pinecone/core-2020-05-10-deduplication huggingface dataset:
Circumvention of Vector-Specific Neutralizing Antibody Response by Alternating Use of Human and Non-Human Adenoviruses: Implications in Gene Therapy

AbstractTo determine whether non-human adenovirus-specific antibodies are cross-neutralizing, rabbit and mouse anti-human adenovirus type 5 (HAd5), anti-bovine adenovirus type 3 (BAd3), and anti-porcine adenovirus type 3 (PAd3) sera were used in cross-virus neutralization assays. Adenovirus neutralizing antibodies were found to be virus-specific, suggesting that virus neutralizing epitope differs significantly in HAd5, BAd3, and PAd3. To further investigate whether immunity to an HAd5-derived vector could be circumvented by the use of non-human adenoviruses in vivo, mice were first immunized either intranasally or intraperitoneally with HAd5, BAd3, PAd3, or BAd3 + PAd3, and after development of adenovirus-specific antibodies, animals were inoculated with the HAd5 recombinant (AdCA36lacZ) containing the bacterial β-galactosidase gene under the control of murine cytomegalovirus immediate-early promoter. Virus-inoculated animals developed virus-specific IgG and IgA antibodies. LacZ expression in animals initially primed with HAd5 was significantly reduced (P < 0.05), suggesting that the immune response against the vector could prevent the transgene expression following subsequent inoculation of the same vector, whereas LacZ expression in mice initially primed with BAd3, PAd3, or BAd3 + PAd3 was significantly higher (P > 0.05) than that obtained in HAd5-primed animals. Our results suggest that HAd5-, BAd3-, or PAd3-based vectors may be used sequentially for human gene therapy or vaccine production as a means to avoid immunity to the vector
A Mutation in the DE Loop of the VP1 Protein That Prevents Polyomavirus Transcription and Replication

AbstractNatural mutants of the DE loop of the Polyomavirus (Py) major coat protein VP1 have been previously shown to display an altered host specificity (L. Ricci, R. Maione, C. Passananti, A. Felsani, and P. Amati, 1992, J. Virol. 66, 7153–7158). To better understand the role of this outfacing loop of the VP1 protein in Py infectivity, we constructed and characterized a Py mutant (Py M17) harboring a deletion of 7 AA within the tip of the DE loop. The mutant virions obtained after DNA transfection were unable to replicate and initiate early transcription in fibroblast cells. Complementation experiments performed to rescue the deficient M17 replication by means of wt functions revealed the cis-dominance of the mutation. In situ cell fractionation experiments demonstrated that the Py mutant, like the Py wt, enters the cells, reaches the nucleus and that both the viral DNA and VP1 protein are found tightly bound to the nuclear matrix. These data suggest that the VP1 protein, associated to the viral DNA, conditions early viral gene expression and that the DE loop of the protein must be involved in this process
Hypervariability in the Envelope Genes of Subgroup J Avian Leukosis Viruses Obtained from Different Farms in the United States

AbstractAvian leukosis virus, subgroup J (ALV-J), has a wide host range, preferentially infecting meat-type birds, and produces a high incidence of myelocytomatosis and nephromas. Using the published sequences from HPRS-103 (ALV-J isolated in 1989 in Great Britain), we designed a set of PCR primers that amplified proviral DNA from nine U.S. field samples. The primers were specific for ALV-J, not amplifying DNA from uninfected cells or cells infected with ALV subgroups A–E. These primers expanded a 2.4-kb fragment that encompasses gp85, gp37, the E element, and most of the 3′ LTR. We also developed a set of PCR primers that amplified a 2.1-kb fragment from ALV-J-infected cells and a 1.6-kb fragment from uninfected ev− chicken embryo fibroblasts (Line 0). Upon cloning and DNA sequencing, we determined that the 2.1- and 1.6-kb fragments contained ALV-J gp85- and gp37-like sequences. Comparison of the amino acid sequences demonstrated that the Line 0 sequences were 97.5% identical with the gp85 and gp37 of HPRS-103 and somewhat less identical with the other nine U.S. isolates. This suggests that the envelope genes of ALV-J may have arisen as a result of a recombination event between exogenous ALV and Line 0-like sequences in the chicken. Phylogenetic analysis also showed that the U.S. field isolates were closely related to one another and more distantly related to the European HPRS-103. The pattern of mutations in the U.S. field isolates suggests that the U.S. strains are slowly drifting away from their progenitor Line 0-like sequences. The development of effective vaccines and diagnostic tests is likely to become more problematic as the viruses continue to mutate
Optimization of Regulated LTR-Mediated Expression

AbstractRetroviral vectors are ideally suited to the study of gene function, allowing efficient, stable expression. Many biological systems (e.g., cell cycle, apoptosis) require the use of regulated expression systems. We therefore developed a regulated retroviral vector system, TRA99, based on a tetracycline transactivator-dependent LTR, where the MMLV enhancer was replaced with a tetracycline-response element. Using fluorescence-activated flow cytometric analysis of a destabilized green fluorescent protein to monitor expression levels, we optimized the minimal promoter configuration with respect to both activated and repressed transcription. The TRA99 vectors demonstrate regulated expression with activated levels comparable to those of standard retroviral vectors and repressed levels indistinguishable from background. This was achieved without using an internal promoter cassette, thus retaining the cis-packaging elements requisite for helper-mediated transfer
Sequencing and Characterization of A-2 Plaque Virus: A New Member of the Picornaviridae Family

AbstractA-2 plaque virus (A2 virus) was originally isolated from the icteric-phase sera of US servicemen with viral hepatitis in the 1960s, but apart from a preliminary characterization little is known about the agent. We have now successfully cloned and sequenced the complete viral genome. A2 viral RNA consists of 7312 nucleotides, excluding the 62 nucleotide poly(A) tract at the 3′ end, with one large open reading frame. Although clearly a member of the Picornaviridae, there is low homology to the available sequences, suggesting it is only loosely related to the classic rhino/enterovirus genus. In addition, there was no reactivity with group specific monoclonal antibody blends against polioviruses, enteroviruses 70 and 71, coxsackievirus B, and echoviruses. Two tamarins were inoculated with A2 virus to study viral pathogenesis. Both animals that received A2 virus became transiently viremic 1 week after the infection, as determined by RT–PCR, and they developed an antibody response to A2 virus. However, no physical signs or biochemical abnormalities, including elevated liver transaminases, were observed. In addition, no liver samples from patients with fulminant hepatitis (n = 7) or controls (n = 7) were positive for A2 viral RNA nor was anti-A2 neutralizing antibody detected in sera from hepatitis patients (n = 14), healthy laboratory donors (n = 14), or US blood donors (n = 33); however, most sera contained antibodies reactive with A2 virus proteins. These results suggest that A2 virus is a new member of the Picornaviridae but that its pathogenicity in nonhuman primates and association with human disease still need to be determined
Different Immunological Requirements for Protection Against Acute versus Persistent Friend Retrovirus Infections

AbstractThe propensity of retroviruses to rapidly establish persistent infections poses a formidable problem in vaccination strategies. In the current study, we use a live attenuated vaccine to study protection against acute and persistent Friend virus infections in mice. Adoptive transfers of immune CD8+ T cells combined with passive immunizations with virus-neutralizing antibodies increased protection against acute disease compared with either treatment alone, but there was no protection against the establishment of persistent infection. In addition, the protection against acute disease elicited by the combination treatment was dependent on endogenous CD4+ T cells as no protection was achieved in CD4+ T-cell-depleted mice. Quantitative studies showed that doubling the numbers of immune lymphocytes used in adoptive transfer experiments increased protection against acute disease depending on the type of lymphocyte subset used in the transfer. CD8+ T cells were the most potent subset for the transfer of such protection. However, even high numbers of immune CD8+ T cells gave no protection against the establishment of persistent infections. The data indicate that strengthening the numbers of specific immune cell subsets may have a beneficial effect on protection against acute disease, but protection from establishment of persistence requires complex immune responses involving multiple lymphocyte subsets
The HIV-1 Env Protein Signal Sequence Retards Its Cleavage and Down-regulates the Glycoprotein Folding

AbstractSecretory proteins and most membrane proteins are synthesized with a signal sequence that is usually cleaved from the nascent polypeptide chain, during its transport, into the lumen of the endoplasmic reticulum (ER). We have analyzed the kinetics of the cleavage of the HIV-1 Env protein signal sequence from gp160 and gp120 in HeLa, BHK, and Jurkat cells. Furthermore, we have determined the effects of this cleavage on the association of the gp160 and gp120 glycoproteins with the ER protein calnexin and the effects of the signal sequence cleavage on protein folding. The cleavage of the HIV-1 Env protein signal sequence on both gp160 and gp120 occurred very slowly in all three cell lines with a t1/2 of 45–60 min. The core glycosylated and signal-sequence-retained forms of gp160 and gp120 associated with calnexin while the signal-sequence-cleaved forms of gp160 and gp120 had disassociated from calnexin and correctly folded as determined by their ability to associate with the CD4 cellular receptor. Further analysis of the folding state of gp160 and gp120 in nonreducing SDS–PAGE revealed that the signal-sequence-retained and calnexin-associated forms of gp160 and gp120 migrated as broad, diffuse bands, whereas the signal-sequence-cleaved or CD4-associated forms of gp160 and gp120 migrated as single sharper bands. The cause of this retardation in the rate of folding and intracellular transport of HIV-1 glycoproteins was localized to their signal sequences by fusing the vesicular stomatitis virus G protein with the HIV-1 Env protein signal sequence and expressing this chimeric protein in mammalian cells. The HIV-1 Env protein signal sequence on the VSV-G protein also confers a reduced rate of cleavage and slow intracellular transport and folding of the chimeric G protein. These results provide direct evidence that in vivo the HIV-1 glycoprotein signal sequence inhibits the folding of HIV-1 Env protein. Our data also suggest a direct correlation between the rate of the signal sequence cleavage and protein folding
Conversion of Vibrio eltor MAK757 to Classical Biotype: Role of Phage PS166

AbstractTemperate phage PS166 infection of Vibrio eltor MAK757 resulted in complete changes in all biotype-specific determinants. About 10% of the PS166 lysogens of MAK757 lost their eltor-specific determinants, namely, the ability to produce soluble hemolysin, cell-associated hemagglutinin for chicken erythrocytes, and resistance to polymyxin B, as well as resistance to Mukherjee's group IV phage and sensitivity to eltor phage e4. These lysogens were found to have acquired the properties of classical strains, most significantly becoming sensitive to group IV phage but resistant to eltor-specific e4. The remainder of these lysogens, however, retained their parental biotype and serotype but acquired auxotrophy for glycine and histidine. The differential behavior of the two types of lysogen was due to the integration of the phage PS166 genome at different locations in the host chromosome. A 800-bp BglII fragment was found to contain the attP site. Phage PS166 has a polyhedral head (95 nm in diameter) and a contractile tail (98 nm in length). The phage chromosome is a linear double-stranded DNA of 110 kb and a G + C content of 58.7%
Human Immunodeficiency Virus Type 1 Nef Epitopes Recognized in HLA-A2 Transgenic Mice in Response to DNA and Peptide Immunization

AbstractWe investigated the immune response against a human immunodeficiency virus type 1 (HIV-1) nef DNA sequence administered epidermally in mice transgenic for the human major histocompatibility complex (MHC) class I molecule HLA-A201. Ten potential HLA-A2 binding 9-mer Nef peptides were identified by a computer-based search algorithm. By a cell surface MHC class I stabilization assay, four peptides were scored as good binders, whereas two peptides bound weakly to HLA-A2. After DNA immunization, cytotoxic T lymphocyte (CTL) responses were predominantly directed against the Nef 44-52, 81-89, and 85-93 peptides. Interestingly, the 44-52 epitope resides outside the regions of Nef where previously described CTL epitopes are clustered. Dominance among Nef-derived peptides did not strictly correlate with HLA-A2 binding, in that only one of the high-affinity binding peptides was targeted in the CTL response. The 44-52, 85-93, and 139-147 peptides also generated specific CTLs in response to peptide immunization. T helper cell proliferation was detected after stimulation with 20-mer peptides in vitro. Three Nef regions (16-35, 106-125, and 166-185) dominated the T helper cell proliferation. The implications of these results for the development of DNA-based vaccines against HIV is discussed
Pathogenesis of Granulocytopenia and Bone Marrow Atrophy during Classical Swine Fever Involves Apoptosis and Necrosis of Uninfected Cells

AbstractGranulocytopenia, a hematological hallmark of classical swine fever, is partially responsible for the suppression of innate immune defenses during classical swine fever. The present report demonstrates that this depletion was apparent as early as 3 days postinfection (p.i.). Both mature peripheral and bone marrow neutrophils were affected, whereas immature neutrophils increased absolutely in the periphery and coincidentally immature myeloid progenitors in the bone marrow. These data suggest that a pathogenic relationship exists between these compartments. The central event was not the arrest of hematopoietic cell proliferation or of the mobilization process, but instead apoptosis and possibly also necrosis were shown to play a role. This increase in apoptotic and dead cells was detected as early as 1–3 days p.i. In contrast, viral RNA in bone marrow hematopoietic cells (BMHC) was first detected 5 days p.i., and significant amounts of infected BMHC were detected only 7 days p.i., with the major target being the myeloid compartment. The increased caspase-3 activity observed supported a role for apoptotic cell death. Furthermore, the elevated caspase-9 activity indicated the involvement of the mitochondrial apoptotic pathway. Taken together, the results demonstrate that granulocytopenia and bone marrow atrophy are mediated by hematopoietic cell death and that indirect virus–host-mediated mechanisms are likely to be responsible
Reovirus μ2 Protein Determines Strain-Specific Differences in the Rate of Viral Inclusion Formation in L929 Cells

AbstractReovirus infection induces the formation of large cytoplasmic inclusions that serve as the major site of viral assembly. Reovirus strains type 3 Dearing (T3D) and type 1 Lang (T1L) differ in the rate of inclusion formation in L929 cells. The median time of inclusion formation is 18 h in cells infected with T3D and 39 h in cells infected with T1L. Using reassortant viruses that contain combinations of gene segments derived from T1L and T3D, we found that the M1 gene, which encodes the μ2 protein, is the primary determinant of the rate of inclusion formation. The S3 gene, which encodes the nonstructural protein ςNS, plays a secondary role in this process. The subcellular location of the μ2 protein was determined by confocal laser scanning microscopy using dual-fluorescence labeling of μ2 and the outer-capsid protein μ1/μ1C. In virus-infected cells, μ2 protein colocalized with other viral proteins in inclusions and was also distributed diffusely in the cytoplasm and nucleus. Expression of recombinant T1L and T3D μ2 proteins resulted in the formation of protein complexes resembling inclusions in both the cytoplasm and the nucleus with kinetics that reflected the strain of origin. The median time of μ2 protein complex formation was 22 h in cells transfected with the T3D M1 gene and 43 h in cells transfected with the T1L M1 gene. These findings suggest that the μ2 protein influences the rate of inclusion formation and contributes to inclusion morphogenesis. The requirement of μ2 protein in inclusion formation was tested by determining the subcellular localization of μ2 in cells infected with temperature-sensitive (ts) mutants that are defective in viral assembly. In contrast to infection with wild-type virus, μ2 did not colocalize with μ1/μ1C protein in subcellular structures that formed in cells infected at nonpermissive temperature with ts mutants tsH11.2, tsC447, and tsG453 with mutations in the M1, S2, and S4 genes, respectively. These results suggest that despite the role of the μ2 protein in controlling the rate of inclusion formation, this process is a concerted function of several reovirus proteins
A Functional NF-κB Binding Site in the Human Papillomavirus Type 16 Long Control Region

AbstractBy computer search, we identified one potential NF-κB binding site in the HPV16 long control region (LCR) at position 7554–7563 having two mismatches in comparison to the consensus NF-κB binding site of the Igκ L promoter. Bandshift experiments with nuclear extracts from HeLa cells or purified glutathione S-transferase–p65 fusion protein clearly demonstrated that NF-κB is able to bind to this region of the LCR. However, in comparison to NF-κB binding on a consensus probe, the affinity of NF-κB for this site is about 250-fold reduced. When mutations were introduced into this NF-κB binding site, the activity of the LCR was increased, strongly suggesting that NF-κB was acting as a transcriptional repressor in the context of the HPV16 LCR. In addition, overexpression of NF-κB p65 repressed the activity of the HPV16 LCR, strengthening this conclusion
Characterization of Human Herpesvirus 7 U27 Gene Product and Identification of Its Nuclear Localization Signal

AbstractA monoclonal antibody, 5H4, that recognizes human herpesvirus 7 (HHV-7) was used in Western analysis to probe HHV-7-infected SupT1 cells. This antibody recognizes a 40-kDa virus-specific polypeptide that is expressed in the absence of viral DNA synthesis. By screening a λgt11 HHV-7 cDNA library, the gene encoding the protein was identified as the U27 open reading frame previously reported [J. Virol. (1996) 70, 5975–5989]. Immunofluorescent studies showed a punctate nuclear localization of the protein in both HHV-7-infected cells and transfected cells. A computer program predicted two classic nuclear localization signals (NLSs) in the middle and C-terminal regions of the protein. A C-terminal deletion mutant of the protein could not enter the nucleus, whereas green fluorescent protein or maltose binding protein fused to the C-terminal region of the protein was transported into the nucleus. These findings demonstrate that the predicted C-terminal, but not middle, NLS of the protein actually function as NLS. In addition, nuclear transport of a maltose binding protein-fusion protein containing the C-terminal NLS of the U27 protein was inhibited by both wheat germ agglutinin and a Q69L Ran-GTP mutant, indicating that the U27 protein is transported into the nucleus from the cytoplasm by means of classic nuclear transport machinery. Interestingly, this NLS motif is highly conserved at the C-termini of all herpesvirus DNA polymerase processivity factors that have been examined
Two Leaky-Late HSV-1 Promoters Differ Significantly in Structural Architecture

AbstractThe HSV-1 VP5 and VP16 transcripts are expressed with leaky-late (γ1) kinetics and reach maximal levels after viral DNA replication. While the minimal VP5 promoter includes only an Sp1 site at −48, a TATA box at −30, and an initiator (Inr) element at the cap site, here we show that elements upstream of −48 can functionally compensate for the mutational loss of the critical Sp1 site at −48. To determine whether this is a general feature of leaky-late promoters, we have carried out a detailed analysis of the VP16 promoter in the context of the viral genome at the gC locus. Sequence analysis suggests a great deal of similarity between the two. Despite this, however, mutational analysis revealed that the 5′ boundary of the VP16 promoter extends to ca. −90. This region includes an Sp1 binding site at −46, CAAT box homology at −77, and “E box” (CACGTG) at −85. Mutational and deletional analyses demonstrate that the proximal Sp1 site plays little or no role in promoter strength; despite this it can be shown to bind Sp1 protein using DNA mobility shift assays. Like the VP5 promoter, the VP16 promoter also requires an initiator element at the cap site. The VP16 Inr element differs in sequence from that of the VP5 promoter, and its deletion or mutation has a significantly smaller effect on promoter strength. The difference between these two Inr elements was confirmed by our finding that the VP16 initiator element binds to the 65-kDa YY1 transcription factor, and the VP5 Inr element competes poorly for the binding between the VP16 element and infected cell proteins in comparative bandshift assays. While the VP16 Inr sequence is identical to that of several murine TATA-less promoters, the VP16 Inr requires a TATA box for measurable activity
The RGD Sequence in the Cytomegalovirus DNA Polymerase Accessory Protein Can Mediate Cell Adhesion

AbstractThe murine cytomegalovirus (MCMV) polymerase processivity factor ppM44 (also referred to as pp50) is an abundant phosphoprotein found in MCMV-infected cells. Sequence analysis of the MCMV M44 open reading frame revealed an “RGD” motif that is also present in the human cytomegalovirus (HCMV) UL44 open reading frame. In this report, histidine-tagged M44 protein produced in Escherichia coli or the vaccinia/T7 expression system was purified to near homogeneity by metal chelation affinity chromatography using His*Bind resins. We demonstrated that recombinant M44 protein could mediate cell adhesion via its conserved “RGD” motif, because a single amino acid change (RGD to RGE) abolished cell attachment. In addition, cell adhesion was abolished in the presence of EDTA. We next showed that recombinant HCMV UL44, but not human herpesvirus type 6 p41, which lacks the RGD motif, could mediate cell adhesion in a similar manner. We also provided evidence that ppM44 was present in the culture medium during virus infection. Thus these results suggested that in addition to its primary role as the polymerase processivity factor, MCMV ppM44 may serve as a substrate for integrin-binding via its conserved RGD motif, with the potential for a novel role in the MCMV replication cycle