bluuebunny/crossref_metadata_2025 huggingface dataset:
Clearance of a persistent paramyxovirus infection is mediated by cellular immune responses but not by serum-neutralizing antibody
Authors: Young D F , Randall R E , Hoyle J A , Souberbielle B E
Published: January 2020

Infection of the lungs of immunodeficient mice with the paramyxovirus simian virus 5 (SV5) was prolonged compared with the time course of infection in immunocompetent mice. Although there was a significant increase in both viral RNA and proteins, little infectious virus was produced. Adoptive transfer of immune lymphocytes (isolated from the spleens of mice previously infected with SV5) but not of nonimmune lymphocytes increased the speed of clearance of virus from the lungs of immunodeficient mice. In contrast, passive transfer of a pool of neutralizing monoclonal antibodies specific for the HN and F glycoproteins of SV5 did not have a significant effect on the speed of clearance of virus. Furthermore, no significant increase in the rate of virus clearance was observed upon adoptive transfer of purified immune B lymphocytes to SV5-infected immunodeficient mice despite production by the mice of high titers of neutralizing antibodies. Evidence is presented that CD8+ effector cells are primarily responsible for the clearance observed. The general significance of these results with respect to immune clearance of persistent virus infections is discussed.
Lymphotoxin activation by human T-cell leukemia virus type I-infected cell lines: role for NF-kappa B
Authors: Paul N L , Lenardo M J , Novak K D , Sarr T , Tang W L , Ruddle N H
Published: January 2020

Human T-cell leukemia virus type I (HTLV-I)-infected T-cell lines constitutively produce high levels of biologically active lymphotoxin (LT; tumor necrosis factor-beta) protein and LT mRNA. To understand the regulation of LT transcription by HTLV-I, we analyzed the ability of a series of deletions of the LT promoter to drive the chloramphenicol acetyltransferase (CAT) reporter gene in HTLV-I-positive MT-2 cells. The smallest LT promoter fragment (-140 to +77) that was able to drive CAT activity contained a site that was similar to the immunoglobulin kappa-chain NF-kappa B-binding site. Since the HTLV-I tax gene activates the nuclear form of NF-kappa B, this finding suggested a possible means of HTLV-I activation of LT production. We found that the LT kappa B-like site specifically formed a complex with NF-kappa B-containing nuclear extract from MT-2, C81-66-45, and other activated T cells. Mutation of the LT kappa B site in the context of the LT promoter (-293 to +77) (mutant M1) reduced the ability of the promoter to drive the CAT gene in HTLV-I-infected and noninfected human T-cell lines. These data suggest a general role for NF-kappa B activation in the induction of LT gene transcription. Activation of LT in HTLV-I-infected cells may explain the pathology associated with HTLV-I infection, including the hypercalcemia that is prevalent in adult T-cell leukemia.
Interaction of virally coded protein and a cell cycle-regulated cellular protein with the bovine parvovirus left terminus ori
Authors: Metcalf J B , Bates R C , Lederman M
Published: January 2020

Replication of parvoviruses requires cis signals located in terminal palindromes that function as origins of replication in conjunction with trans-acting viral and cellular proteins. A gel retardation assay was used to identify proteins in crude nuclear extracts of bovine parvovirus (BPV)-infected bovine fetal lung cells that interact with the hairpinned left end (3' OH terminus of the viral minus strand in the flop conformation) of BPV. Three specific DNA-protein complexes formed. One complex was shown to involve a BPV structural protein(s) by inhibiting its formation when antiserum specific for these BPV proteins was used. By specific competition with serum containing antibodies against the BPV nonstructural proteins, a second complex was shown to involve a BPV nonstructural protein. A third complex contained protein of cellular origin and was also formed with extracts of uninfected bovine fetal lung cells. DNA competition assays suggest that the viral proteins do not bind to the right hairpin, which differs in sequence and secondary structure from the left terminus, or to a BPV terminus that lacks the first 52 nucleotides, preventing formation of the stem of the hairpin. The cellular protein is regulated in a cell cycle-dependent fashion, with its binding activity increased in uninfected, actively dividing cells compared with contact-inhibited cells. Since autonomous parvovirus replication requires an S-phase factor for progeny formation, the terminal binding protein demonstrated here is a candidate for this factor.
Transcription factors NFI and NFIII/oct-1 function independently, employing different mechanisms to enhance adenovirus DNA replication
Authors: Mul Y M , Verrijzer C P , van der Vliet P C
Published: January 2020

Initiation of adenovirus DNA replication is strongly enhanced by two transcription factors, nuclear factor I (NFI) and nuclear factor III (NFIII/oct-1). These proteins bind to two closely spaced recognition sequences in the origin. We produced NFI and NFIII/oct-1, as well as their biologically active, replication-competent DNA-binding domains (NFI-BD and the POU domain), in a vaccinia virus expression system and purified these polypeptides to apparent homogeneity. By DNase I footprinting and gel retardation, we show that the two proteins, as well as their purified DNA-binding domains, bind independently and without cooperative effects to their recognition sequences. By using a reconstituted system consisting of the purified viral proteins (precursor terminal protein-DNA polymerase complex (pTP-pol) and DNA-binding protein, we show that NFIII/oct-1 or the POU domain stimulates DNA replication in the absence of NFI or NFI-BD and vice versa. When added together, the enhancing effect of the two transcription factors was independent and nonsynergistic. Interestingly, stimulation by NFI or NFI-BD was strongly dependent on the concentration of the pTP-pol complex. At low pTP-pol concentrations, NFI or NFI-BD stimulated up to 50-fold, while at high concentrations, the stimulation was less than twofold, indicating that the need for NFI can be overcome by high pTP-pol concentrations. In contrast, stimulation by NFIII/oct-1 or the POU domain was much less dependent on the pTP-pol concentration. These data support a model in which NFI enhances initiation through an interaction with pTP-pol. Glutaraldehyde cross-linking experiments indicate contacts between pTP-pol and NFI but not NFIII/oct-1. The site of interaction is located in the NFI-BD domain.
Major histocompatibility complex-conferred resistance to Theiler's virus-induced demyelinating disease is inherited as a dominant trait in B10 congenic mice
Authors: Patick A K , Pease L R , David C S , Rodriguez M
Published: January 2020

Intracerebral inoculation of Theiler's murine encephalomyelitis virus into susceptible strains of mice produces chronic demyelinating disease in the central nervous system characterized by persistent viral infection. Immunogenetic data suggest that genes from both major histocompatibility complex (MHC) and non-MHC loci are important in determining susceptibility or resistance to demyelination. The role of the MHC in determining resistance or susceptibility to disease can be interpreted either as the presence of antigen-presenting molecules that confer resistance to viral infection or as the ability of MHC products to contribute to pathogenesis by acting as viral receptors or by mediating immune attack against virally infected cells. These alternatives can be distinguished by determining whether the contribution of the MHC to resistance is inherited as a recessive or dominant trait. Congenic mice with different MHC haplotypes on identical B10 backgrounds were crossed and quantitatively analyzed for demyelination, infectious virus, and local virus antigen production. F1 hybrid progeny derived from resistant B10 (H-2b), B10.D2 (H-2d), or B10.K (H-2k) and susceptible B10.R111 (H-2r), B10.M (H-2f), or B10.BR (H-2k) parental mice exhibited no or minimal demyelination, indicating that on a B10 background, resistance is inherited as a dominant trait. Although infectious virus, as measured by viral plaque assay, was cleared inefficiently from the central nervous systems of resistant F1 hybrid progeny mice, we found a direct correlation between local viral antigen production and demyelination. These data are consistent with our hypothesis that the immunological basis for resistance is determined by efficient presentation of the viral antigen to the immune system, resulting in local virus clearance and absence of subsequent demyelination.
Localization of the herpes simplex virus type 1 65-kilodalton DNA-binding protein and DNA polymerase in the presence and absence of viral DNA synthesis
Authors: Goodrich L D , Schaffer P A , Dorsky D I , Crumpacker C S , Parris D S
Published: January 2020

Using indirect immunofluorescence, well-characterized monoclonal and polyclonal antibodies, and temperature-sensitive (ts) mutants of herpes simplex virus type 1, we demonstrated that the 65-kilodalton DNA-binding protein (65KDBP), the major DNA-binding protein (infected cell polypeptide 8 [ICP8]), and the viral DNA polymerase (Pol) colocalize to replication compartments in the nuclei of infected cells under conditions which permit viral DNA synthesis. When viral DNA synthesis was blocked by incubation of the wild-type virus with phosphonoacetic acid, the 65KDBP, Pol, and ICP8 failed to localize to replication compartments. Instead, ICP8 accumulated nearly exclusively to prereplication sites, while the 65KDBP was only diffusely localized within the nuclei. Although some of the Pol accumulated in prereplication sites occupied by ICP8 in the presence of phosphonoacetic acid, a significant amount of Pol also was distributed throughout the nuclei. Examination by double-labeling immunofluorescence of DNA- ts mutant virus-infected cells revealed that the 65KDBP also did not colocalize with ICP8 to prereplication sites at temperatures nonpermissive for virus replication. These results are in disagreement with the hypothesis that ICP8 is the major organizational protein responsible for attracting other replication protein to prereplication sites in preparation for viral DNA synthesis (A. de Bruyn Kops and D. M. Knipe, Cell 55:857-868, 1988), and they suggest that other viral proteins, perhaps in addition to ICP8, or replication fork progression per se are required to organize the 65KDBP.
Analysis of sequence diversity in hypervariable regions of the external glycoprotein of human immunodeficiency virus type 1
Authors: Simmonds P , Balfe P , Ludlam C A , Bishop J O , Brown A J
Published: January 2020

Nucleotide sequences in three hypervariable regions of the human immunodeficiency virus type 1 (HIV-1) env gene were obtained by sequencing provirus present in peripheral blood mononuclear cells of HIV-infected individuals. Single molecules of target sequences were isolated by limiting dilution and amplified in two stages by the polymerase chain reaction, using nested primers. The product was directly sequenced to avoid errors introduced by Taq polymerase during the amplification process. There was extensive variation between sequences from the same individual as well as between sequences from different individuals. Interpatient variability was markedly less in individuals infected from a common source. A high proportion of amino acid substitutions in the hypervariable regions altered the number and positions of potential N-linked glycosylation sites. Sequences in two hypervariable regions frequently contained short (3- to 15-bp) duplications or deletions, and by amplifying peripheral blood mononuclear cell DNA containing 10(2) or 10(3) proviral molecules and analyzing the product by high-resolution electrophoresis, the total number and abundance of distinct length variants within an individual could be estimated, providing a more comprehensive analysis of the variants present than would be obtained by sequencing alone. Sequences from many individuals showed frequent amino acid substitutions at certain key positions for neutralizing-antibody and cytotoxic T-cell recognition in the immunodominant loop. The rates of synonymous and nonsynonymous nucleotide substitution in the region of this and flanking regions indicate that strong positive selection for amino acid change is operating in the generation of antigenic diversity.
Two DNA polymerases may be required for synthesis of the lagging DNA strand of simian virus 40
Authors: Nethanel T , Kaufmann G
Published: January 2020

Agents discriminating between DNA polymerase alpha and DNA polymerases of class delta (polymerase delta or epsilon) were used to characterize steps in the synthesis of the lagging DNA strand of simian virus 40 during DNA replication in isolated nuclei. The synthesis of lagging-strand intermediates below 40 nucleotides, termed DNA primers (T. Nethanel, S. Reisfeld, G. Dinter-Gottlieb, and G. Kaufmann, J. Virol. 62:2867-2873, 1988), was selectively inhibited by butylphenyl dGTP or by neutralizing DNA polymerase alpha monoclonal antibodies. The synthesis of longer lagging chains of up to 250 nucleotides (Okazaki pieces) was affected to a lesser extent, possibly indirectly, by these agents. Aphidicolin, which inhibits both alpha- and delta-class enzymes, elicited the opposite pattern: DNA primers accumulated in its presence and were not converted into Okazaki pieces. These and previous data suggest that DNA polymerase alpha primase synthesizes DNA primers, whereas another DNA polymerase, presumably DNA polymerase delta or epsilon, mediates the conversion of DNA primers into Okazaki pieces.
The herpes simplex virus type 1 UL42 gene product: a subunit of DNA polymerase that functions to increase processivity
Authors: Gottlieb J , Marcy A I , Coen D M , Challberg M D
Published: January 2020

Genetic experiments have shown that the products of the herpes simplex virus type 1 (HSV-1) DNA polymerase (UL30) and UL42 genes are both required for viral DNA replication, and a number of studies have suggested that these two proteins specifically interact. We have confirmed and extended these findings. The viral DNA polymerase from HSV-1-infected cells has been purified as a complex containing equimolar quantities of the UL30 (Pol, the catalytic subunit) and UL42 polypeptides. Sedimentation and gel filtration analyses of this complex are consistent with the idea that the complex consists of a heterodimer of Pol and UL42. A complex with identical physical and functional properties was also purified from insect cells coinfected with recombinant baculoviruses expressing the two polypeptides. Therefore, the formation of the Pol-UL42 complex does not require the participation of any other HSV-encoded protein. We have compared the catalytic properties of the Pol-UL42 complex with those of the isolated subunits of the enzyme purified from recombinant baculovirus-infected insect cells. The specific activity of the catalytic subunit alone was nearly identical to that of the complex when assayed on activated DNA. When assayed on a defined template such as singly primed M13 DNA, however, the combination of Pol and UL42 utilized fewer primers and formed larger products than Pol alone. Template challenge experiments demonstrated that the Pol-UL42 complex was more highly processive than Pol alone. Our data are consistent with the idea that the UL42 polypeptide is an accessory subunit of the DNA polymerase that acts to increase the processivity of polymerization.
Chemical synthesis of human papillomavirus type 16 E7 oncoprotein: autonomous protein domains for induction of cellular DNA synthesis and for trans activation
Authors: Rawls J A , Pusztai R , Green M
Published: January 2020

The human papillomavirus type 16 E7 protein belongs to a family of nuclear oncoproteins that share amino acid sequences and functional homology. To localize biochemical activities associated with E7, we chemically synthesized the full-length 98-amino-acid polypeptide and several deletion mutant peptides. We show that the E7 polypeptide is biologically active and possesses at least two functional domains; the first induces cellular DNA synthesis in quiescent rodent cells, and the second trans activates the adenovirus E1A-inducible early E2 promoter and binds zinc. Further, each domain is autonomous and can function on separate peptides. DNA synthesis induction activity maps within the N-terminal portion of the molecule, which contains sequences related to adenovirus E1A conserved domains 1 and 2 required for cell transformation and binding of the retinoblastoma gene product. trans-Activation and Zn-binding activities map within the C-terminal portion of the molecule, a region which contains Cys-X-X-Cys motifs. trans Activation does not require protein synthesis, implying a mechanism that involves interaction with a preexisting cellular factor(s). E7 trans activates the adenovirus E2 promoter but not other E1A-inducible viral promoters, suggesting the possibility that E7 trans activation involves interaction, directly or indirectly, with cellular transcription factor E2F.
Overlapping cytotoxic T-lymphocyte and B-cell antigenic sites on the influenza virus H5 hemagglutinin
Authors: Hioe C E , Dybdahl-Sissoko N , Philpott M , Hinshaw V S
Published: January 2020

To define the recognition site of cytotoxic T lymphocytes (CTLs) on influenza virus H5 hemagglutinin (HA), an H5 HA-specific CTL clone was examined for the ability to recognize monoclonal antibody-selected HA variants of influenza virus A/Turkey/Ontario/7732/66 (H5N9). On the basis of 51Cr release assays with the variants, a CTL epitope was located near residue 168 of H5 HA. To define the epitope more precisely, a series of overlapping peptides corresponding to this region was synthesized and tested for CTL recognition. The minimum peptide recognized by the CTL clone encompassed residues 158 to 169 of H5 HA. Relative to the H3 HA three-dimensional structure, this CTL epitope is located near the distal tip of the HA molecule, also known as a major B-cell epitope on H3 HA. A single mutation at residue 168 (Lys to Glu) in the H5 HA variants abolished CTL recognition; this same amino acid was shown previously to be critical for B-cell recognition (M. Philpott, C. Hioe, M. Sheerar, and V. S. Hinshaw, J. Virol. 64:2941-2947, 1990). Additionally, mutations within this region of the HA molecule were associated with attenuation of the highly virulent A/Turkey/Ontario/7732/66 (H5N9) (M. Philpott, B. C. Easterday, and V.S. Hinshaw, J. Virol. 63:3453-3458, 1989). When tested for recognition of other H5 viruses, the CTL clone recognized the HA of A/Turkey/Ireland/1378/83 (H5N8) but not that of A/Chicken/Pennsylvania/1370/83 (H5N2), even though these viruses contain identical HA amino acid 158-to-169 sequences. These results suggest that differences outside the CTL epitope affected CTL recognition of the intact HA molecule. The H5 HA site defined in these studies is, therefore, important in both CTL and B-cell recognition, as well as the pathogenesis of the virus.
Genetic analysis of the enhancer requirements for polyomavirus DNA replication in mice
Authors: Rochford R , Campbell B A , Villarreal L P
Published: January 2020

In this report, we describe the first systematic analysis of the genetic requirements for polyomavirus (Py) enhancer-activated viral DNA replication during the acute phase of infection in mice. Four mutants were made which substituted XhoI sites for conserved enhancer consensus sequences (adenovirus type 5 E1A, c-fos, simian virus 40, and a glucocorticoidlike consensus sequence). Viral DNA replication in infected mouse organs was measured by DNA blot analysis. Only the loss of the glucocorticoidlike consensus sequence element significantly reduced Py DNA replication in the kidneys, the primary target organ for viral replication. The loss of the c-fos, adenovirus type 5 E1A, or simian virus 40 consensus sequences, however, expanded organ-specific viral DNA replication, relative to wild-type Py, by allowing high-level replication in the pancreas or heart or both. Analysis of Py variants selected for replication in undifferentiated embryonal carcinoma cell lines (PyF441, PyF111) showed that there was little change in levels of viral DNA replication in kidneys and other organs as compared with those in the wild-type virus. If the entire B enhancer is deleted, only low overall levels of viral replication are observed. Wild-type levels of replication in the kidneys can be reconstituted by addition of a single domain from within the A enhancer (nucleotides 5094 to 5132) to the B enhancer deletion virus, suggesting that a single domain from the A enhancer can functionally substitute for the entire B enhancer. This also indicates that the determinants for kidney-specific replication are not found in the B enhancer.
Solubility of vesicular stomatitis virus M protein in the cytosol of infected cells or isolated from virions
Authors: McCreedy B J , McKinnon K P , Lyles D S
Published: January 2020

The peripheral membrane M protein of vesicular stomatitis virus purified by detergent extraction of virions and ion-exchange chromatography was determined to be a monomer in the absence of detergent at high salt concentrations. Reduction of the ionic strength below 0.2 M resulted in a rapid aggregation of M protein. This self-association was reversible by the detergent Triton X-100 even in low salt. However, aggregation was not reversible by high salt concentration alone. M protein is initially synthesized as a soluble protein in the cytosol of infected cells, thus raising the question of how the solubility of M protein is maintained at physiological ionic strength. Addition of radiolabeled M protein purified from virions to unlabeled cytosol from either infected or uninfected cells inhibited the self-association reaction. Cytosolic fractions from infected or uninfected cells were equally effective at preventing the self-association of M protein. Self-association could also be prevented by an irrelevant protein such as bovine serum albumin. Sedimentation velocity analysis indicated that most of the newly synthesized M protein is monomeric, suggesting that the solubility of M protein in the cytosol is maintained by either low-affinity interaction with macromolecules in the cytosol or interaction of a small population of M-protein molecules with cytosolic components.
The herpes simplex virus 1 gene for ICP34.5, which maps in inverted repeats, is conserved in several limited-passage isolates but not in strain 17syn+
Authors: Chou J , Roizman B
Published: January 2020

In a previous study, it was reported that herpes simplex virus 1 (HSV-1) strain F contains a transcribed open reading frame situated in the inverted repeats of the L component between the terminal a sequence and the open reading frame that encodes the alpha 0 gene (J. Chou and B. Roizman, J. Virol. 57: 629-637, 1986). By means of an antibody to repeats of the trimer Ala-Thr-Pro predicted to be specified by the open reading frame, it was shown that the open reading frame specifies a protein (M. Ackermann, J. Chou, M. Sarmiento, R. A. Lerner, and B. Roizman, J. Virol. 58: 843-850, 1986). This open reading frame is absent from the reported sequence of HSV-1(17)syn+ (D. J. McGeoch, M. A. Dalrymple, A. J. Davison, A. Dolan, M. C. Frame, D. McNab, L. J. Perry, J. E. Scott, and P. Taylor, J. Gen. Virol. 69: 1531-1574, 1988; L. J. Perry and D. J. McGeoch, J. Gen. Virol. 69: 2831-2846, 1988). To define the extent of variability in this open reading frame, we compared the sequences of the ICP34.5-encoding open reading frames of the genomes of three strains characterized by limited passage in cell culture with that of the HSV-1(17)syn+ strain. Furthermore, to establish unambiguously that the antibody to the Ala-Thr-Pro repeats reacts with the product of this open reading frame, we inserted a short sequence that encodes a known epitope in frame at the 5' terminus of the coding domain. Our results indicate that with minor variations, the open reading frame is conserved in the three HSV-1 genomes analyzed but not in HSV-1(17)syn+. Thus, two strains contain an inserted amino acid and one strain, isolated from a case of human encephalitis, lacks a seven-amino-acid sequence. The recombinant virus carrying the foreign epitope expressed a slightly slower-migrating protein which reacted with both the rabbit polyclonal antibody to the Ala-Thr-Pro trimer repeats and the monoclonal antibody to the inserted epitope. The implications of the results are discussed.
Normal and scrapie-associated forms of prion protein differ in their sensitivities to phospholipase and proteases in intact neuroblastoma cells
Authors: Caughey B , Neary K , Buller R , Ernst D , Perry L L , Chesebro B , Race R E
Published: January 2020

Previous studies have indicated that scrapie infection results in the accumulation of a proteinase K-resistant form of an endogenous brain protein generally referred to as prion protein (PrP). The molecular nature of the scrapie-associated modification of PrP accounting for proteinase K resistance is not known. As an approach to understanding the cellular events associated with the PrP modification in brain tissue, we sought to identify proteinase K-resistant PrP (PrP-res) in scrapie-infected neuroblastoma cells in vitro and to compare properties of PrP-res with those of its normal proteinase K-sensitive homolog, PrP-sen. PrP-res was detected by immunoblot in scrapie-infected but not uninfected neuroblastoma clones. Densitometry of immunoblots indicated that there was two- to threefold more PrP-res than PrP-sen in one infected clone. Metabolic labeling and membrane immunofluorescence experiments indicated that PrP-sen was located on the cell surface and could be removed from intact cells by phosphatidylinositol-specific phospholipase C and proteases. In contrast, PrP-res was not removed after reaction with these enzymes. Thus, either the scrapie-associated PrP-res was not on the cell surface or it was there in a form that is resistant to these hydrolytic enzymes. Attempts to detect intracellular PrP-res by immunofluorescent staining of fixed and permeabilized cells revealed that PrP was present in discrete perinuclear Golgi-like structures. However, the staining pattern was similar in both scrapie-infected and uninfected clones, and thus the intracellular staining may have represented only PrP-sen. Analysis of scrapie infectivity in cells treated with extracellular phospholipase, proteinase K, and trypsin indicated that, like PrP-res, the scrapie agent was not removed from the infected cells by any of these enzymes.
Immunogenic domains of hepatitis delta virus antigen: peptide mapping of epitopes recognized by human and woodchuck antibodies
Authors: Wang J G , Jansen R W , Brown E A , Lemon S M
Published: January 2020

Hepatitis delta virus (HDV) is a defective RNA virus which is dependent on hepatitis B virus for essential helper functions. Only a single highly basic phosphoprotein, HDV antigen (HDAg), is expressed by the HDV genome during infection in humans. Antibody directed to HDAg is important in the diagnosis of HDV infection, and it is likely but not yet proven that the immune response to HDAg provides significant protection against subsequent exposures to HDV. In an effort to map the antigenic domains of HDAg, 209 overlapping hexapeptides, spanning the entire 214 amino acid residues of the protein, were synthesized on polyethylene pins and probed by enzyme-linked immunosorbent assay with sera containing high titers of anti-HD antibodies. Domains recognized by antibodies present in serum from human chronic carriers of this virus included residues 2 to 7, 63 to 74, 86 to 91, 94 to 100, 159 to 172, 174 to 195, and 197 to 207. Antibody from an acutely superinfected woodchuck recognized similar epitopes, as well as a domain spanning residues 121 to 128. Together, residues in these antigenic domains constitute 41% of the HDAg molecule. Oligopeptides 15 to 29 residues in length and representing epitopes of HDAg found to be dominant in humans (residues 2 to 17, 156 to 184, and 197 to 211) were synthesized in bulk and found to possess significant antigenic activity by microdilution enzyme-linked immunosorbent assay. The reactivity of peptide 197-211 with human sera confirms that the entire 214 amino acids of HDAg are expressed during infection in vivo. In addition, these results suggest that synthetic peptides may be useful reagents for development of new and improved diagnostic tests for HDV infection.
Identification of phorbol ester response elements in the promoter of Epstein-Barr virus putative lytic switch gene BZLF1
Authors: Flemington E , Speck S H
Published: January 2020

The product of the Epstein-Barr virus BZLF1 gene encodes a protein which is related to c-fos, it has been shown to bind specifically to a consensus AP-1 site, and its expression in latently Epstein-Barr virus-infected lymphocytes is sufficient to trigger the viral lytic cycle. We identified several elements within the BZLF1 promoter (Zp) which are responsive to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), an inducer of the viral lytic cycle. These elements fall into two classes based on the factors which bind to these sequences and their resulting functional behavior. Four of the elements are homologous (ZI elements) and share homology to a protein-binding domain in the promoter region of the coordinately expressed BRLF1 gene. When cloned upstream of heterologous promoters, the ZI elements function as silencers which exhibit TPA-inducible enhancer activity. A distinct TPA-responsive element (ZII) is located near the TATA box and shares homology with the AP-1-binding site in the c-jun promoter. A synthetic oligonucleotide with a sequence corresponding to the ZII element effectively competes for binding of nuclear factors to the c-jun AP-1 site. Furthermore, we found that a complex of c-jun and c-fos bound to the ZII domain.
Molecular basis for linkage of a continuous and discontinuous neutralization epitope on the structural polypeptide VP2 of poliovirus type 1
Authors: Wiegers K J , Wetz K , Dernick R
Published: January 2020

We obtained neutralizing monoclonal antibodies against a continuous neutralization epitope on VP2 of poliovirus type 1 strain Mahoney by using a combined in vivo-in vitro immunization procedure. The antibody-binding site was mapped to amino acid residues within the peptide segment (residues 164 through 170) of VP2 by competition with synthetic peptide and sequencing of resistant mutants. Cross-neutralization of these mutants with another neutralizing monoclonal antibody revealed a linkage of the continuous epitope and a discontinuous neutralization epitope involving both loops of the double-loop structure of VP2 at the twofold axis on the surface of the virion.
Passively transferred monoclonal antibody to the M2 protein inhibits influenza A virus replication in mice
Authors: Treanor J J , Tierney E L , Zebedee S L , Lamb R A , Murphy B R
Published: January 2020

The M2 protein of influenza A virus is expressed on the surfaces of infected cells, and a monoclonal antibody to this protein inhibits plaque enlargement of sensitive influenza A viruses without reducing plaque titer (S.L. Zebedee and R.A. Lamb, J. Virol. 62:2762-2772, 1988). In the current study, passively transferred monoclonal antibody to M2 reduced the level of replication of influenza A virus but not of influenza B virus in the lungs of mice. These experiments demonstrated that antibody to a protein conserved among influenza A virus subtypes inhibits virus growth in vivo.
CD4-independent, productive infection of a neuronal cell line by human immunodeficiency virus type 1
Authors: Li X L , Moudgil T , Vinters H V , Ho D D
Published: January 2020

One neuronal cell line (SK-N-MC) was found to be susceptible to productive infection by multiple isolates of the human immunodeficiency virus type 1 (HIV-1). Characterization of SK-N-MC cells showed that these cells are neuroectodermal in origin in that they express dopamine hydroxylase, catecholamines, neuron-specific enolase, and neurofilaments. Despite their susceptibility to HIV-1 infection, SK-N-MC cells had no detectable CD4 and this infection was not blocked by anti-CD4 monoclonal antibodies (OKT4A, Leu3A) or recombinant soluble CD4. These experiments demonstrated that certain cells of neuroectodermal origin are susceptible to infection in vitro by HIV-1 via a CD4-independent mechanism.
Limited expression of poliovirus by vaccinia virus recombinants due to inhibition of the vector by proteinase 2A
Authors: Jewell J E , Ball L A , Rueckert R
Published: January 2020

A recombinant vaccinia virus was constructed that expressed poliovirus coat precursor protein P1 fused to about two-thirds of the 2A proteinase. The truncated 2A segment could be cleaved away from the P1 region by coinfecting with poliovirus type 1, 2, or 3 or with human rhinovirus 14 but not with encephalomyocarditis virus. Further cleavage of the vector-derived P1 to yield mature poliovirus capsid proteins was not observed. Attempts to isolate vaccinia virus recombinants containing portions of the poliovirus genome that encompassed the complete gene for proteinase 2A were unsuccessful, unless expression of functional 2A was abolished by insertion of a frameshift mutation. We conclude that an activity of the 2A proteinase, probably its role in translational inhibition, prevented isolation of vaccinia virus recombinants that expressed 2A.
Epstein-Barr virus (EBV) antigens processed and presented by B cells, B blasts, and macrophages trigger T-cell-mediated inhibition of EBV-induced B-cell transformation
Authors: Bejarano M T , Masucci M G , Morgan A , Morein B , Klein G , Klein E
Published: January 2020

The ability of B cells, B blasts, and macrophages to present Epstein-Barr virion antigens to autologous T cells and trigger their capacity to inhibit Epstein-Barr virus-induced B-cell transformation was tested. Macrophages were as efficient as B cells and B blasts in presenting the virus to T lymphocytes. This function required antigen processing, because it was inhibited by chloroquine treatment and by fixation of the antigen-presenting cells immediately after viral exposure but not 18 h later. T cells exposed to the purified Epstein-Barr virus envelope antigen gp350 coupled to immunostimulating complexes also showed inhibitory function. These results suggest that recognition of processed virion antigens elicits the generation of T-cell-mediated inhibition of Epstein-Barr virus-induced B-cell transformation.
Structural requirements for trans activation of human immunodeficiency virus type 1 long terminal repeat-directed gene expression by tat: importance of base pairing, loop sequence, and bulges in the tat-responsive sequence
Authors: Roy S , Parkin N T , Rosen C , Itovitch J , Sonenberg N
Published: January 2020

In order to elucidate the molecular mechanisms of action of the tat-responsive sequence, mutational analysis of the tat-responsive sequence was carried out. The most critical region comprised nucleotides +18 to +44 and included the 3-nucleotide bulge at positions +23 to +25, the loop sequence, and an intact stem. In addition, base pairing up to nucleotide +52 was required for the full magnitude of the trans-activation response. Single-nucleotide bulges at positions +5 to +17 were dispensable. Analysis of truncated and full-length transcripts demonstrated that a transcriptional antitermination model does not fully account for trans activation.
Early death after feline infectious peritonitis virus challenge due to recombinant vaccinia virus immunization
Authors: Vennema H , de Groot R J , Harbour D A , Dalderup M , Gruffydd-Jones T , Horzinek M C , Spaan W J
Published: January 2020

The gene encoding the fusogenic spike protein of the coronavirus causing feline infectious peritonitis was recombined into the genome of vaccinia virus. The recombinant induced spike-protein-specific, in vitro neutralizing antibodies in mice. When kittens were immunized with the recombinant, low titers of neutralizing antibodies were obtained. After challenge with feline infectious peritonitis virus, these animals succumbed earlier than did the control group immunized with wild-type vaccinia virus (early death syndrome).
Enhanced infectivity of herpes simplex virus type 1 viral DNA in a cell line expressing the trans-inducing factor Vmw65
Authors: Werstuck G , Bilan P , Capone J P
Published: January 2020

Vmw65 is a structural component of herpes simplex virus (HSV) which is involved in transactivating the expression of the viral immediate-early (IE) genes. To gain further insight into the function of this protein, a cell line, BSV65, was established which expresses biologically active Vmw65 under control of the Moloney leukemia virus long terminal repeat. This cell line was shown to specifically activate IE genes as demonstrated by transient transfection assays with reporter genes linked to HSV IE or delayed-early promoter-regulatory regions. Furthermore, by using mobility shift assays, cell extracts were shown to be capable of forming a Vmw65-containing complex with oligonucleotides that contained a TAATGARAT motif, a conserved cis-acting IE regulatory element which is required for Vmw65-mediated trans induction. BSV65 cells were able to complement HSV type 1 in 1814, a mutant which is unable to trans-induce IE gene expression and whose growth is impaired at low multiplicities of infection. Transfection of purified HSV type 1 viral DNA into BSV65 cells resulted in an approximately 200-fold increase in virus production compared with the parental cell line. In addition, in comparison to wild-type cells, infectious virus production occurred sooner and efficiency of plaque formation was higher in BSV65 cells following transfection of viral DNA but not following infection with virus. Northern (RNA) dot blot analysis of cells transfected with viral DNA showed that transcription of the IE gene Vmw175 was approximately 10-fold greater in BSV65 cells compared with wild-type cells. These results indicate that, in the presence of functional Vmw65, there is a greater probability that transfected viral DNA will lead to a productive infection.
Serum enhancement of human immunodeficiency virus (HIV) infection correlates with disease in HIV-infected individuals
Authors: Homsy J , Meyer M , Levy J A
Published: January 2020

The sera from 16 individuals infected with the human immunodeficiency virus (HIV) at different clinical stages were evaluated for antibody-dependent neutralization and/or enhancement of infectivity by HIV. The HIV isolate from each individual (homotypic) and established laboratory strains showing broad cellular host range and cytopathicity were used. All sera could neutralize one of the laboratory-passaged isolates, whereas only two could neutralize the corresponding homotypic strain. Seven homotypic isolates were enhanced by serum from the respective individual. This activity was primarily observed in patients with acquired immune deficiency syndrome. Moreover, the tropism for macrophages of four of these seven viral isolates was found to be enhanced by the homotypic sera. Finally, sequential pairs of HIV and sera obtained from five HIV-infected individuals with different clinical progression were studied over time. The enhancing activity of three of the five sera appeared to increase over time, indicating changes in both the host virus population and the type of antibodies produced. These results suggest that enhancing antibodies contribute to the spread and pathogenesis of HIV in vivo. They emphasize the necessity of studying further the association of enhancing antibodies and disease progression in infected individuals.
Newcastle disease virus fusion protein expressed in a fowlpox virus recombinant confers protection in chickens
Authors: Taylor J , Edbauer C , Rey-Senelonge A , Bouquet J F , Norton E , Goebel S , Desmettre P , Paoletti E
Published: January 2020

A cDNA copy of the RNA encoding the fusion (F) protein of Newcastle disease virus (NDV) strain Texas, a velogenic strain of NDV, was obtained and the sequence was determined. The 1,792-base-pair sequence encodes a protein of 553 amino acids which has essential features previously established for the F protein of virulent NDV strains. These include the presence of three strongly hydrophobic regions and pairs of dibasic amino acids in the pentapeptide Arg-Arg-Gln-Arg-Arg preceding the putative cleavage site. When inserted into a fowlpox virus vector, a glycosylated protein was expressed and presented on the surface of infected chicken embryo fibroblast cells. The F protein expressed by the recombinant fowlpox virus was cleaved into two polypeptides. When inoculated into susceptible birds by a variety of routes, an immunological response was induced. Ocular or oral administration of the recombinant fowlpox virus gave partial protection, whereas both intramuscular and wing-web routes of inoculation gave complete protection after a single inoculation.
Evolution of the nucleoprotein gene of influenza A virus
Authors: Gorman O T , Bean W J , Kawaoka Y , Webster R G
Published: January 2020

Nucleotide sequences of 24 nucleoprotein (NP) genes isolated from a wide range of hosts, geographic regions, and influenza A virus serotypes and 18 published NP gene sequences were analyzed to determine evolutionary relationships. The phylogeny of NP genes was determined by a maximum-parsimony analysis of nucleotide sequences. Phylogenetic analysis showed that NP genes have evolved into five host-specific lineages, including (i) Equine/Prague/56 (EQPR56), (ii) recent equine strains, (iii) classic swine (H1N1 swine, e.g., A/Swine/Iowa/15/30) and human strains, (iv) gull H13 viruses, and (v) avian strains (including North American, Australian, and Old World subgroups). These NP lineages match the five RNA hybridization groups identified by W. J. Bean (Virology 133:438-442, 1984). Maximum nucleotide differences among the NPs was 18.5%, but maximum amino acid differences reached only 10.8%, reflecting the conservative nature of the NP protein. Evolutionary rates varied among lineages; the human lineage showed the highest rate (2.54 nucleotide changes per year), followed by the Old World avian lineage (2.17 changes per year) and the recent equine lineage (1.22 changes per year). The per-nucleotide rates of human and avian NP gene evolution (1.62 x 10(-3) to 1.39 x 10(-3) changes per year) are lower than that reported for human NS genes (2.0 x 10(-3) changes per year; D. A. Buonagurio, S. Nakada, J. D. Parvin, M. Krystal, P. Palese, and W. M. Fitch, Science 232:980-982, 1986). Of the five NP lineages, the human lineage showed the greatest evolution at the amino acid level; over a period of 50 years, human NPs have accumulated 39 amino acid changes. In contrast, the avian lineage showed remarkable conservatism; over the same period, avian NP proteins changed by 0 to 10 amino acids. The specificity of the H13 NP in gulls and its distinct evolutionary separation from the classic avian lineage suggests that H13 NPs may have a large degree of adaptation to gulls. The presence of avian and human NPs in some swine isolates demonstrates the susceptibility of swine to different virus strains and supports the hypothesis that swine may serve as intermediates for the introduction of avian influenza virus genes into the human virus gene pool. EQPR56 is relatively distantly related to all other NP lineages, which suggests that this NP is rooted closest to the ancestor of all contemporary NPs. On the basis of estimation of evolutionary rates from nucleotide branch distances, current NP lineages are at least 100 years old, and the EQPR56 NP is much older.(ABSTRACT TRUNCATED AT 400 WORDS)
Expression strategy of a phlebovirus: biogenesis of proteins from the Rift Valley fever virus M segment
Authors: Suzich J A , Kakach L T , Collett M S
Published: January 2020

The middle (M) RNA segment of Rift Valley fever virus (RVFV) encodes four proteins: the major viral glycoproteins G2 and G1, a 14-kilodalton (kDa) protein, and a 78-kDa protein. These proteins are derived from a single large open reading frame (ORF) present in the virus-complementary M-segment mRNA. We used recombinant vaccinia viruses in which sequences representing the M-segment ORF were engineered as a surrogate system to study phlebovirus protein expression. To investigate the translational initiation codon requirements for synthesis of these proteins, we constructed a series of vaccinia virus recombinants containing specific sequence changes which eliminated select ATG codons found in the region of the ORF preceding the mature glycoprotein-coding sequences (the preglycoprotein region). Examination of phleboviral proteins synthesized in cells infected with these vaccinia virus recombinants clearly showed that the first ATG of the ORF was required for the production of the 78-kDa protein, while synthesis of the 14-kDa protein was absolutely dependent on the second in-phase ATG codon. Efficient biosynthesis of glycoprotein G2 was shown to depend on one or more ATG codons within the preglycoprotein region, but not the first one of the ORF. Synthesis of about one-half of the total glycoprotein G1 was affected by the amino acid changes that eliminated ATG codons, while production of the remainder appeared to be independent of all ATG codons in the preglycoprotein region. These data indicated that the means for glycoprotein G1 biosynthesis was distinct from those of the other three M-segment gene products. The results presented herein suggest that a surprisingly complex expression strategy is employed by the RVFV M segment. Although the full nature of the mechanisms involved in the biogenesis of the four RVFV M-segment proteins remains unclear, it does involve the use of at least two (ATG codons 1 and 2), and likely more, distinct translation start sites within the same ORF to produce its complete complement of gene products.
Mutation of the serine 312 phosphorylation site does not alter the ability of mouse p53 to inhibit simian virus 40 DNA replication in vivo
Authors: Meek D W , Eckhart W
Published: January 2020

Two mutations were introduced into the wild-type mouse p53 gene by oligonucleotide-directed mutagenesis. These mutations substituted alanine or aspartic acid for serine at position 312, which is constitutively phosphorylated. Phosphopeptide mapping of the mutant proteins, expressed in COS cells, confirmed the loss of phosphorylation at position 312. There were no changes in the ability of the mutant p53s to express the conformation-dependent epitope for monoclonal antibody PAb246 or to participate in complexes with the simian virus 40 (SV40) large T antigen. Replication of a plasmid containing the SV40 origin of replication was inhibited in COS cells by wild-type p53 and both of the phosphorylation site mutants with equal efficiency. A transforming mutant of p53, encoding valine at position 135, did not inhibit SV40 DNA replication in COS cells.