increased risk of developing tropical spastic paraparesis/HTLV-I-associated myelopathy (TSP/HAM). In this study, a rabbit model of HTLV-I infection was used to determine whether anti-Tax antibody responses could predict the presence of virus with the potential to replicate. Seven of 14 HTLV-I-infected rabbits developed anti-Tax antibody responses. The onset of Tax reactivity was variable, but once detected remained constant throughout the remainder of the 60-week course of the study. All anti-Tax antibody positive rabbits produced virus as measured by p19 expression upon coculture, while p19 was detected in only one of the Tax antibody negative animals. Thus the presence of an anti-Tax antibody response correlates with p19 expression following cocultivation, and may be a useful predictor of virus replication in HTLV-I infected individuals
Immune Responses but No Protection against SHIV by Gene-Gun Delivery of HIV-1 DNA Followed by Recombinant Subunit Protein Boosts

AbstractThe efficacy of combining immunization with human immunodeficiency vitus type 1 (HIV-1) DNA and HIV-1 recombinant proteins to obtain protection from chimeric simian/human immunodeficiency virus (SHIV) was determined. Four cynomolgus monkeys received four gene-gun immunizations intraepidermally of plasmid DNA encoding HIV-1laienv(gp160),gag, tat, nef,andrevproteins. Ten micrograms of DNA was used per immunization. The animals were boosted twice intramuscularly with 50 μg of HIV-1laiEnv (MicroGeneSys), Gag, Tat, Nef, and Rev recombinant proteins mixed in Ribi adjuvant. The antibody responses were amplified following the administration of the recombinant subunit boosts. One month after the final subunit immunization, the vaccinated animals together with four control animals were challenged intravenously with 10 monkey infectious doses of SHIV that expresses theenv, tatandrevgenes of HIV-1 and gag and nef from SIV. However, only low titers of neutralizing antibodies were present at the day of challenge. The consecutive HIV-1 DNA and recombinant protein immunizations induced B- and T-cell responses but not protection against SHIV replication nor reduction of the viral load
Hepatitis C Virus E1 Protein Induces Modification of Membrane Permeability inE. coliCells

AbstractThe E1 gene of hepatitis C virus (HCV) has been cloned and expressed in BL21(DE3)pLysEscherichia colistrain by pET3a vector to analyze changes in membrane permeability produced by this protein. We showed that the expression of E1 (aa 192–383), as well as of two C-terminal fragments (aa 331–383 and aa 341–383) corresponding to the transmembrane (TM) region of this protein, induced a rapid lysis of cells. On the contrary, the expression of a mutant of E1 (aa 192–340), lacking the last 40 amino acids, did not cause cell lysis. The analysis of permeability changes revealed that modification of membrane permeability to several compounds were observed only in clones expressing E1 and C-terminal fragments, while the synthesis of the C-terminal-deleted mutant had little or no effect on permeability. These findings demonstrate that the TM domain of E1 protein has membrane-active properties that may be involved in some aspects of virus-cell interaction
SH3-Domain Binding Function of HIV-1 Nef Is Required for Association with a PAK-Related Kinase

AbstractHIV-1 Nef has previously been shown to bind to Src homology-3 (SH3) domains of a subset of Src family tyrosine kinases. In addition, Nef has been reported to coprecipitate with a serine/threonine kinase activity termed NAK (for Nef-associated kinase). The identity of NAK remains uncertain, but it has been suggested to represent a novel member of the p21-activated kinase (PAK) family. We report here that NAK autophosphorylation is increased not only by an activated form of the p21-family GTPase cdc42 but also by a plasma membrane-targeted fragment of the adapter protein Nck, thus providing further evidence that NAK is related to PAKs. A detailed structure-based mutational analysis of Nef revealed that all amino acid changes that inhibited the Nef/Hck-SH3 interaction, as measured by surface-plasmon resonance, also abolished coprecipitation of NAK. As PAK family proteins do not contain SH3 domains, these observations are best explained by a protein complex in which Nef, NAK, and an SH3-protein all contact each other. In addition, a number of conserved amino acids in Nef that are not involved in SH3 binding were also found to be crucial for association with NAK. Molecular modeling suggests that these residues are involved in formation of an adjacent binding surface for NAK or another critical component of the NAK/Nef complex
Human Serum Attenuates the Activity of Protease Inhibitors toward Wild-Type and Mutant Human Immunodeficiency Virus

AbstractThe potency of therapeutic regimens containing human immunodeficiency virus (HIV) protease inhibitors is related to the ability to maintain concentrations of drug in the plasma of patients that are sufficient for blocking viral replication. The estimation of concentrations required forin vivoactivity usingin vitroassays is complicated by the fact that extensive binding of many protease inhibitors to serum proteins attenuates their antiviral potency. To provide insight into the relativein vivopotency of current protease inhibitors, we assayed theirin vitroactivity against wild-type and mutant HIV in the presence of human serum (HS). Using this assay, ABT-378, a new protease inhibitor with trough levels in humans far in excess of the EC50in the presence of 50% HS, was identified. The antiviral activity of ABT-378 was only modestly attenuated by HS, in contrast to ritonavir, saquinavir, and nelfinavir. Examination of the effect of individual serum components suggested that the activity of ABT-378 is affected predominantly by binding to α1-acid glycoprotein (AGP) while the activity of ritonavir is modulated by both AGP and albumin. The method described here may provide insight into thein vivopotency of protease inhibitors and be useful for the preclinical evaluation and selection of new protease inhibitors for clinical studies
Phosphorylation of the M2 Protein of Influenza A Virus Is Not Essential for Virus Viability

AbstractM2 is a minor component of the influenza A virus envelope. The cytoplasmic tail of the M2 protein is posttranslationally modified in the infected cell by palmitylation and phosphorylation. The primary site for phosphorylation of the M2 cytoplasmic tail is serine 64, which is highly conserved yet not required for the activity of the M2 ion channel. Using an exogenous incorporation assay, we have shown that incorporation of M2 into virus particles is type-specific and does not require phosphorylation of the cytoplasmic tail. In addition, phosphorylation of the cytoplasmic tail is not required for the directional transport of M2 in polarized MDCK cells. Using a reverse genetics and reassortment procedure, we generated a virus (Ra) specifically mutated in segment 7 such that the M2 cytoplasmic tail could no longer be phosphorylated. The virus was found to grow as well as wild-type virus in tissue culture and in eggs, was stable on passage in these systems, and possessed no second-site mutations in the engineered RNA segment.In vivoRa replicated in Balb/c mice at least as well as the parent strain A/WSN/33. These studies indicate that phosphorylation of the M2 cytoplasmic tail is not required forin vitroorin vivoreplication of influenza A virus
Human Cytomegalovirus Infection Downregulates Expression of the Cellular Aminopeptidases CD10 and CD13

AbstractDuring the course of a productive infection, human cytomegalovirus (HCMV) has a sophisticated relationship with its host cell. An increasing number of virus-encoded genes are being identified which act specifically to usurp or modulate functions in the host cell associated with transcriptional control, cell signalling, and protein synthesis. While HCMV infection is associated with a general upregulation of cellular gene expression, the expression a small subset of cellular proteins, including the MHC-1 heavy chain and fibronectin, is downregulated. This study now identifies two additional cellular proteins, aminopeptidase N (CD13) and neutral endopeptidase (CD10), that are downregulated during HCMV infection. While aminopeptidase N and neutral endopeptidase exhibit no significant sequence homology, both are expressed on the cell surface and have very similar enzymatic properties. HCMV infection was associated with reduced surface expression and enzyme activity of CD13 and CD10, an apparent decrease in the rate of synthesis of both proteins in metabolic-labelling experiments, and inhibited glycosylation of the nascent CD13 and CD10 polypeptide chains that were synthesized. Levels of CD10 poly A+RNA were suppressed efficiently at all stages of virus infection; however, the reduction in CD13 poly A+RNA levels was much less pronounced. This differential effect suggests that HCMV may be downregulating expression of CD10 and CD13 by independent mechanisms. Indeed, treatment of cells with an inhibitor of viral DNA synthesis blocks downregulation of CD13, whilst downregulation of CD10 is unaffected. While it is not yet clear what advantage is bestowed on the virus by downregulating expression of CD13 and CD10, aminopeptidases are known to have a role in peptide processing in both the MHC class I the MHC class II antigen presentation pathways
Electrostatic Interactions Drive Scaffolding/Coat Protein Binding and Procapsid Maturation in Bacteriophage P22

AbstractThe first step in assembly of the bacteriophage P22 is the formation of a T=7 icosahedral “procapsid,” the major components of which are the coat protein and an inner core composed of the scaffolding protein. Although not present in the mature virion, the scaffolding protein is required for procapsid assembly. Eleven amino-acid residues at the extreme carboxyl terminus of the scaffolding protein are required for binding to the coat protein, and upon deletion of these residues, approximately 20 additional residues become disordered. Sequence analysis and NMR data suggest that the 30 residues at the carboxyl terminus form a helix-loop-helix motif which is stabilized by interhelical hydrophobic interactions. This “coat protein recognition domain” presents an unusually high number of positively charged residues on one face, suggesting that electrostatic interactions between this domain and the coat protein may contribute to recognition and binding. We report here that high ionic strength (1 M NaCl) completely inhibited procapsid assemblyin vitro.When scaffolding protein was added to empty procapsid “shells” of coat protein, 1 M NaCl partially inhibited the binding of scaffolding protein to the shells. This suggests that the positively charged coat protein recognition domain at the carboxyl terminus of the scaffolding protein binds to a negatively charged region on the coat protein. During DNA packaging, the scaffolding protein exits the procapsid; scaffolding protein exit is followed by the expansion of the procapsid into a mature capsid. Procapsid shells can be induced to undergo a similar expansion reactionin vitroby heating (45–70°C); this process was also inhibited by 1 M NaCl. These results are consistent with a model in which negatively charged scaffold protein-binding domains in the coat proteins move apart during procapsid expansion; this relief of electrostatic repulsion could provide a driving force for expansion and subsequent maturation. High-salt concentrations would screen this repulsion, while packaging of DNA (a polyanion)in vivomay increase the instability of the procapsid enough to trigger its expansion
A Short Noncoding Viral DNA Element Showing Characteristics of a Replication Origin Confers Bacteriophage Resistance toStreptococcus thermophilus

AbstractA 302-bp noncoding DNA fragment from the DNA replication module of phage φSfi21 was shown to protect theStreptococcus thermophilusstrainSfi1 from infection by 17 of 25 phages. The phage-inhibitory DNA possesses two determinants, each of which individually mediated phage resistance. The phage-inhibitory activity was copy number dependent and operates by blocking the accumulation of phage DNA. Furthermore, when cloned on a plasmid, the φSfi21 DNA acts as an origin of replication driven by phage infection. Protein or proteins in the φSfi21-infected cells were shown to interact with this phage-inhibitory DNA fragment, forming a retarded protein–DNA complex in gel retardation assays. A model in which phage proteins interact with the inhibitory DNA such that they are no longer available for phage propagation can be used to explain the observed bacteriophage resistance. Genome analysis of φSfi19, a phage that is insensitive to the inhibitory activity of the φSfi21-derived DNA, led to the characterisation of a variant putative phage replication origin that differed in 14 of 302 nucleotides from that of φSfi21. The variant origin was cloned and exhibited an inhibitory activity toward phages that were insensitive to the φSfi21-derived DNA
The Nonstructural Small Glycoprotein sGP of Ebola Virus Is Secreted as an Antiparallel-Orientated Homodimer

AbstractThe nonstructural small glycoprotein sGP, which unlike the transmembrane GP is synthesized from primary nonedited mRNA species, is secreted from infected cells as a disulfide-linked homodimer. Site-directed mutagenesis of all cysteine residues revealed that dimerization is due to an intermolecular disulfide linkage between cysteine residues at positions 53 and 306. Formic acid hydrolysis of sGP demonstrated that sGP dimers consist of monomers in antiparallel orientation. Another editing product of the GP gene of Ebola virus (ssGP), which shares 295 amino-terminal amino acid residues with sGP, is secreted from cells in a monomeric form due to the lack of the carboxyl-terminal part (present in sGP), including cysteine at position 306
Domain Structure, Intracellular Trafficking, and β2-Microglobulin Binding of a Major Histocompatibility Complex Class I Homolog Encoded by Molluscum Contagiosum Virus

AbstractThe MC80R gene of molluscum contagiosum virus (MCV) type 1 encodes a major histocompatibility complex (MHC) class I homolog that lacks several amino-acid residues critical for peptide binding by MHC molecules, contains an unusually long N-terminal hydrophobic domain possibly derived by triplication of a signal peptide, and has a C-terminal transmembrane domain with two glutamate residues. All of these features were present in the orthologous gene of MCV type 2. The MC80R gene was expressed as two glycosylated polypeptides of Mr 47,000 and 42,000. Pulse-chase experiments indicated that the larger polypeptide was a precursor of the shorter one and that the entire N-terminal domain was slowly removed, consistent with its function as a long signal peptide. The protein was largely sequestered in the endoplasmic reticulum and Golgi membranes, remained endoglycosidase-H sensitive, and was not detected on the cell surface. In addition, a genetically modified form of the MC80R protein lacking the transmembrane and cytoplasmic domains was not secreted. The roles of the MC80R protein domains were investigated by constructing chimera between the viral protein and the MHC class I protein HLA-A2. Expression studies confirmed that the N- and C-terminal hydrophobic regions of the MC80R protein served as signal and transmembrane domains, respectively. The central portion of the MC80R protein, corresponding to the α1–α3 extracellular domains of HLA-A2, was largely responsible for sequestering the protein in the endoplasmic reticulum or Golgi compartments. The MC80R protein, as well as HLA-A2 chimera with the central region of MC80R, formed stable intracellular complexes with β2-microglobulin. Complex formation, however, was detected only by overexpression of the MC80R protein or β2-microglobulin