bluuebunny/crossref_metadata_2025 huggingface dataset:
Activation of endothelial cells via antibody-enhanced dengue virus infection of peripheral blood monocytes
Authors: Anderson R , Wang S , Osiowy C , Issekutz A C
Published: January 2020

Although endothelial cells have been speculated to be a target in the pathogenesis of dengue hemorrhagic fever (DHF), there has been little evidence linking dengue virus infection to any alteration in endothelial cell function. In this study, we show that human umbilical vein endothelial cells become activated when exposed to culture fluids from dengue virus-infected peripheral blood monocytes. Maximum activation was achieved with culture fluids from monocytes in which virus infection was enhanced by the addition of dengue virus-immune serum, thus correlating with epidemiological evidence that prior immunity to dengue virus is a major risk factor for DHF. Activation was strongest for endothelial cell expression of VCAM-1 and ICAM-1. In contrast, activation of endothelial cell E-selectin expression appeared to be more transient, as indicated by its detection at 3 h, but not at 16 h, of treatment. Treatment of monocyte culture fluids with anti-tumor necrosis factor alpha (TNF-alpha) antibody largely abolished the activation effect (as measured by endothelial cell expression of ICAM-1), whereas treatment with IL-1beta receptor antagonist had a much smaller inhibitory effect on activation. Endothelial cells inoculated directly with dengue virus or with virus-antibody combinations were poorly infectable (compared to Vero cells or peripheral blood monocytes), and virus-inoculated endothelial cells showed no increased expression of VCAM-1, ICAM-1, or E-selectin. Taken together, the results strongly indicate that dengue virus can modulate endothelial cell function by an indirect route, in which a key intermediary is TNF-alpha released from virus-infected monocytes.
Efficient encapsidation of human immunodeficiency virus type 1 vectors and further characterization of cis elements required for encapsidation
Authors: McBride M S , Schwartz M D , Panganiban A T
Published: January 2020

To determine whether there is a cis-acting effect of translational expression of gag on RNA encapsidation, we compared the encapsidation of wild-type RNA with that of a mutant in which the translation of gag was ablated. This comparison indicated that there is not such a cis effect. To determine what is necessary and sufficient for encapsidation, we measured the relative encapsidation efficiencies of human immunodeficiency virus type 1 vector RNAs containing mutations in domains proximal to the canonical encapsidation signal or containing large deletions in the remainder of the genome. These data indicate that TAR and two additional regions are required for encapsidation and that the 5' end of the genome is sufficient for encapsidation. The Rev-responsive element is required mainly for efficient RNA transport from the nucleus to the cytoplasm. A foreign sequence was found to have a negative effect on encapsidation upon placement within the parental vector. Interestingly, this negative effect was compounded by multiple copies of the sequence.
Role of the first and third extracellular domains of CXCR-4 in human immunodeficiency virus coreceptor activity
Authors: Brelot A , Heveker N , Pleskoff O , Sol N , Alizon M
Published: January 2020

The CXCR-4 chemokine receptor and CD4 behave as coreceptors for cell line-adapted human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) and for dual-tropic HIV strains, which also use the CCR-5 coreceptor. The cell line-adapted HIV-1 strains LAI and NDK and the dual-tropic HIV-2 strain ROD were able to infect CD4+ cells expressing human CXCR-4, while only LAI was able to infect cells expressing the rat homolog of CXCR-4. This strain selectivity was addressed by using human-rat CXCR-4 chimeras. All chimeras tested mediated LAI infection, but only those containing the third extracellular domain (e3) of human CXCR-4 mediated NDK and ROD infection. The e3 domain might be required for the functional interaction of NDK and ROD, but not LAI, with CXCR-4. Alternatively, LAI might also interact with e3 but in a different way. Monoclonal antibody 12G5, raised against human CXCR-4, did not stain cells expressing rat CXCR-4. Chimeric human-rat CXCR-4 allowed us to map the 12G5 epitope in the e3 domain. The ability of 12G5 to neutralize infection by certain HIV-1 and HIV-2 strains is also consistent with the role of e3 in the coreceptor activity of CXCR-4. The deletion of most of the amino-terminal extracellular domain (e1) abolished the coreceptor activity of human CXCR-4 for ROD and NDK but not for LAI. These results indicate that HIV strains have different requirements for their interaction with CXCR-4. They also suggest differences in the interaction of dual-tropic HIV with CCR-5 and CXCR-4.
Herpes simplex virus type 1 prereplicative sites are a heterogeneous population: only a subset are likely to be precursors to replication compartments
Authors: Lukonis C J , Burkham J , Weller S K
Published: January 2020

When herpes simplex virus type 1 (HSV-1) DNA replication is blocked by viral polymerase inhibitors, such as phosphonoacetic acid (PAA) or acyclovir (ACV), UL29 (ICP8) localizes to numerous punctate nuclear foci which are called prereplicative sites. Since this pattern can form in cells infected with mutants which are defective in UL5, UL8, UL9, or UL52 in the presence of polymerase inhibitors (C. J. Lukonis and S. K. Weller, J. Virol. 70:1751-1758, 1996; L. M. Liptak, S. L. Uprichard, and D. M. Knipe, J. Virol. 70:1759-1767, 1996), we previously proposed that it is unlikely that these numerous UL29 foci actually represent a functional subassembly of viral replication proteins that could lead to the formation of replication compartments (C. J. Lukonis and S. K. Weller, J. Virol. 70:1751-1758, 1996). In this paper, we have investigated the requirement for formation of the prereplicative site pattern by using double mutants of HSV. From the analysis of mutants lacking both UL5 and UL9, we conclude that neither viral helicase is required for the prereplicative site pattern to form as long as a polymerase inhibitor is present. From the analysis of mutants defective in both UL30 and UL5, we suggest that the prereplicative site pattern can form under conditions in which viral and/or cellular polymerases are inhibited. Furthermore, reexamination of the UL29 staining pattern in cells infected with wild-type virus in the presence of PAA reveals that at least two different UL29 staining patterns can be detected in these cells. One population of cells contains numerous (greater than 20) punctate UL29 foci which are sites of cellular DNA synthesis. In another population of cells, fewer punctate foci (less than 15) are detected, and these structures do not colocalize with sites of cellular DNA synthesis. Instead, they colocalize with PML, a component of nuclear matrix structures known as ND10. We propose that ND10-associated UL29 sites represent domains at which replication compartments form.
Selective targeting of human cells by a chimeric adenovirus vector containing a modified fiber protein
Authors: Stevenson S C , Rollence M , Marshall-Neff J , McClelland A
Published: January 2020

The adenovirus fiber protein is responsible for attachment of the virion to unidentified cell surface receptors. There are at least two distinct adenovirus fiber receptors which interact with the group B (Ad3) and group C (Ad5) adenoviruses. We have previously shown by using expressed adenovirus fiber proteins that it is possible to change the specificity of the fiber protein by exchanging the head domain with another serotype which recognizes a different receptor (S. C. Stevenson et al., J. Virol. 69:2850-2857, 1995). A chimeric fiber cDNA containing the Ad3 fiber head domain fused to the Ad5 fiber tail and shaft was incorporated into the genome of an adenovirus vector with E1 and E3 deleted encoding beta-galactosidase to generate Av9LacZ4, an adenovirus particle which contains a chimeric fiber protein. Western blot analysis of the chimeric fiber vector confirmed expression of the chimeric fiber protein and its association with the adenovirus capsid. Transduction experiments with fiber protein competitors demonstrated the altered receptor tropism of the chimeric fiber vector compared to that of the parental Av1LacZ4 vector. Transduction of a panel of human cell lines with the chimeric and parental vectors provided evidence for a different cellular distribution of the Ad5 and Ad3 receptors. Three cell lines (THP-1, MRC-5, and FaDu) were more efficiently transduced by the vector containing the Ad3 fiber head than by the Ad5 fiber vector. In contrast, human coronary artery endothelial cells were transduced more readily with the vector containing the Ad5 fiber than with the chimeric fiber vector. HeLa and human umbilical vein endothelial cells were transduced at equivalent levels compared with human diploid fibroblasts, which were refractory to transduction with both vectors. These results provide evidence for the differential expression of the Ad5 and Ad3 receptors on human cell lines derived from clinically relevant target tissues. Furthermore, we show that exchange of the fiber head domain is a viable approach to the production of adenovirus vectors with cell-type-selective transduction properties. It may be possible to extend this approach to the use of ligands for a range of different cellular receptors in order to target gene transfer to specific cell types at the level of transduction.
Diminishing adenovirus gene expression and viral replication by promoter replacement
Authors: Fang B , Koch P , Roth J A
Published: January 2020

The adenovirus E4 promoter was replaced by a synthetic promoter composed of a minimal TATA box and five consensus 17-mer yeast GAL4-binding-site elements. The viral vectors, which also contained human factor IX (hFIX) cDNA driven by Rous sarcoma virus long terminal repeat in the E1 region, were then constructed and expanded in 293 cells permanently expressing GAL4/VP16 fusion protein. Viral replication and expression of adenovirus E4 genes and late genes (hexon and fiber) were evaluated in vitro in the human lung carcinoma cell line H1299. Viral replication and viral gene expression were dramatically reduced in the cells transduced by vectors with a replaced E4 promoter compared to the levels in the cells transduced by vectors with the wild-type E4 promoter. The levels of transgene (hFIX) expression remained similar between vectors with or without E4 promoter replacement. These results indicate that diminution of viral gene expression and viral replication is achievable by promoter replacement.
Association of ERK2 mitogen-activated protein kinase with human immunodeficiency virus particles
Authors: Cartier C , Deckert M , Grangeasse C , Trauger R , Jensen F , Bernard A , Cozzone A , Desgranges C , Boyer V
Published: January 2020

Here we report the presence of a protein kinase activity associated with human immunodeficiency virus type 1 (HIV-1) particles. We observed phosphorylation of five major proteins by the endogenous protein kinase activity. Phosphoamino acid analysis revealed phosphorylated serine and threonine residues. In addition, we observed autophosphorylation of two proteins in the presence of gamma-ATP in an in-gel phosphorylation assay. These two proteins are not linked by a disulfide bond, suggesting that two different protein kinases are associated with HIV-1 virions. Our results indicate the presence of ERK2 mitogen-activated protein kinase and of a 53,000-molecular-weight protein kinase associated with virions. Moreover, the use of different HIV strains derived from T cells and promonocytic cells, as well as the use of human T-cell leukemia virus type 1 particles, demonstrates that ERK2 is strongly associated with retrovirus particles in a cell-independent manner. Exogenous substrates, such as histone proteins, and a viral substrate, such as Gag protein, are phosphorylated by virus-associated protein kinases.
In vivo sequence variability of human immunodeficiency virus type 1 envelope gp120: association of V2 extension with slow disease progression
Authors: Shioda T , Oka S , Xin X , Liu H , Harukuni R , Kurotani A , Fukushima M , Hasan M K , Shiino T , Takebe Y , Iwamoto A , Nagai Y
Published: January 2020

According to the rate of depletion of CD4 cell counts, we grouped 12 cases of human immunodeficiency virus type 1 (HIV-1) infection as 6 rapid (21.0 to 33.8 cells per microl per month) and 6 slow (0.9 to 7.9 cells per microl per month) progressors and determined the individual viral quasispecies patterns by sequencing the genome region encoding the V1, V2, and V3 loops of envelope protein. Although the quasispecies structures varied widely from one individual to another, a strong correlation was observed between a low rate of disease progression and a high degree of genetic diversity of HIV-1. Furthermore, the V2 loop extension was observed specifically in individuals with slow or no disease progression, whereas basic amino acid substitutions in V3 characteristic of a viral phenotype shift from non-syncytium inducing to syncytium inducing were observed in patients with advanced stages of disease regardless of their rate of disease progression. Studies with recombinant viruses suggested that elongation of V2 potentially restricts the capacity of HIV-1 to replicate in macrophages. Thus, our results suggest the association of distinct sequence features of both V3 and V2 with particular patterns of disease progression. Elongation of the V2 loop may be a good predictor of slow disease progression, while basic substitutions of V3 without elongation of V2 are characteristic of rapid progression.
Brain-infiltrating cytolytic T lymphocytes specific for Theiler's virus recognize H2Db molecules complexed with a viral VP2 peptide lacking a consensus anchor residue
Authors: Borson N D , Paul C , Lin X , Nevala W K , Strausbauch M A , Rodriguez M , Wettstein P J
Published: January 2020

Mice expressing the H2b haplotype are resistant to infection with Theiler's murine encephalomyelitis virus (TMEV), which causes chronic demyelination in susceptible mice. The prominent cytolytic T-lymphocyte (CTL) response to the VP2 antigen encoded by TMEV led us to the identification of a class I-binding peptide derived from the VP2 antigen. Escherichia coli transformants overexpressing a series of 11 overlapping VP2 protein fragments were subjected to lysis and alkali digestion, and the resultant peptide pools were tested for their abilities to sensitize RMA-S targets for lysis by CTLs. The source of effector CD8+ T cells for the assays was either freshly harvested central nervous system-infiltrating lymphocytes (CNS-IL) or CNS-IL-derived VP2-specific CTL clones and lines. A 10-residue peptide at VP2 positions 121 to 130 (VP2(121-130)) (FHAGSLLVFM) was identified that sensitized targets for lysis and formed stable complexes with H2Db class I molecules. The VP2(121-130) peptide sensitized target cells for lysis by freshly harvested CNS-IL CTLs at femtomolar concentrations. Despite its relative high level of biological activity, the VP2(121-130) peptide is distinguished from other Db-binding peptides by its lack of an asparagine residue at position five, which had been previously proposed to be a requirement for Db-peptide complexing.
Gene expression during reactivation of herpes simplex virus type 1 from latency in the peripheral nervous system is different from that during lytic infection of tissue cultures
Authors: Tal-Singer R , Lasner T M , Podrzucki W , Skokotas A , Leary J J , Berger S L , Fraser N W
Published: January 2020

Herpes simplex virus (HSV) replicates in peripheral tissues and forms latent infections in neurons of the peripheral nervous system. It can be reactivated from latency by various stimuli to cause recurrent disease. During lytic infection in tissue culture cells, there is a well-described temporal pattern of (i) immediate-early, (ii) early, and (iii) late gene expression. However, latency is characterized by little if any expression of genes of the lytic cycle of infection. During reactivation, the pattern of gene expression is presumed to be similar to that during the lytic cycle in tissue culture, though recent work of W. P. Halford et al. (J. Virol. 70:5051-5060, 1996) and P. F. Nichol et al. (J. Virol. 70:5476-5486, 1996) suggests that it is modified in neuronal cell cultures. We have used the mouse trigeminal ganglion explant model and reverse transcription-PCR to determine the pattern of viral and cellular gene expression during reactivation. Surprisingly, the pattern of viral gene expression during lytic infection of cell cultures is not seen during reactivation. During reactivation, early viral transcripts were detected before immediate-early transcripts. The possibility that a cellular factor upregulates early genes during the initial reactivation stimulus is discussed.
Immunogenicity of a contiguous T-B synthetic epitope of the A/PR/8/34 influenza virus
Authors: Brumeanu T D , Casares S , Bot A , Bot S , Bona C A
Published: January 2020

A contiguously linked T-B synthetic viral epitope (110HA120-150HA159,T-B) was investigated for its potency in inducing humoral and cellular immune responses in vivo. The T-cell epitope 110HA120 corresponds to the site 1 hemagglutinin (HA) of the A/PR/8/34 (PR8) influenza virus and is recognized by CD4 T cells in association with I-Ed class II major histocompatibility complex molecules. The 150HA159 represents a major B-cell epitope of the HA protein. T-B dipeptide emulsified in Freund's complete adjuvant was able to induce strong antiviral antibody titers and a high frequency of specific T-cell precursors after a single inoculation in BALB/c mice. In contrast, immunization under identical conditions with equimolar mixtures of T and B peptides did not elicit antibody titers or a cellular immune response. As indicated by the isotypes of antiviral antibodies, the T-B dipeptide preferentially induced a Th1-like immune response. Challenge with T-B dipeptide, but not with T or B peptide alone, stimulated peptide-specific T memory cells in mice previously primed with PR8 virus or with T-B dipeptide. As a consequence, 71 and 57% of these mice, respectively, survived infection with two 100% lethal doses of PR8 virus. Our results suggest that, inasmuch as contiguity between T- and B-cell epitopes provides enough signaling capacity to trigger the mechanisms of T-B-cell cooperation in vivo, a T-B contiguous epitope may well represent a minimal built-in subunit vaccine. Aside from their potential bioavailability, the T-B contiguous epitopes may also represent attractive tools for investigating the molecular mechanisms of T-B-cell cooperation responsible for antiviral protection.
Molecular analysis of the feline immunodeficiency virus protease: generation of a novel form of the protease by autoproteolysis and construction of cleavage-resistant proteases
Authors: Laco G S , Fitzgerald M C , Morris G M , Olson A J , Kent S B , Elder J H
Published: January 2020

The feline immunodeficiency virus (FIV) protease is essential for virion maturation and subsequent viral replication in that it cleaves the Gag and Gag/Pol polyproteins at eight sites to release the respective structural proteins and enzymes. During purification of a recombinant FIV protease (PR), we noted that it underwent autoproteolysis (autolysis) to give discrete cleavage products. These additional PR cleavage sites were defined using N-terminal amino acid sequence analysis and mass spectrometry. Protease breakdown products were also found in FIV virions and were of the same apparent molecular weights as the in vitro autolysis products. Four primary PR autolysis sites were blocked via substitution of either the P1 amino acid with a beta-branched amino acid or the P1' amino acid with lysine. Cleavage-resistant PRs which had Km and k(cat) values similar to those of FIV PR were constructed. An autolysis time course determined that blocking all four primary autolysis sites yielded a cleavage-resistant PR which was enzymatically stable. Concomitant with autolysis is the generation of an N-terminally truncated form of the PR (Thr6/PR) which has enhanced stability with respect to that of FIV PR. A structural basis for the Thr6/PR activity is presented, as are the possible roles of autolysis in the viral replication cycle.
Apoptosis plays an important role in experimental rabies virus infection
Authors: Jackson A C , Rossiter J P
Published: January 2020

Cultured rat prostatic adenocarcinoma (AT3) cells infected with the challenge virus standard (CVS) strain of fixed rabies virus showed characteristic morphologic features of apoptosis, evidence of oligonucleosomal DNA fragmentation, and expression of the Bax protein. CVS-infected Bcl-2-transfected AT3 cells did not demonstrate these features. Adult ICR mice inoculated intracerebrally with CVS showed morphologic changes of apoptosis, DNA fragmentation, and increased Bax expression in neurons, with changes most marked in the hippocampus and cerebral cortex. Ultrastructurally, some neurons demonstrated morphologic features more typical of necrosis. These studies provide evidence that apoptosis plays an important role in the pathogenesis of rabies virus infection.
VP5 of infectious bursal disease virus is not essential for viral replication in cell culture
Authors: Mundt E , Köllner B , Kretzschmar D
Published: January 2020

Infectious bursal disease virus (IBDV), a member of the Birnaviridae family, encodes in its bisegmented double-stranded RNA genome four structural virion proteins, VP1, VP2, VP3, and VP4, as well as a nonstructural protein, VP5. Recently, the establishment of an infectious cRNA system for IBDV has been described (E. Mundt and V. N. Vakharia, Proc. Natl. Acad. Sci. USA 93:11131-11136, 1996). Here, we report the isolation of a VP5- IBDV mutant constructed by site-directed mutagenesis of the methionine start codon of VP5, followed by cRNA transfection. The resulting virus mutant was replication competent in cell culture, which indicates that VP5 is not required for productive replication of IBDV. Absence of VP5 expression was verified by lack of reactivity with newly established anti-VP5 monoclonal antibodies and polyclonal sera. VP5- IBDV exhibited a delay in replication in chicken embryo cells compared to the VP5+ parental virus. However, final yields were similar. Our results thus show that VP5 is nonessential for IBDV replication, which makes it a prime candidate for the construction of deleted, marked vaccines.
Human immunodeficiency virus type 1 and 2 envelope glycoproteins oligomerize through conserved sequences
Authors: Center R J , Kemp B E , Poumbourios P
Published: January 2020

Hetero-oligomerization between human immunodeficiency virus type 2 (HIV-2) envelope glycoprotein (Env) truncation mutants and epitope-tagged gp160 is dependent on the presence of gp41 transmembrane protein (TM) amino acids 552 to 589, a putative amphipathic alpha-helical sequence. HIV-2 Env truncation mutants containing this sequence were also able to form cross-type hetero-oligomers with HIV-1 Env. HIV-2/HIV-1 hetero-oligomerization was, however, more sensitive to disruption by mutagenesis or increased temperature. The conservation of the Env oligomerization function of the HIV-1 and HIV-2 alpha-helical sequences suggests that retroviral TM alpha-helical motifs may have a universal role in oligomerization.
Infection of primary cells by adeno-associated virus type 2 results in a modulation of cell cycle-regulating proteins
Authors: Hermanns J , Schulze A , Jansen-Db1urr P , Kleinschmidt J A , Schmidt R , zur Hausen H
Published: January 2020

It has been demonstrated that infection of primary human cells with adeno-associated viruses (AAV) leads to a decrease in cellular proliferation and to growth arrest. We analyzed the molecular basis of this phenomenon and observed that infection with AAV type 2 (AAV2) had an effect on several factors engaged in the control of the mammalian cell cycle. In particular, all of the pRB family members, pRB, p107, and p130, which are involved in G1 cell cycle checkpoint control, were affected. After infection, a shift from hyper- to hypophosphorylated forms was observed. Cyclins A and B1, which are required for G1/S transition and progression into mitosis, respectively, were downregulated at the transcriptional level as well as at the protein level, whereas the G1 cyclins D1 and E remained unaffected. In addition, the steady-state levels of cyclin-dependent kinases CDK1 and CDK2 and of transcription factor E2F-1 were diminished. Of all the factors known to be involved in phosphorylation of pRB family proteins, only the CDK inhibitor p21WAF1 exhibited a response to AAV2 infection. p21WAF1 mRNA was quickly and progressively upregulated in a p53-independent manner over at least 72 h. Consistent with the increased p21WAF1 protein levels, cyclin E- and cyclin A-dependent kinase activities declined to low levels and E2F-p130-cyclin-CDK2 complexes were disrupted. From these data, we conclude that the major effect of AAV2 infection on primary human fibroblasts appears to be upregulation of p21WAF1 gene expression and thus cell cycle arrest by the suppression of pRB family protein phosphorylation.
Pathogenesis of simian immunodeficiency virus encephalitis: viral determinants of neurovirulence
Authors: Mankowski J L , Flaherty M T , Spelman J P , Hauer D A , Didier P J , Amedee A M , Murphey-Corb M , Kirstein L M , Muñoz A , Clements J E , Zink M C
Published: January 2020

To examine the relationship between macrophage tropism and neurovirulence, macaques were inoculated with two recombinant hybrid viruses derived from the parent viruses SIVmac239, a lymphocyte-tropic, non-neurovirulent clone, and SIV/17E-Br, a macrophage-tropic, neurovirulent virus strain. The first recombinant, SIV/17E-Cl, contained the portion of the env gene that encodes the surface glycoprotein and a short segment of the transmembrane glycoprotein of SIV/17E-Br in the backbone of SIVmac239. Unlike SIVmac239, SIV/17E-Cl replicated productively in macrophages, demonstrating that sequences in the surface portion of env determine macrophage tropism. None of five macaques inoculated with SIV/17E-Cl developed simian immunodeficiency virus (SIV) encephalitis. The second recombinant, SIV/17E-Fr, which contained the entire env and nef genes and the 3' long terminal repeat of SIV/17E-Br in the SIVmac239 backbone, was also macrophage tropic. Six of nine macaques inoculated with SIV/17E-Fr developed SIV encephalitis ranging from mild to moderate in severity, indicating a significant (P = 0.031) difference in the neurovirulence of the two recombinants. In both groups of macaques, CD4+ cell counts declined gradually during infection and there was no significant difference in the rate of the decline between the two groups of macaques. This study demonstrated that macrophage tropism alone is not sufficient for the development of neurological disease. In addition, it showed that while sequences in the surface portion of the envelope gene determine macrophage tropism, additional sequences derived from the transmembrane portion of envelope and/or nef confer neurovirulence.
Posttranslational processing and identification of a neutralization domain of the GP4 protein encoded by ORF4 of Lelystad virus
Authors: Meulenberg J J , van Nieuwstadt A P , van Essen-Zandbergen A , Langeveld J P
Published: January 2020

GP4 is a minor structural glycoprotein encoded by ORF4 of Lelystad virus (LV). When it was immunoprecipitated from cell lysates and extracellular virus of CL2621 cells infected with LV, it was shown to have an apparent molecular mass of approximately 28 and 31 kDa, respectively. This difference in size occurred because its core N-glycans were modified to complex type N-glycans during the transport of the protein through the endoplasmic reticulum and Golgi compartment. A panel of 15 neutralizing monoclonal antibodies (MAbs) reacted with the native GP4 protein expressed by LV and the recombinant GP4 protein expressed in a Semliki Forest virus expression system. However, these MAbs did not react with the GP4 protein of U.S. isolate VR2332. To map the binding site of the MAbs, chimeric constructs composed of ORF4 of LV and VR2332 were generated. The reactivity of these constructs indicated that all the MAbs were directed against a region spanning amino acids 40 to 79 of the GP4 protein of LV. Six MAbs reacted with solid-phase synthetic dodecapeptides. The core of this site consists of amino acids 59 to 67 (SAAQEKISF). Comparison of the amino acid sequences of GP4 proteins from various European and North American isolates indicated that the neutralization domain spanning amino acids 40 to 79 is the most variable region of GP4. The neutralization domain of GP4, described here, is the first identified for LV.
Tiny T antigen: an autonomous polyomavirus T antigen amino-terminal domain
Authors: Riley M I , Yoo W , Mda N Y , Folk W R
Published: January 2020

Three mRNAs from the murine polyomavirus early region encode the three well-characterized tumor antigens. We report the existence of a fourth alternatively spliced mRNA which encodes a fourth tumor antigen, tiny T antigen, which comprises the amino-terminal domain common to all of the T antigens but is extended by six unique amino acid residues. The amount of tiny T antigen in infected cells is small because of its short half-life. Tiny T antigen stimulates the ATPase activity of Hsc70, most likely because of its DnaJ-like motif. The common amino-terminal domain may interface with chaperone complexes to assist the T antigens in carrying out their diverse functions of replication, transcription, and transformation in the appropriate cellular compartments.
Probing the substrate specificity of hepatitis C virus NS3 serine protease by using synthetic peptides
Authors: Zhang R , Durkin J , Windsor W T , McNemar C , Ramanathan L , Le H V
Published: January 2020

We probed the substrate specificity of a recombinant noncovalent complex of the full-length hepatitis C virus (HCV) NS3 serine protease and NS4A cofactor, using a series of small synthetic peptides derived from the three trans-cleavage sites of the HCV nonstructural protein sequence. We observed a distinct cleavage site preference exhibited by the enzyme complex. The values of the turnover number (k(cat)) for the most efficient NS4A/4B, 4B/5A, and 5A/5B peptide substrates were 1.6, 11, and 8 min(-1), respectively, and the values for the corresponding Michaelis-Menten constants (Km) were 280, 160, and 16 microM, providing catalytic efficiency values (k(cat)/Km) of 92, 1,130, and 8,300 M(-1) s(-1). An alanine-scanning study for an NS5A/5B substrate (P6P4') revealed that P1 Cys and P3 Val were critical. Finally, substitutions at the scissile P1 Cys residue by homocysteine (Hcy), S-methylcysteine (Mcy), Ala, S-ethylcysteine (Ecy), Thr, Met, D-Cys, Ser, and penicillamine (Pen) produced progressively less efficient substrates, revealing a stringent stereochemical requirement for a Cys residue at this position.
Transformation and tumorigenic properties of a mutant polyomavirus containing a middle T antigen defective in Shc binding
Authors: Yi X , Peterson J , Freund R
Published: January 2020

Polyomavirus middle T antigen is phosphorylated on several tyrosine residues which act as binding sites for cellular proteins, including phosphatidylinositol 3-kinase, Shc, and phospholipase C-gamma. In this report we describe the transforming properties and tumor-inducing ability of a polyomavirus that contains a single-site mutation in middle T antigen which changes a tyrosine residue at amino acid position 250 to serine. This mutation disrupts the association of middle T with the transforming protein Shc. The mutant virus is weakly transforming, inducing foci which are smaller and of different morphology than those of the wild type. Although the virus induced tumors in close to 100% of inoculated mice, the spectrum of tumors and their morphology were altered compared to those of wild-type virus. The mutant virus induced a reduced frequency of kidney and thymic tumors. Both the mammary gland and the thymic tumors that were induced were histologically distinct from those induced by wild-type polyomavirus. These results demonstrate that the signal transduction pathway that is deregulated by the middle T-Shc association is important for full transformation of cells in culture and for tumor induction in some target tissues in the mouse-polyomavirus system.
Isolation and characterization of human papillomavirus type 6-specific T cells infiltrating genital warts
Authors: Hong K , Greer C E , Ketter N , Van Nest G , Paliard X
Published: January 2020

The potential role of T cells in the control of human papillomavirus type 6 (HPV-6) infections is an appealing premise, but their actual role has been sparsely investigated. Since HPV-6 infections are confined to the epithelium, such an investigation should focus on the T cells present at the site of infection (i.e., the warts). Therefore, we isolated wart-infiltrating lymphocytes (WIL) from patients with clinically diagnosed anogenital warts. These WIL were characterized by their phenotype and their specificity for E7 and L1 proteins of HPV-6. The phenotype of WIL varied drastically from patient to patient, as determined by their expression of CD4, CD8, T-cell receptor alpha/beta chain (TCR alpha beta), and TCR gamma delta. Despite this heterogeneity in phenotype, HPV-6 E7 and/or L1-specific WIL, as determined by lymphoproliferation, could be isolated from more than 75% of the patients studied. Among all L1 peptides recognized by WIL, peptides 311-330 and 411-430 were the most consistently detected, with seven of nine patients for whom L1 peptide reactivity was observed responding to at least one of them. Moreover, the HPV-6 epitopic peptides recognized by WIL differed to some extent from those recognized by peripheral T cells.
Modulation of RANTES production by human cytomegalovirus infection of fibroblasts
Authors: Michelson S , Dal Monte P , Zipeto D , Bodaghi B , Laurent L , Oberlin E , Arenzana-Seisdedos F , Virelizier J L , Landini M P
Published: January 2020

Chemokines play a major role in inflammatory responses and affect hematopoiesis both negatively and positively. We show that fresh isolates and laboratory strains (Towne and Ad-169) of human cytomegalovirus (HCMV) induce production of the CC chemokine RANTES in fibroblasts. Induction of extracellular RANTES production occurred as early as 8 h after infection, peaked around 24 h after infection, and was almost undetectable by 48 and 72 h. Upregulation occurred in the absence of viral DNA synthesis, suggesting that it was due to immediate-early-early HCMV gene expression. CMV infection stimulated RANTES transcription, since reverse transcription-PCR detected a sharp increase in RANTES RNA which persisted even when extracellular RANTES was no longer detected. Induction of RANTES in fibroblasts was not due to prior induction of tumor necrosis factor alpha or interleukin 1 beta. Down-regulation required an active viral genome. Decrease of RANTES in culture supernatants may be associated with the appearance of the HCMV CC chemokine receptor US28, since we show that this gene is transcribed as early as 8 h after infection. Modulation of CC chemokine production early during CMV infection might have a regulatory effect on viral replication, as well as affect immune surveillance.
Molecular characterization of the type-specific gamma-determinant located on the adenovirus fiber
Authors: Eiz B , Pring-Akerblom P
Published: January 2020

The fiber knob carries the type-specific gamma-antigen which can be demonstrated in hemagglutination inhibition tests. In order to characterize the gamma-determinant we selected subgenus DI adenovirus serotypes 9 and 19 (Ad9 and Ad19) which exhibited 29 amino acid exchanges in the knob domain. Like all subgenus DI adenoviruses they showed a complete hemagglutination pattern with rat and human erythrocytes. We constructed a total of 14 chimeric Ad9/Ad19 and Ad19/Ad9 fiber proteins, which possessed fiber knobs with progressively exchanged Ad9 and Ad19 amino acids. Furthermore, we created 39 fiber proteins with distinct amino acid exchanges in the knob regions by primer-directed mutagenesis. The proteins were expressed in Escherichia coli and tested in hemagglutination and hemagglutination inhibition tests. From our results we can conclude that the type-specific gamma-determinant is not restricted to a distinct region on the adenovirus fiber knob but is composed of at least 17 amino acids. Most of the amino acids contributing to the Ad9 and Ad19 gamma-determinants are located on the fiber knob loops.
Specific interaction of polypyrimidine tract-binding protein with the extreme 3'-terminal structure of the hepatitis C virus genome, the 3'X
Authors: Tsuchihara K , Tanaka T , Hijikata M , Kuge S , Toyoda H , Nomoto A , Yamamoto N , Shimotohno K
Published: January 2020

We previously identified a highly conserved 98-nucleotide (nt) sequence, the 3'X, as the extreme 3'-terminal structure of the hepatitis C virus (HCV) genome (T. Tanaka, N. Kato, M.-J. Cho, and K. Shimotohno, Biochem. Biophys. Res. Commun. 215:744-749, 1995). Since the 3' end of positive-strand viral RNA is the initiation site of RNA replication, the 3'X should contribute to HCV negative-strand RNA synthesis. Cellular factors may also be involved in this replication mechanism, since several cellular proteins have been shown to interact with the 3'-end regions of other viral genomes. In this study, we found that both 38- and 57-kDa proteins in the human hepatocyte line PH5CH bound specifically to the 3'-end structure of HCV positive-strand RNA by a UV-induced cross-linking assay. The 57-kDa protein (p57), which had higher affinities to RNA probes, recognized a 26-nt sequence including the 5'-terminal 19 nt of the 3'X and 7 flanking nt, designated the transitional region. This sequence contains pyrimidine-rich motifs and shows similarity to the consensus binding sequence of the polypyrimidine tract-binding protein (PTB), which has been implicated in alternative pre-mRNA splicing and cap-independent translation. We found that this 3'X-binding p57 is identical to PTB. The 3'X-binding p57 was immunoprecipitated by anti-PTB antibody, and recombinant PTB bound to the 3'X RNA. In addition, p57 bound solely to the 3'-end region of positive-strand RNA, not to this region of negative-strand RNA. We suggest that 3'X-PTB interaction is involved in the specific initiation of HCV genome replication.
The UL84 protein of human cytomegalovirus acts as a transdominant inhibitor of immediate-early-mediated transactivation that is able to prevent viral replication
Authors: Gebert S , Schmolke S , Sorg G , Flöss S , Plachter B , Stamminger T
Published: January 2020

The 86-kilodalton immediate-early (IE) 2 protein (IE2-p86) of human cytomegalovirus (HCMV) is a multifunctional regulator of HCMV gene expression which appears to be essential for triggering the lytic replicative cycle. IE2-p86 functions as a promiscuous transactivator of both viral and cellular gene expression and can repress transcription from its own promoter. In this study we demonstrate that a viral early protein, termed pUL84, which is able to interact with IE2-p86 both in vivo and in vitro, modulates IE2-p86 in a specific manner. First, pUL84 acts as a transdominant inhibitor of IE2-p86-mediated transactivation of both homologous and heterologous promoters. Second, negative autoregulation by IE2-p86 is augmented in the presence of pUL84. Using two in vivo assays, we obtained evidence that expression of pUL84 during the IE phase of the viral replicative cycle leads to an inhibition of viral early gene expression which prevents replication of HCMV and results in a persistent infection of UL84-positive cell lines. Transdominant inhibition of a viral IE function by a protein expressed during the later phases of replication appears to be a novel principle used by herpesviruses which could account for the slow replication of HCMV and may be useful in the development of new antiviral strategies.
Ribonucleic Acid Polymerase Activity in Sendai Virions and Nucleocapsid
Authors: Robinson William S.
Published: January 2020

After dissociation of purified Sendai virus with the neutral detergent Nonidet P-40 and 2-mercaptoethanol, it catalyzed the incorporation of ribonucleoside triphosphates into an acid-insoluble product. The enzyme activity was associated with viral nucleocapsid as well as whole virions. The reaction product was ribonucleic acid (RNA) which annealed specifically with virion RNA. Sedimentation of the
3
H-RNA reaction product revealed two components, a 45
S
component with properties of double-stranded RNA and 4 to 6
S
component which appeared to be mostly single-stranded RNA.
Enlargement of
Escherichia coli
After Bacteriophage Infection I. Description of the Phenomenon
Authors: Freedman M. L. , Krisch R. E.
Published: January 2020

Escherichia coli
B/r and B
s−1
ceased division and increased in mean cell volume soon after infection with T-even phage. The effect was obtained with wild-type or rapid lysis mutants, as well as with ultraviolet light-killed phage and with bacteriophage ghosts which lack deoxyribonucleic acid. The cell response did not require the presence of phage genetic material or the production of progeny phage. A Poisson distribution of the fraction of adsorbed phage at different multiplicites of infection indicates that one phage per bacterium will produce maximum increase in cell volume. T-even phage-resistant
E. coli
mutants showed no enlargement response, and phage T1, T3, and T7 elicited neither abrupt termination of cell division nor host cell enlargement. Infection with baseplate-defective T4D 12
−
amber mutants, which bind reversibly to but do not penetrate the bacterium, also had no effect. In vitro restoration of normal baseplate function in these defective viruses allowed phage adsorption and penetration and caused host cell division arrest and enlargement. These findings indicate that arrest of division and increase in mean cell volume occur together when a sensitive strain of
E. coli
is infected with T-even phage that adsorb and penetrate normally.
Genetic Assay for Small Fragments of Bacteriophage φX174 Deoxyribonucleic Acid
Authors: Hutchison Clyde A. , Edgell Marshall H.
Published: January 2020

The double-stranded replicative form deoxyribonucleic acid (RF-DNA) of bacteriophage φX174 was fragmented by pancreatic deoxyribonuclease, and the complementary strand fragments were then annealed to intact viral single strands. When such complexes infected
Escherichia coli
spheroplasts, some of the progeny virus bore genetic markers derived from the RF-DNA fragments. In this way, genetic markers have been salvaged from DNA fragments less than 50 nucleotides in length. This method is potentially useful as a specific assay to aid in the purification of genetically defined DNA fragments and also as a mechanism for the incorporation of small chemically synthesized DNA sequences into viral genomes.
Unilateral Synthesis of Reovirus Double-Stranded Ribonucleic Acid by a Cell-Free Replicase System
Authors: Sakuma S. , Watanabe Y.
Published: January 2020

A large-particle fraction obtained from reovirus-infected L cells contained both replicase and transcriptase activity. The in vitro replicase reaction slowed down soon after initiation, whereas the transcriptase reaction proceeded at an unabated rate. The replicase and transcriptase were both template-bound and could be separated from one another by controlled chymotryptic digestion followed by centrifugation in a CsCl gradient. The transcriptase was recovered as a sharp band (ρ = 1.43) and resembled virus core derived from mature virions. In contrast, replicase activity was distributed throughout the gradient, indicating that replicase is associated with structures of various density in CsCl. In subsequent experiments, the replicase product was found to be indistinguishable from the double-stranded ribonucleic acid (RNA) reovirus genome with respect to its buoyant density in cesium-salt gradients and denaturation-annealing characteristics. A “hybridization-competition” experiment in which the replicase product was denatured and annealed in the presence of an excess of plus-RNA indicated that the in vitro replicase reaction proceeded by means of a unilateral synthesis of minus-RNA upon a preexisting plus-RNA template, presumably of single-stranded form.
Studies of Nondefective Adenovirus 2-Simian Virus 40 Hybrid Viruses IV. Characterization of the Simian Virus 40 Ribonucleic Acid Species Induced by Wild-Type Simian Virus 40 and by the Nondefective Hybrid Virus, Ad2
+
ND
1
Authors: Oxman Michael N. , Levine Arthur S. , Crumpacker Clyde S. , Levin Myron J. , Henry Patrick H. , Lewis Andrew M.
Published: January 2020

Ad2
+
ND
1
, a nondefective adenovirus 2 (Ad2)-simian virus 40 (SV40) hybrid virus, has been previously shown to contain a small segment of the SV40 genome covalently linked to Ad2 deoxyribonucleic acid (DNA). The SV40 portion of this hybrid virus has been characterized by relating the SV40-specific ribonucleic acid (RNA) sequences transcribed from the Ad2
+
ND
1
DNA to those transcribed from the DNA of SV40 itself. RNA-DNA hybridization-competition studies indicate that the SV40 component of Ad2
+
ND
1
consists of some, but not all, of that part of the SV40 genome which is transcribed early, i.e., prior to viral DNA replication, in SV40 lytic infection.
Comparison of
Bacillus cereus
Bacteriophages CP-51 and CP-53
Authors: Yelton David B. , Thorne Curtis B.
Published: January 2020

Transducing bacteriophages CP-51 and CP-53 were compared. Unlike CP-51, CP-53 appeared to be a lysogenizing phage. CP-51 gave greater frequencies of co-transduction for linked markers than did CP-53. CP-51 was found to be a larger phage which carried more deoxyribonucleic acid (DNA) than CP-53. CP-51 DNA contained about 43% guanine plus cytosine and in addition contained 5-hydroxymethyluracil in place of thymine. CP-53 DNA contained no unusual bases; its guanine plus cytosine content was 37%.
Association of 4
S
Ribonucleic Acid with Oncornavirus Ribonucleic Acids
Authors: Erikson E. , Erikson R. L.
Published: January 2020

Oncornavirus 60 to 70
S
ribonucleic acids (RNA), such as those from avian myeloblastosis virus, Schmidt-Ruppin virus, or mouse sarcoma-mouse leukemia viruses, isolated by conventional techniques, contain 4
S
transferlike RNA molecules that are released upon dissociation of the 60 to 70
S
RNA with heat. The 4
S
RNA represents 2.5 to 3.0% of the RNA in the 65
S
aggregate or 4 to 5 molecules per molecule of 35
S
RNA formed.
Ultrastructural Study of Long-Term Measles Infection in Cultures of Hamster Dorsal-Root Ganglion
Authors: Raine Cedric S. , Feldman Lawrence A. , Sheppard Rachel D. , Bornstein Murray B.
Published: January 2020

The morphogenesis of the Edmonston strain of measles is described in cultures of hamster dorsal-root ganglion maintained for as long as 63 days postinoculation. The patterns observed confirmed those previously reported in both neural and non-neural tissue. However, in the present tissue, the development of viral material could be followed chronologically within different cell types such as neurons and Schwann cells. Active replication was visualized up to 63 days postinoculation. The appearance of cytoplasmic nucleocapsid preceded that of intranuclear nucleocapsid, the latter occurring after 14 days. These intranuclear inclusions were formed after the transformation of the nucleoli into bizarre pleomorphic bodies which eventually segregated into clumps of nucleocapsid. These intranuclear inclusions mimic those seen in subacute sclerosing panencephalitis, now known to be etiologically related to a measles-like virus.
Superinfection Exclusion by P22 Prophage and the Replication Complex
Authors: Darlington Oliver F. , Levine Myron
Published: January 2020

Wild-type
sie
+
P22 prophage converted
Salmonella typhimurium
lysogens to exclude deoxyribonucleic acid (DNA) injected by superinfecting phage. DNA from a P22 superinfecting virulent phage associated with the replication complex in a
sie
−
lysogen but not in a
sie
+
lysogen.