pinecone/core-2020-05-10-deduplication huggingface dataset:
Antigenic Variation of SIV: Mutations in V4 Alter the Neutralization Profile

AbstractAntigenic variation is a characteristic feature of lentiviral infection. The SIV/macaque model of AIDS provides an ideal system in which to investigate the molecular basis of antigenic variation. The purpose of this study was to genetically map the nucleotide changes inenvthat alter the neutralization phenotype of SIV. Serum taken from an SIVmac239-infected macaque (2D) at 30 weeks postinoculation was found to neutralize the input virus (SIVmac239) and an isolate, P9, obtained at 10 weeks p.i., but did not neutralize two other isolates, P13 and P23, obtained at 20 and 52 weeks, respectively. Sequence analysis of these virus variants revealed clustered amino acid changes in V1 and single base pair changes in V2–V4 of P13 and P23. Infectious recombinant viruses in which the V1 and V1–V3 sequences of SIVmac239 were replaced with those of P13 or P23 retained the neutralization profile of SIVmac239; both were neutralized by macaque 2D serum. Recombinants containing the entire surface glycoprotein (gp120) (V1–V5) and the 5′ portion of gp41 of P13 and P23 and those containing gp120 sequences from V4 through the 5′ portion of the transmembrane glycoprotein (gp41) were not neutralized by 2D serum. Using a panel of monoclonal antibodies in radioimmunoprecipitation assays, P23 and recombinants containing V4 and V5 of P23 were shown to be antigenically distinct from P13 and SIVmac239. The majority of the amino acid changes in the antigenically distinct viruses were clustered in V4 (amino acids 413–418) and these changes created new potential N-linked glycosylation sites. This study demonstrates that a small number of specific amino acid changes (amino acids 412 to 418 in theenvgene) in the V4 region of the SIV envelope glycoprotein can alter antibody recognition and neutralization and that these phenotypic changes may be associated with altered glycosylation of the envelope
Assembly and Release of SIV Env Proteins with Full-Length or Truncated Cytoplasmic Domains

AbstractWe have used recombinant vaccinia viruses expressing full-length or truncatedgagorenvgenes of SIVmac239 to investigate the requirements for assembly of SIV proteins. We observed that assembly of virus-like particles (VLPs) was found to be 3- to 5-fold higher with full-length Env than with the truncated forms, or than VLPs containing only Gag proteins, in primary monkey cells or various human cell lines. When cells expressing Env proteins in the absence of Gag were examined by immunoelectron microscopy, clusters of Env protein and membrane vesicles containing Env proteins were observed at cell surfaces. A low level of vesicles was released from cells expressing full-length Env, but about a 10-fold higher level was released in cells expressing a truncated form of Env [Env733(t)] in which the cytoplasmic domain is only 17 amino acids in length. Another truncated protein, Env718(t), with a short cytoplasmic tail of 3 aa, was also incorporated into VLPs at a 10-fold higher level than the full-length Env protein and was more efficiently released in vesicles. The mature SU and TM proteins were predominantly incorporated into VLPs with full-length Env, but both cleaved and uncleaved precursor proteins were present in VLPs with truncated Env as well as in Env and Env(t) vesicles. A more prominent layer of spikes was seen by electron microscopy in VLPs with truncated Env than in VLPs containing full-length Env. These results indicate that truncated Env proteins have the ability to self-associate on the cell surface and are assembled into a more closely packed array than full-length Env, which could explain the preferential incorporation of Env proteins with short cytoplasmic tails into virions
Genetic Analysis of the Bovine Papillomavirus E2 Transcriptional Activation Domain

AbstractThe bovine papillomavirus type 1 E2 transactivator has a large amino-terminal 215-residue transcriptional activation domain (TAD) that is active inSaccharomyces cerevisiaeand higher eukaryotic cells. Comparison to other transcriptional activators suggests that its functions may be mediated in part through two acidic regions, A1 and A2, in this domain. We have characterized the functional elements within the E2 TAD using LexA–E2 fusions and by screening randomly generated libraries of E2 mutations for transcriptional activation in yeast. The A1 region was highly sensitive to substitutions that reduce negative charge, although there was not a perfect correlation between overall charge and transcriptional activity. Mutations were isolated within a hydrophobic amino acid motif that overlaps the A2 region and resembles elements described in other viral and cellular transactivation domains. When fused to the LexA DNA binding domain, this hydrophobic motif within the acidic A2 region was unable to activate transcription inS. cerevisiae.Multiple highly defective mutations primarily altering hydrophobic amino acids were identified in the distal third of the E2 TAD. The transcription phenotype of many of these E2 TAD mutations was similar in yeast and COS cells
The Transactivation and DNA Binding Domains of the BPV-1 E2 Protein Have Different Roles in Cooperative Origin Binding with the E1 Protein

AbstractThe bovine papillomavirus E2 transactivator protein enhances the ability of the E1 protein to bind to the viral origin of replication which contains an E1 binding site flanked by two E2 binding sites. To determine which regions and functions of the E2 protein are important for this cooperative interaction, a series of mutated E2 proteins were assayed for their ability to enhance E1 origin-specific binding. Cooperative origin binding required at least one E2 DNA binding site, an intact functional E2 DNA binding domain, and an intact transactivation domain. The hinge region of the E2 proteins was dispensable for this activity. To further examine the role of the E2 C-terminal domain, a series of chimeric proteins were generated that substituted the yeast GAL4 DNA binding domain for the E2 DNA binding domain. These chimeric proteins were able to cooperatively bind to a hybrid origin that contained GAL4 binding sites in place of the E2 binding sites. These studies indicate that the E2 transactivation domain is sufficient for interaction with the E1 protein and that the E2 DNA binding domain is required for interaction with origin DNA sequences
In VitroStudies on the Activation of the Hepatitis C Virus NS3 Proteinase by the NS4A Cofactor

AbstractProteolytic processing of the nonstructural proteins of the hepatitis C virus (HCV) is mediated by two viral proteinases: the NS2-3 proteinase cleaving at the NS2/3 junction and the NS3 serine-type proteinase responsible for processing at the NS3/4A, NS4A/B, NS4B/5A, and NS5A/B sites. Activity of the NS3 proteinase is modulated by NS4A. In the absence of this cofactor processing at the NS3-dependent sites does not occur or, in the case of the NS5A/B junction, is poor but increased when NS4A is present. Although recent studies demonstrated that proteinase activation requires direct interaction between NS3 and NS4A, the mechanism by which NS4A exerts the activation function is not known. To further analyze the conditions of proteinase activation and to characterize the NS3 sequences important for complex formation and activation we used anin vitroassay in which radiolabeled HCV substrates were mixed with NS3 proteinase and synthetic NS4A peptides. We found that microsomal membranes are not required for proteinase activation. However, they are important for efficient accessibility of the NS4A/B site but not the other trans-cleavage sites. Studies with NS3 deletion mutants identified a region between amino acids 15 and 22 which is essential for proteinase activation. Results obtained with several mutations introduced into this sequence show that a weak overall association between NS3 and NS4A is sufficient for proteinase activation and suggest that a beta-sheet at the NS3 amino terminus plays an important role. Although not essential for proteinase activation the amino terminal 14 NS3 residues were found to have an auxilliary function probably by stabilizing the NS3/4A interaction. Finally, we could demonstrate intracellular, peptide-mediated modulation of proteinase activity providing the basis for the development of a novel therapeutic concept
Modulation of Bacteriophage T4 Capsid Size

AbstractBacteriophage T4 capsid assembly requires the vertex protein (gp24). Mutations that bypass this requirement are found in gene 23, which produces the major capsid protein (gp23). The latter were used to study the role of gp24 in head length control. We found that gp24 is no longer present in the capsids of several gp24 bypass mutants. We measured the capsid lengths of several of these bypass mutants, because gp24 had been reported to be implicated in headlength control. One bypass mutant (reported in 1977) produced 40–60% short headed (“petite”) phage in the presence of wild-type amounts of gp24. The bypass mutations, when combined withambermutations in gene 24, produced normal size heads in either suppressor or nonsuppressor host bacteria. When several known bypass mutations were back-crossed with wild-type phage, one-third of thebyp/24wt mutants isolated produced large amounts of petite phage, indicating that the ability to produce petite phage is a general property of the bypass mutations. Sequencing several of these bypass mutants showed that those that produced petite phage contained at least one additional missense mutation in gene 23. This suggests that gp24 itself has no direct role in head length regulation, but that in the presence of bypass 24 mutations and certain easily acquired gene 23 mutations (calledtrb) the gp23–gp24 interactions can modulate head length
Expression of Bovine Interleukin-1β in a Bovine Herpesvirus-1 Vector:In VitroAnalysis

AbstractIn order to evaluate whether bovine herpesvirus-1 (BHV-1) could be used as a live viral vector for the expression of cytokines, we constructed a recombinant BHV-1 expressing bovine interleukin-1β (boIL-1β). The boIL-1β coding sequence, corresponding to the cleaved mature product, was fused with the BHV-1 glycoprotein C (gC) signal peptide sequence; the resultant gC-boIL-1β fusion gene was recombined into the gC locus of the BHV-1 genome. Southern blot analysis confirmed the proper genomic configuration of the recombinant virus. Results from transcript analysis showed that boIL-1β was expressed in infected cells with kinetics similar to that of gC. Indirect immunofluorescence and immunoprecipitation assays showed that the recombinant protein was produced in both cell-associated and secreted forms. Western blot analysis detected a 19.3-kDa protein. Further analysis, using an IL-1β bioassay demonstrated that both the cellular and secreted forms of recombinant boIL-1β possessed biological activity. The expression of the boIL-1β protein did not affect thein vitrogrowth efficiency of the virus, which exhibited similar growth kinetics to that of a simple gC deletion mutant. The results from this study demonstrate that BHV-1 can be used to express a functional cytokine, thereby establishing the basis to further study recombinant BHV-1 expressing cytokines as an alternative means to attenuate the virus and also as a potentialin situcytokine delivery system to modulate immune responses against BHV-1 and other cattle pathogens
Palmitoylation of the Murine Leukemia Virus Envelope Glycoprotein Transmembrane Subunits

AbstractThe envelope protein of Friend murine leukemia virus is modified by fatty acylation of the transmembrane (TM) protein subunit. The labeling by [3H]palmitic acid was found to be sensitive to treatment with the reducing reagents 2-mercaptoethanol and hydroxylamine, indicating the presence of a thioester linkage. Pulse-chase experiments showed that the precursor protein can be labeled by [3H]palmitic acid prior to its cleavage into the surface and TM subunits. By using site-directed mutagenesis, we determined that palmitoylation occurs on a cysteine residue, Cys 606, located in the transmembrane domain. A thin-layer chromatography assay after acid hydrolysis showed that incorporated label comigrated with palmitic acid. When another cysteine residue was introduced into the cytoplasmic tail 22 amino acids from the transmembrane domain, no palmitoylation was observed to occur on this cysteine residue, demonstrating the importance of the position of the cysteine residue for palmitoylation. Sequence comparison revealed that most retrovirus envelope proteins have one or two conserved cysteine residues in their transmembrane domain. Mutations that change the palmitoylation state of the murine leukemia virus envelope protein did not affect its transport, processing, surface expression, or cell fusion activity. The palmitate-deficient viral envelope proteins were incorporated into virus particles, and replication of the virusin vitrowas not affected significantly by the mutation of the palmitoylation site
Intracellular Synthesis, Processing, and Transport of Proteins Encoded by ORFs 5 to 7 of Porcine Reproductive and Respiratory Syndrome Virus

AbstractPorcine Reproductive and Respiratory Syndrome Virus (PRRSV), a small enveloped virus containing a positive-strand RNA genome, possesses at least three major structural proteins designated N, M, and E. The N protein is considered as the major component of the nucleocapsid, whereas M and E are membrane-associated. Previous studies using peptide-specific antibodies assigned these proteins to ORFs 7, 6, and 5, respectively. In the present report, monospecific antisera raised againstEscherichia coli-expressed ORFs 5, 6, and 7 products were used to study the synthesis and processing of PRRSV structural proteins in the highly permissive MARC-145 cell line. Treatment of viral proteins with various glycosidases showed that only E was modified by N-linked glycans. Pulse–chase experiments revealed that intracellular transport of the major envelope glycoprotein was delayed in the premedial Golgi compartment. During the first 30 min of chase, E undergoes a gradual downward shift of its apparent molecular weight, thought to result from trimming of the mannose-rich glycan structures. Once E is transported to the medial Golgi or proximal elements, some molecules undergo complete processing of all their high-mannose N-linked oligosaccharides to complex type, while in other molecules only a fraction of N-linked glycans are terminally glycosylated. These two differentially glycosylated forms of E were found to be incorporated into extracellular virions. In cells and virions, both M and E were shown to occur in heterodimeric complexes linked by disulfide bonds. The oligomerization process, as analyzed from pulse–chase experiments, showed that M and E are incorporated into M-E complexes with different kinetics and efficiencies, in a fashion similar to their counterparts in equine arteritis virus. Apparently, all steps of E protein N-glycans processing proceed after its association with M which occurs in the endoplasmic reticulum (ER). In the infected cells, E and M appear highly membrane-associated, while N is predominantly cytosolic
Interferon Induction by HIV-1-Infected Cells: A Possible Role of Sulfatides or Related Glycolipids

AbstractWe have investigated the mechanism of interferon (IFN) induction in peripheral blood mononuclear cells by HIV-1(IIIB)-infected H9 cells or by recombinant gp120. A monoclonal antibody specific for the galactosylsphingosinyl moiety in galactocerebrosides and sulfatides inhibited IFN induction in a dose-dependent manner. Furthermore, exogenous sulfatides inhibited with an ID50of approximately 1 μM,whereas galactocerebrosides were not inhibitory at 40 times higher concentrations. These studies suggest that sulfate containing galactolipids such as sulfatides on responder cells may be part of the gp120–membrane complex that initiates the induction of IFN. A partial homology of an epitope on the V3 loop of gp120 with a previously suggested binding domain for sulfated glycoconjugates supports this conclusion
Ligation of Double-Stranded and Single-Stranded [Oligo(dT)] DNA by Vaccinia Virus DNA Ligase

AbstractVaccinia virus DNA ligase has been expressed inEscherichia coli,purified, and biochemically characterized. The enzyme ligates double-stranded (ds) DNA substrates with either cohesive or blunt-end termini and the latter reaction is stimulated by PEG. Vaccinia virus DNA ligase can also ligate oligo(dT) when annealed to either a poly(dA) or a poly(rA) backbone and, remarkably, free oligo(dT). This ligation of a single-stranded (ss) substrate is unique among eukaryotic DNA ligases. The enzyme requires high ATP concentrations with aKmfor the overall ligation of a ssDNA substrate of 0.8 mM.The salt, divalent cation, temperature, and pH requirements of the enzyme for the optimal ligation of ss and ds substrate are described
Reexamination of the Sendai Virus P Protein Domains Required for RNA Synthesis: A Possible Supplemental Role for the P Protein

AbstractThe Sendai virus P protein plays a central role in viral genome amplification and expression, forming complexes with the viral L protein to generate the polymerase (P-L) and unassembled N (P-N°). This latter complex prevents N from selfassembling illegitimately, i.e., independently of the concurrent assembly of a nascent viral genome, and is thought to represent the functional form of N in nucleocapsid assembly. Based upon earlier functional studies using anin vitrotranscription/replication system in which the P, L, and N proteins were coexpressed, we identified two regions of the P protein required for RNA synthesis, namely, the C-terminal 40% of the protein, and a second, apparently redundant domain near the N-terminus (either amino acids 1–77 or 78–145). The lack of sequence conservation in this second region, apart from overall negative charge, was reminiscent of the acidic activation domains of cellular transcription factors. However, we recently mapped a chaperone domain at the N-terminal of P (aa 33–41), which is required for stable complex formation with unassembled N° and, thus, for assembly and genome replication. In this present study we show that coexpression of N protein with P deletion mutants lacking this region (e.g., PΔ1–324) results in the sequestration of the mutant P by the illegitimately assembled form of N. As a consequence, P protein is unavailable for RNA synthesis from bona fide templates. We also find that in the absence of coexpressed N protein, the entire N-terminal 60% of the P protein is not required for mRNA synthesis. During these studies, a supplemental role for the P protein in viral RNA synthesis, independent of stable complex formation with L, was observed. This function involves, at least in part, the binding of additional copies of P to the N:RNA template
Poliovirus Neurovirulence Correlates with the Presence of a Cryptic AUG Upstream of the Initiator Codon

AbstractPoliovirus mutants with extended (>150-nt) deletions in the 5′-untranslated region between the internal ribosome entry site and the initiator codon have been selected previously (Pilipenkoet al., Cell68, 119–131, 1992; Gmylet al., J. Virol.67, 6309–6316, 1993). These deletions were transferred into the genome of a mouse-pathogenic poliovirus strain and found to be strongly attenuating. The deletions can be considered as covering three structural elements, a stem–loop (domain E) with a conserved cryptic AUG and two spacers, upstream and downstream of it. In an attempt to identify putative essential determinants of neurovirulence in these individual structural elements, appropriate mutants were engineered. The results demonstrated that neither of the above elements is essential for neurovirulence. The results strongly suggested that the presence of a cryptic AUG in the oligopyrimidine/AUG tandem followed, at a sufficient distance, by the initiator codon was necessary to ensure the neurovirulent phenotype of our constructs. On the other hand, the attenuated phenotype appeared to correlate with the occurrence of the initiator AUG as a moiety of the oligopyrimidine/AUG tandem. Possible mechanisms underlying these effects are discussed. Identification of the cryptic AUG as an essential determinant for neurovirulence provides a rational basis for the design of genetically stable attenuated poliovirus variants
An Early Gene of the Chlorella Virus PBCV-1 Encodes a Functional Aspartate Transcarbamylase

AbstractPBCV-1 belongs to a family of large viruses that replicate in the exsymbiont green algaeChlorellastrain NC64A. The viral, 330-kb DNA genome encodes a relatively large number of functionally active proteins including restriction and modification enzymes, DNA polymerase, glycosylation, and cell wall degrading enzymes. Sequencing of the viral DNA, now in progress, revealed many major open reading frames (ORF), which resemble known genes in sequence data bases and which have not previously been found in viral genomes. Here we report on the identification and characterization of one such gene, aspartate transcarbamylase (ATCase), an enzyme that catalyzes the committing step in thede novobiosynthetic pathway of pyrimidines. The cloned gene is highly homologous to a variety of plant ATCases and includes the typical ATCase catalytic motif. When cloned into the pGEX-2T expression vector, a fusion protein with ATCase activity could be demonstrated and distinguished from the host ATCase activity. The viral enzyme is expressed early and transiently in the infection. To our knowledge, this is the first virus known to encode and express its ownde novonucleotide precursors’ synthetic enzymes
A Sendai Virus Vector Leading to the Efficient Expression of Mutant M Proteins Interfering with Virus Particle Budding

AbstractA Sendai virus expression vector in the form of a transcribing copy-back defective interfering RNA was constructed and shown to efficiently express a tagged matrix protein in the only context of a Sendai virus infection. In an attempt to identify relevant M protein domains involved in viral assembly and budding, a series of deletion mutants were tested for their ability to bind to cellular membrane fractions. The deletion of a region spanning amino acids 105–137 significantly decreased this binding when the protein was expressed in a system driven by the T7 RNA polymerase away from any other viral proteins. Plus or minus charges were introduced in the hydrophobic portion of a predicted amphiphilic helix in this region, and M proteins with altered membrane binding properties were produced. The genes encoding these mutant M proteins were then inserted in the Sendai virus vector and shown to be expressed at levels similar to that of the endogenous wild-type M protein. The presence of a negative charge in the hydrophobic region of the putative amphiphilic helix prevented the incorporation of the mutant protein into virus particles and appeared to decrease the efficiency of virus particle budding. In contrast, the introduction of a positive charge appeared to increase the M mutant uptake into virions. The use a Sendai virus vector has therefore been shown instrumental in the identification of mutant M proteins interfering with the viral assembly-budding process
293 Cell Lines That Inducibly Express High Levels of Adenovirus Type 5 Precursor Terminal Protein

Abstract293 cell lines that inducibly express high levels of adenovirus type 5 precursor terminal protein (pTP) under the control of a tetracycline-dependent promoter were constructed. To construct the cell lines expressing pTP, 293 cells were stably transfected with a plasmid encoding the tetracycline repressor/VP16 transactivator protein (tTA) using selection with hygromycin. Cell lines that expressed high levels of tTA activity were then stably transfected with plasmids in which pTP expression is directed by the tTA-dependent promoter from either a cDNA or a modified genomic construct using selection with G418. Cell lines that expressed high, inducible levels of pTP efficiently complemented a temperature-sensitive pTP mutant virus for growth and plaque formation at the nonpermissive temperature