bluuebunny/crossref_metadata_2025 huggingface dataset:
Protection of Macaques against Intrarectal Infection by a Combination Immunization Regimen with Recombinant Simian Immunodeficiency Virus SIVmne gp160 Vaccines
Authors: Polacino Patricia , Stallard Virginia , Montefiori David C. , Brown Charles R. , Richardson Barbra A. , Morton William R. , Benveniste Raoul E. , Hu Shiu-Lok
Published: December 2019

We previously reported that immunization with recombinant simian immunodeficiency virus SIVmne envelope (gp160) vaccines protected macaques against intravenous challenge by the cloned homologous virus E11S but that this protection was only partially effective against the uncloned virus, SIVmne. In the present study, we examine the protective efficacy of this immunization regimen against infection by a mucosal route. We found that the same gp160-based vaccines were highly effective against intrarectal infection not only with the E11S clone but also with the uncloned SIVmne. Protection against mucosal infection is therefore achievable by parenteral immunization with recombinant envelope vaccines. Protection appears to correlate with high levels of SIV-specific antibodies and, in animals protected against the uncloned virus, the presence of serum-neutralizing activities. To understand the basis for the differential efficacies against the uncloned virus by the intravenous versus the intrarectal routes, we examined viral sequences recovered from the peripheral blood mononuclear cells of animals early after infection by both routes. We previously showed that the majority (85%) of the uncloned SIVmne challenge stock contained V1 sequences homologous to the molecular clone from which the vaccines were made (E11S type), with the remainder (15%) containing multiple conserved changes (the variant types). In contrast to intravenously infected animals, from which either E11S-type or the variant type V1 sequences could be recovered in significant proportions, animals infected intrarectally had predominantly E11S-type sequences. Preferential transmission or amplification of the E11S-type viruses may therefore account in part for the enhanced efficacy of the recombinant gp160 vaccines against the uncloned virus challenge by the intrarectal route compared with the intravenous route.
Recombinant Duck Interferon Gamma Inhibits Duck Hepatitis B Virus Replication in Primary Hepatocytes
Authors: Schultz Ursula , Chisari Francis V.
Published: December 2019

Interferon gamma (IFN-γ), which has been cloned in several mammalian species and recently in birds, plays a critical role in modulating immune system function. IFN-γ and tumor necrosis factor alpha (TNF-α) have been shown to be crucial in the pathogenesis of viral hepatitis and in the transient disappearance of hepatitis B virus (HBV) from the liver after adoptive transfer of HBV-specific cytotoxic T lymphocytes into HBV-transgenic mice. Similar studies in the natural animal hosts of related hepadnaviruses have been limited because the corresponding probes and recombinant cytokines were not available. For this reason, we initiated studies to clone and characterize cytokines from the duck, the natural host of the duck hepatitis B virus (DHBV). We describe here the cDNA cloning and initial characterization of the IFN-γ homologue of ducks (DuIFN-γ). The DuIFN-γ cDNA codes for a predicted mature protein of 145 amino acids with a molecular mass of 16.6 kDa. The precursor protein has 67% identity with the previously cloned chicken IFN-γ and 21 to 34% identity with mammalian IFN-γ. Recombinant DuIFN-γ induces the transcription of several IFN-inducible genes including IFN regulatory factor 1 and guanylate-binding protein, and it exhibits antiviral activity that protects duck cells from vesicular stomatitis virus-mediated lysis. Importantly, treatment of primary duck hepatocytes with recombinant DuIFN-γ inhibits DHBV replication in a dose-dependent fashion. Time course analysis revealed that IFN-γ treatment does not affect initial covalently closed circular DNA (cccDNA) conversion but inhibits the synthesis of progeny cccDNA by amplification.
Transcriptional Repression of Human Hepatitis B Virus Genes by a bZIP Family Member, E4BP4
Authors: Lai Chao-Kuen , Ting Ling-Pai
Published: December 2019

Box α is an essential element of both the upstream regulatory sequence of the core promoter and the second enhancer, which positively regulate the transcription of human hepatitis B virus (HBV) genes. In this paper, we describe the cloning and characterization of a box α binding protein, E4BP4. E4BP4 is a bZIP type of transcription factor. Overexpression of E4BP4 represses the stimulating activity of box α in the upstream regulatory sequence of the core promoter and the second enhancer in differentiated human hepatoma cell lines. E4BP4 can also suppress the transcription of HBV genes and the production of HBV virions in a transient-transfection system that mimics the viral infection in vivo. Expression of an E4BP4 antisense transcript can, instead, elevate the transcription of the core promoter. A low abundance of E4BP4 protein and mRNA in differentiated human hepatoma cell lines is detected, and E4BP4 is not a major component of box α binding proteins in untransfected differentiated human hepatoma cell lines. C/EBPα and C/EBPβ, in contrast, are major components of the box α binding activity present in nuclear extracts. E4BP4 has a stronger binding affinity towards box α than the endogenous box α binding activity present in nuclear extracts. Structure and function analysis of E4BP4 reveals that DNA binding activity is sufficient to confer the negative regulatory function of E4BP4. These results indicate that binding site occlusion is the mechanism whereby E4BP4 suppresses transcription in HBV.
Differentiation of M1 Myeloid Precursor Cells into Macrophages Results in Binding and Infection by Theiler’s Murine Encephalomyelitis Virus and Apoptosis
Authors: Jelachich M. L. , Bramlage C. , Lipton H. L.
Published: December 2019

Infection of susceptible mouse strains with BeAn, a less virulent strain of Theiler’s murine encephalomyelitis virus (TMEV), results in immune system-mediated demyelinating lesions in the central nervous system (CNS) similar to those in multiple sclerosis. Since macrophages appear to carry the major detectable antigen burden in vivo, and purification of sufficient cell numbers from the CNS for detailed analysis is difficult, macrophage-like cell lines provide an accessible system with which to study virus-macrophage interactions. The myeloid precursor cell line M1 differentiates in response to cytokines and expresses many characteristics of tissue macrophages. Incubation of TMEV with undifferentiated M1 cells produced neither infection nor apoptosis, whereas differentiated M1 (M1-D) cells developed a restricted virus infection and changes indicative of apoptosis. Virus binding and RNA replication as well as cellular production of alpha/beta interferons increased with differentiation. Although the amount of infectious virus was highly restricted, BeAn-infected M1-D cells synthesized and appropriately processed virus capsid proteins at levels comparable to those for permissive BHK-21 cells. Analysis of Bcl-2 protein family expression in undifferentiated and differentiated cells suggests that susceptibility of M1-D cells to apoptosis may be controlled, in part, by expression of the proapoptotic α isoform of Bax and Bak. These data suggest that macrophage differentiation plays a role in susceptibility to TMEV infection and apoptosis.
Macrophages Are the Major Reservoir of Latent Murine Gammaherpesvirus 68 in Peritoneal Cells
Authors: Weck Karen E. , Kim Susanne S. , Virgin Herbert W. , Speck Samuel H.
Published: December 2019

B cells have previously been identified as the major hematopoietic cell type harboring latent gammaherpesvirus 68 (γHV68) (N. P. Sunil-Chandra, S. Efstathiou, and A. A. Nash, J. Gen. Virol. 73:3275–3279, 1992). However, we have shown that γHV68 efficiently establishes latency in B-cell-deficient mice (K. E. Weck, M. L. Barkon, L. I. Yoo, S. H. Speck, and H. W. Virgin, J. Virol. 70:6775–6780, 1996), demonstrating that B cells are not required for γHV68 latency. To understand this dichotomy, we determined whether hematopoietic cell types, in addition to B cells, carry latent γHV68. We observed a high frequency of cells that reactivate latent γHV68 in peritoneal exudate cells (PECs) derived from both B-cell-deficient and normal C57BL/6 mice. PECs were composed primarily of macrophages in B-cell-deficient mice and of macrophages plus B cells in normal C57BL/6 mice. To determine which cells in PECs from C57BL/6 mice carry latent γHV68, we developed a limiting-dilution PCR assay to quantitate the frequency of cells carrying the γHV68 genome in fluorescence-activated cell sorter-purified cell populations. We also quantitated the contribution of individual cell populations to the total frequency of cells carrying latent γHV68. At early times after infection, the frequency of PECs that reactivated γHV68 correlated very closely with the frequency of PECs carrying the γHV68 genome, validating measurement of the frequency of viral-genome-positive cells as a measure of latency in this cell population. F4/80-positive macrophage-enriched, lymphocyte-depleted PECs harbored most of the γHV68 genome and efficiently reactivated γHV68, while CD19-positive, B-cell-enriched PECs harbored about a 10-fold lower frequency of γHV68 genome-positive cells. CD4-positive, T-cell-enriched PECs contained only a very low frequency of γHV68 genome-positive cells, consistent with previous analyses indicating that T cells are not a reservoir for γHV68 latency (N. P. Sunil-Chandra, S. Efstathiou, and A. A. Nash, J. Gen. Virol. 73:3275–3279, 1992). Since macrophages are bone marrow derived, we determined whether elicitation of a large inflammatory response in the peritoneum would recruit additional latent cells into the peritoneum. Thioglycolate inoculation increased the total number of PECs by about 20-fold but did not affect the frequency of cells that reactivate γHV68, consistent with a bone marrow reservoir for latent γHV68. These experiments demonstrate γHV68 latency in two different hematopoietic cell types, F4/80-positive macrophages and CD19-positive B cells, and argue for a bone marrow reservoir for latent γHV68.
Irreversible Inhibition of Human Immunodeficiency Virus Type 1 Integrase by Dicaffeoylquinic Acids
Authors: Zhu Kai , Cordeiro Mara L. , Atienza Jocelyn , Robinson W. Edward , Chow Samson A.
Published: December 2019

Human immunodeficiency virus type 1 (HIV-1) and other retroviruses require integration of a double-stranded DNA copy of the RNA genome into the host cell chromosome for productive infection. The viral enzyme, integrase, catalyzes the integration of retroviral DNA and represents an attractive target for developing antiretroviral agents. We identified several derivatives of dicaffeoylquinic acids (DCQAs) that inhibit HIV-1 replication in tissue culture and catalytic activities of HIV-1 integrase in vitro. The specific step at which DCQAs inhibit the integration in vitro and the mechanism of inhibition were examined in the present study. Titration experiments with different concentrations of HIV-1 integrase or DNA substrate found that the effect of DCQAs was exerted on the enzyme and not the DNA. In addition to HIV-1, DCQAs also inhibited the in vitro activities of MLV integrase and truncated variants of feline immunodeficiency virus integrase, suggesting that these compounds interacted with the central core domain of integrase. The inhibition on retroviral integrases was relatively specific, and DCQAs had no effect on several other DNA-modifying enzymes and phosphoryltransferases. Kinetic analysis and dialysis experiments showed that the inhibition of integrase by DCQAs was irreversible. The inhibition did not require the presence of a divalent cation and was unaffected by preassembling integrase onto viral DNA. The results suggest that the irreversible inhibition by DCQAs on integrase is directed toward conserved amino acid residues in the central core domain during catalysis.
Rapid Evolution of H5N1 Influenza Viruses in Chickens in Hong Kong
Authors: Zhou Nan Nan , Shortridge Kennedy F. , Claas Eric C. J. , Krauss Scott L. , Webster Robert G.
Published: December 2019

The H5N1 avian influenza virus that killed 6 of 18 persons infected in Hong Kong in 1997 was transmitted directly from poultry to humans. Viral isolates from this outbreak may provide molecular clues to zoonotic transfer. Here we demonstrate that the H5N1 viruses circulating in poultry comprised two distinguishable phylogenetic lineages in all genes that were in very rapid evolution. When introduced into new hosts, influenza viruses usually undergo rapid alteration of their surface glycoproteins, especially in the hemagglutinin (HA). Surprisingly, these H5N1 isolates had a large proportion of amino acid changes in all gene products except in the HA. These viruses maybe reassortants each of whose HA gene is well adapted to domestic poultry while the rest of the genome arises from a different source. The consensus amino acid sequences of “internal” virion proteins reveal amino acids previously found in human strains. These human-specific amino acids may be important factors in zoonotic transmission.
Distinct Human Immunodeficiency Virus Strains in the Bone Marrow Are Associated with the Development of Thrombocytopenia
Authors: Voulgaropoulou Frosso , Tan Benjamin , Soares Marcelo , Hahn Beatrice , Ratner Lee
Published: December 2019

We analyzed bone marrow and blood from human immunodeficiency virus type 1 (HIV-1)-infected individuals and described the HIV-1 quasispecies in these cellular compartments. HIV-1 isolates from the bone marrow of thrombocytopenic patients contained distinct amino acids in the V3 loop and infected T-cell lines, implicating this virus in the development of thrombocytopenia.
Effectiveness of Polyene Antibiotics in Treatment of Transmissible Spongiform Encephalopathy in Transgenic Mice Expressing Syrian Hamster PrP Only in Neurons
Authors: Demaimay Remi , Race Richard , Chesebro Bruce
Published: December 2019

To date very few drugs have favorably influenced the course of transmissible spongiform encephalopathies. In previous studies, the polyene antibiotics amphotericin B (AmB) and MS-8209 prolonged the incubation time in Syrian hamsters of the 263K strain of scrapie, but AmB had no effect against other scrapie strains in Syrian hamsters. In the present experiments using transgenic mice expressing Syrian hamster PrP in neurons only, MS-8209 extended the life spans of animals infected with the 263K strain but not the DY strain. AmB was effective against both 263K and DY and prevented death in 18% of DY-infected animals. The AmB effect against strain 263K was more prominent in mice whose endogenous PrP gene had been inactivated by homologous recombination. It was unclear whether this difference was due to a change in the duration of the disease or to possible interactive effects between the mouse PrP gene and the drugs themselves. The effectiveness of treatment after intracerebral scrapie infection in transgenic mice expressing PrP only in neurons suggested that neurons are important sites of action for these drugs.
Role of Rubella Virus Glycoprotein Domains in Assembly of Virus-Like Particles
Authors: Garbutt Mike , Law Lok Man J. , Chan Honey , Hobman Tom C.
Published: December 2019

Rubella virus is a small enveloped positive-strand RNA virus that assembles on intracellular membranes in a variety of cell types. The virus structural proteins contain all of the information necessary to mediate the assembly of virus-like particles in the Golgi complex. We have recently identified intracellular retention signals within the two viral envelope glycoproteins. E2 contains a Golgi retention signal in its transmembrane domain, whereas a signal for retention in the endoplasmic reticulum has been localized to the transmembrane and cytoplasmic domains of E1 (T. C. Hobman, L. Woodward, and M. G. Farquhar, Mol. Biol. Cell 6:7–20, 1995; T. C. Hobman, H. F. Lemon, and K. Jewell, J. Virol. 71:7670–7680, 1997). In the present study, we have analyzed the role of these retention signals in the assembly of rubella virus-like particles. Deletion or replacement of these domains with analogous regions from other type I membrane glycoproteins resulted in failure of rubella virus-like particles to be secreted from transfected cells. The E1 transmembrane and cytoplasmic domains were not required for targeting of the structural proteins to the Golgi complex and, surprisingly, assembly and budding of virus particles into the lumen of this organelle; however, the resultant particles were not secreted. In contrast, replacement or alteration of the E2 transmembrane or cytoplasmic domain, respectively, abrogated the targeting of the structural proteins to the budding site, and consequently, no virion formation was observed. These results indicate that the transmembrane and cytoplasmic domains of E2 and E1 are required for early and late steps respectively in the viral assembly pathway and that rubella virus morphogenesis is very different from that of the structurally similar alphaviruses.
Sequence and Structural Elements at the 3′ Terminus of Bovine Viral Diarrhea Virus Genomic RNA: Functional Role during RNA Replication
Authors: Yu Haiying , Grassmann Claus W. , Behrens Sven-Erik
Published: December 2019

Bovine viral diarrhea virus (BVDV), a member of the genusPestivirusin the familyFlaviviridae, has a positive-stranded RNA genome consisting of a single open reading frame and untranslated regions (UTRs) at the 5′ and 3′ ends. Computer modeling suggested the 3′ UTR comprised single-stranded regions as well as stem-loop structures—features that were suspected of being essentially implicated in the viral RNA replication pathway. Employing a subgenomic BVDV RNA (DI9c) that was shown to function as an autonomous RNA replicon (S.-E. Behrens, C. W. Grassmann, H. J. Thiel, G. Meyers, and N. Tautz, J. Virol. 72:2364–2372, 1998) the goal of this study was to determine the RNA secondary structure of the 3′ UTR by experimental means and to investigate the significance of defined RNA motifs for the RNA replication pathway. Enzymatic and chemical structure probing revealed mainly the conserved terminal part (termed 3′C) of the DI9c 3′ UTR containing distinctive RNA motifs, i.e., a stable stem-loop, SL I, near the RNA 3′ terminus and a considerably less stable stem-loop, SL II, that forms the 5′ portion of 3′C. SL I and SL II are separated by a long single-stranded intervening sequence, denoted SS. The 3′-terminal four C residues of the viral RNA were confirmed to be single stranded as well. Other intramolecular interactions, e.g., with upstream DI9c RNA sequences, were not detected under the experimental conditions used. Mutagenesis of the DI9c RNA demonstrated that the SL I and SS motifs do indeed play essential roles during RNA replication. Abolition of RNA stems, which ought to maintain the overall folding of SL I, as well as substitution of certain single-stranded nucleotides located in the SS region or SL I loop region, gave rise to DI9c derivatives unable to replicate. Conversely, SL I stems comprising compensatory base exchanges turned out to support replication, but mostly to a lower degree than the original structure. Surprisingly, replacement of a number of residues, although they were previously defined as constituents of a highly conserved stretch of sequence of the SS motif, had little effect on the replication ability of DI9c. In summary, these results indicate that RNA structure as well as sequence elements harbored within the 3′C region of the BVDV 3′ UTR create a commoncis-acting element of the replication process. The data further point at possible interaction sites of host and/or viral proteins and thus provide valuable information for future experiments intended to identify and characterize these factors.
Spliced mRNA Encoding the Murine Cytomegalovirus Chemokine Homolog Predicts a β Chemokine of Novel Structure
Authors: MacDonald Margaret R. , Burney Mary W. , Resnick Stuart B. , Virgin Herbert W.
Published: December 2019

A viral mRNA of the late kinetic class expressed by murine cytomegalovirus (MCMV) contains an open reading frame (ORF) whose predicted protein, designated MCK-1, has homology to β chemokines (M. R. MacDonald, X.-Y. Li, and H. W. Virgin IV, J. Virol. 71:1671–1678, 1997). The present study analyzed further the structure of the transcript in infected fibroblast cells. A splicing event removed the MCK-1 stop codon, bringing a downstream ORF into frame with the chemokine homolog and demonstrating that the MCK-1 ORF was an exon of a larger gene. The predicted 31.4-kDa protein, designated MCK-2, contains a putative amino-terminal signal sequence and a β chemokine domain, followed by a carboxyl-terminal domain without significant homology to known proteins. Quantitative analysis of mRNA forms in MCMV-infected fibroblast cells at late times after infection indicated that the viral chemokine RNA was predominantly spliced. There was no evidence for expression of RNA encoding either MCK-1 or MCK-2 at immediate early or early times after infection with MCMV. Monoclonal antibodies generated against bacterially expressed MCK-2 recognized multiple proteins in the range of ∼30 to ∼45 kDa in Western blot analysis of MCK-2 expressed in transfected COS cells. The monoclonal antibodies immunoprecipitated a similar group of proteins in transfected COS cells metabolically labeled with radioactive cysteine. Radiolabelled protein of apparent higher molecular mass was immunoprecipitated from culture medium overlying the transfected cells, suggesting that posttranslationally modified MCK-2 can be secreted. Two proteins with apparent molecular mass suggestive of posttranslational modification were detected by Western blot analysis of cells harvested at late times after infection with MCMV. These studies show that MCMV encodes and expresses a β chemokine homolog with a novel predicted structure.
Identification of a Receptor-Binding Pocket on the Envelope Protein of Friend Murine Leukemia Virus
Authors: Davey Robert A. , Zuo Yi , Cunningham James M.
Published: December 2019

Based on previous structural and functional studies, a potential receptor-binding site composed of residues that form a pocket at one end of the two long antiparallel helices in the receptor-binding domain of Friend 57 murine leukemia virus envelope protein (RBD) has been proposed. To test this hypothesis, directed substitutions for residues in the pocket were introduced and consequences for infection and for receptor binding were measured. Receptor binding was measured initially by a sensitive assay based on coexpression of receptor and RBD in
Xenopus
oocytes, and the findings were confirmed by using purified proteins. Three residues that are critical for both binding and infection (S84, D86, and W102), with side chains that extend into the pocket, were identified. Moreover, when mCAT-1 was overexpressed, the infectivity of Fr57-MLV carrying pocket substitutions was partially restored. Substitutions for 18 adjacent residues and 11 other previously unexamined surface-exposed residues outside of the RBD pocket had no detectable effect on function. Taken together, these findings support a model in which the RBD pocket interacts directly with mCAT-1 (likely residues, Y235 and E237) and multiple receptor-envelope complexes are required to form the fusion pore.
Host-Specific Modulation of the Selective Constraints Driving Human Immunodeficiency Virus Type 1
env
Gene Evolution
Authors: Bagnarelli Patrizia , Mazzola Francesca , Menzo Stefano , Montroni Maria , Butini Luca , Clementi Massimo
Published: December 2019

To address the evolution of human immunodeficiency virus type 1 (HIV-1) within a single host, we analyzed the HIV-1 C2-V5
env
regions of both cell-free genomic-RNA- and proviral-DNA-derived clones. Sequential samples were collected over a period of 3 years from six untreated subjects (three typical progressors [TPs] and three slow progressors [SPs], all with a comparable length of infection except one. The evolutionary analysis of the C2-V5
env
sequences performed on 506 molecular clones (253 RNA- and 253 DNA-derived sequences) highlighted a series of differences between TPs and SPs. In particular, (i) clonal sequences from SPs (DNA and RNA) showed lower nucleotide similarity than those from TPs (
P
= 0.0001), (ii) DNA clones from SPs showed higher intra- and intersample nucleotide divergence than those from TPs (
P
< 0.05), (iii) higher host-selective pressure was generally detectable in SPs (DNA and RNA sequences), and (iv) the increase in the genetic distance of DNA and RNA sequences over time was paralleled by an increase in both synonymous (Ks) and nonsynonymous (Ka) substitutions in TPs but only in nonsynonymous substitutions in SPs. Several individual peculiarities of the HIV-1 evolutionary dynamics emerged when the V3, V4, and V5
env
regions of both TPs and SPs were evaluated separately. These peculiarities, probably reflecting host-specific features of selective constraints and their continuous modulation, are documented by the dynamics of Ka/Ks ratios of hypervariable
env
domains.
Modulation of Phosphate Uptake and Amphotropic Murine Leukemia Virus Entry by Posttranslational Modifications of PIT-2
Authors: Rodrigues Pierre , Heard Jean Michel
Published: December 2019

PIT-2 is a type III sodium phosphate cotransporter and the receptor for amphotropic murine leukemia viruses. We have investigated the expression and the functions of a tagged version of PIT-2 in CHO cells. PIT-2 remained equally abundant at the cell surface within 6 h following variation of the phosphate supply. In contrast, the efficiency of phosphate uptake and retrovirus entry was inversely related to the extracellular phosphate concentration, indicating that PIT-2 activities are modulated by posttranslational modifications of cell surface molecules induced by phosphate. Conformational changes of PIT-2 contribute to both activities, as shown by the inhibitory effect of sulfhydryl reagents known as inhibitors of type II cotransporters. A physical association of PIT-2 with actin was demonstrated. Modifications of the actin network were induced by variations of the concentrations of extracellular phosphate, cytochalasin D, or lysophosphatidic acid. They revealed that the formation of actin stress fibers determines the cell surface distribution of PIT-2, the internalization of the receptor in response to virus binding, and the capacity to process retrovirus entry. Thus, the presence of PIT-2 at the cell surface is not sufficient to ensure phosphate transport and susceptibility to amphotropic retrovirus infection. Further activation of cell surface PIT-2 molecules is required for these functions.
Expression and Characterization of a Novel Structural Protein of Human Cytomegalovirus, pUL25
Authors: Battista Maria Concetta , Bergamini Giovanna , Boccuni Maria Cristina , Campanini Fabio , Ripalti Alessandro , Landini Maria Paola
Published: December 2019

Human cytomegalovirus (HCMV) UL25 has recently been found to encode a new structural protein that is present in both virion and defective viral particles (C. J. Baldick and T. Shenk, J. Virol. 70:6097–6105, 1996). In the present work a polyclonal antibody was raised against a prokaryotic pUL25 fusion protein in order to investigate the biosynthesis and localization of the UL25 product (pUL25) during HCMV replication in human fibroblasts. Furthermore, pUL25 was transiently expressed in its native form and fused to the FLAG epitope, in COS7 and U373MG cells, in order to compare the properties of the isolated protein and that produced during infection. Immunoblotting analysis revealed a group of polypeptides, ranging from 80 to 100 kDa, in both transfected and infected cells; in vivo labeling experiments with infected cells demonstrated they are posttranslationally modified by phosphorylation. The transcriptional analysis of the UL25 open reading frame combined with the study of pUL25 biosynthesis showed true late kinetics for this protein in infected human fibroblasts. By indirect immunofluorescence both recombinant and viral pUL25 were detected exclusively in the cytoplasm of transfected or infected cells. Interestingly, pUL25 was shown to localize in typical condensed structures in the perinuclear region as already observed for other HCMV tegument proteins. Colocalization of ppUL99 in the same vacuoles suggests that these structure are endosomal cisternae, which are proposed to be a preferential site of viral particle envelopment. Our data suggest that pUL25 is most likely a novel tegument protein and possibly plays a key role in the process of envelopment.
Highly Diverse Intergenic Regions of the Paramyxovirus Simian Virus 5 Cooperate with the Gene End U Tract in Viral Transcription Termination and Can Influence Reinitiation at a Downstream Gene
Authors: Rassa John C. , Parks Griffith D.
Published: December 2019

A dicistronic minigenome containing the M-F gene junction was used to determine the role of the simian virus 5 (SV5) intergenic regions in transcription. The M-F junction differs from the other SV5 junctions by having a short M gene end U tract of only four residues (U4 tract) and a 22-base M-F intergenic sequence between the M gene end and F gene start site. Replacing the 22-base M-F intergenic region with nonviral sequences resulted in a minigenome template (Rep 22) that was defective in termination at the end of the M gene. Efficient M gene termination could be restored to the mutant Rep 22 template in either of two ways: by increasing the U tract length from four to six residues or by restoring a G residue immediately downstream of the wild-type (WT) U4 tract. In a dicistronic SH-HN minigenome, a U4-G combination was functionally equivalent to the naturally occurring SH U6-A gene end in directing SH transcription termination. In addition to affecting termination, the M-F intergenic region also influenced polymerase reinitiation. In the context of the WT U4-G M gene end, substituting nonviral sequences into the M-F intergenic region had a differential effect on F gene reinitiation, where some but not all nonviral sequences inhibited reinitiation. The inhibition of F gene reinitiation correlated with foreign sequences having a high C content. Deleting 6 bases or inserting 18 additional nucleotides into the middle of the 22-base M-F intergenic segment did not influence M gene termination or F gene reinitiation, indicating that M-F intergenic length per se is not a important factor modulating the SV5 polymerase activity. Our results suggest that the sequence diversity at an SV5 gene junction reflects specific combinations which may differentially affect SV5 gene expression and provide an additional level of transcriptional control beyond that which results from the distance of a gene from the 3′ end promoter.
Cooperative Interaction of CI Protein Regulates Lysogeny of
Lactobacillus casei
by Bacteriophage A2
Authors: García Pilar , Ladero Victor , Alonso Juan C. , Suárez Juan E.
Published: December 2019

The temperate bacteriophage A2 forms stable lysogens in
Lactobacillus casei
. The A2-encoded
cI
product (CI), which is responsible for maintaining the A2 prophage in the lysogenic state, has been purified. The CI protein, which is a monomer of 25.3 kDa in solution, specifically binds to a 153-bp DNA fragment that contains two divergent promoters, P
L
and P
R
. These promoters mediate transcription from
cI
and a putative
cro
, respectively. Three similar, although not identical, 20-bp inverted repeated DNA segments (operator sites O
1
, O
2
, and O
3
) were found in this segment. CI selectively interacts with O
1
, which is placed downstream from the transcription start point of the
cro
gene, and with O
2
and O
3
, which overlap with the −35 region of the two promoters. Using a heterologous RNA polymerase, we have determined the transcription start points of P
L
and P
R
. CI exerts a negative effect on the in vitro transcription of P
R
by repositioning the RNA polymerase in a concentration-dependent manner. CI, when bound to O
1
and O
2
, enhances the positioning of the RNA polymerase with the P
L
promoter. Our data indicate that the CI protein regulates the lytic and lysogenic pathways of the A2 phage.
In Vitro Recoating of Reovirus Cores with Baculovirus-Expressed Outer-Capsid Proteins μ1 and ς3
Authors: Chandran Kartik , Walker Stephen B. , Chen Ya , Contreras Carlo M. , Schiff Leslie A. , Baker Timothy S. , Nibert Max L.
Published: December 2019

Reovirus outer-capsid proteins μ1, ς3, and ς1 are thought to be assembled onto nascent core-like particles within infected cells, leading to the production of progeny virions. Consistent with this model, we report the in vitro assembly of baculovirus-expressed μ1 and ς3 onto purified cores that lack μ1, ς3, and ς1. The resulting particles (recoated cores, or r-cores) closely resembled native virions in protein composition (except for lacking cell attachment protein ς1), buoyant density, and particle morphology by scanning cryoelectron microscopy. Transmission cryoelectron microscopy and image reconstruction of r-cores confirmed that they closely resembled virions in the structure of the outer capsid and revealed that assembly of μ1 and ς3 onto cores had induced rearrangement of the pentameric λ2 turrets into a conformation approximating that in virions. r-cores, like virions, underwent proteolytic conversion to particles resembling native ISVPs (infectious subvirion particles) in protein composition, particle morphology, and capacity to permeabilize membranes in vitro. r-cores were 250- to 500-fold more infectious than cores in murine L cells and, like virions but not ISVPs or cores, were inhibited from productively infecting these cells by the presence of either NH
4
Cl or E-64. The latter results suggest that r-cores and virions used similar routes of entry into L cells, including processing by lysosomal cysteine proteinases, even though the former particles lacked the ς1 protein. To examine the utility of r-cores for genetic dissections of μ1 functions in reovirus entry, we generated r-cores containing a mutant form of μ1 that had been engineered to resist cleavage at the δ:φ junction during conversion to ISVP-like particles by chymotrypsin in vitro. Despite their deficit in δ:φ cleavage, these ISVP-like particles were fully competent to permeabilize membranes in vitro and to infect L cells in the presence of NH
4
Cl, providing new evidence that this cleavage is dispensable for productive infection.
Complete Sequence of Enzootic Nasal Tumor Virus, a Retrovirus Associated with Transmissible Intranasal Tumors of Sheep
Authors: Cousens Christina , Minguijon Esmeralda , Dalziel Robert G. , Ortin Aurora , Garcia Mercedes , Park Jane , Gonzalez Lorenzo , Sharp J. Michael , de las Heras Marcelo
Published: December 2019

The sequence of the complete genome of ovine enzootic nasal tumor virus, an exogenous retrovirus associated exclusively with contagious intranasal tumors of sheep, was determined. The genome is 7,434 nucleotides long and exhibits a genetic organization characteristic of type B and D oncoviruses. Enzootic nasal tumor virus is closely related to the Jaagsiekte sheep retrovirus and to sheep endogenous retroviruses.
Jaagsiekte Retrovirus Is Widely Distributed both in T and B Lymphocytes and in Mononuclear Phagocytes of Sheep with Naturally and Experimentally Acquired Pulmonary Adenomatosis
Authors: Holland Martin J. , Palmarini Massimo , Garcia-Goti Mercedes , Gonzalez Lorenzo , McKendrick Iain , de las Heras Marcelo , Sharp J. Michael
Published: December 2019

Jaagsiekte sheep retrovirus (JSRV) is a type D retrovirus specifically associated with a contagious lung tumor of sheep, sheep pulmonary adenomatosis (SPA). JSRV replicates actively in the transformed epithelial cells of the lung, and JSRV DNA and RNA have been detected in lymphoid tissues of naturally affected animals. To determine the lymphoid target cells of JSRV, CD4+T cells, CD8+T cells, B lymphocytes, and adherent cell (macrophage/monocyte) populations were isolated from the mediastinal lymph nodes of naturally affected sheep and lambs inoculated with JSRV. Cells were enriched to high purity and then analyzed for JSRV proviral DNA by heminested PCR, and the proviral burden was quantitated by limiting dilution analysis. JSRV proviral DNA was found in all subsets examined but not in appropriate negative controls. In sheep naturally affected with SPA, JSRV proviral burden was greatest in the adherent cell population. In the nonadherent lymphocyte population, surface immunoglobulin-positive B cells contained the greatest proviral burden, while CD4+and CD8+T cells contained the lowest levels of JSRV proviral DNA. In most of the cases (5 of 8), provirus also could be detected in the peripheral blood mononuclear cell (PBMC) population. A kinetic study of JSRV infection in the mediastinal lymphocyte population of newborn lambs inoculated with JSRV found that JSRV proviral DNA could be detected as early as 7 days postinoculation before the onset of pulmonary adenomatosis, although the proviral burden was greatly reduced compared to adult natural cases. This was reflected in the levels found in PBMC since proviral DNA was detected in 2 of 13 animals. At the early time points studied (7 to 28 days postinoculation) no one subset was preferentially infected. These data indicate that JSRV can infect lymphoid and phagocytic mononuclear cells of sheep and that dissemination precedes tumor formation. Infection of lymphoid tissue, therefore, may play an important role in the pathogenesis of SPA.
Protection of Macaques against Pathogenic Simian/Human Immunodeficiency Virus 89.6PD by Passive Transfer of Neutralizing Antibodies
Authors: Mascola John R. , Lewis Mark G. , Stiegler Gabriela , Harris Dawn , VanCott Thomas C. , Hayes Deborah , Louder Mark K. , Brown Charles R. , Sapan Christine V. , Frankel Sarah S. , Lu Yichen , Robb Merlin L. , Katinger Hermann , Birx Deborah L.
Published: December 2019

The role of antibody in protection against human immunodeficiency virus (HIV-1) has been difficult to study in animal models because most primary HIV-1 strains do not infect nonhuman primates. Using a chimeric simian/human immunodeficiency virus (SHIV) based on the envelope of a primary isolate (HIV-89.6), we performed passive-transfer experiments in rhesus macaques to study the role of anti-envelope antibodies in protection. Based on prior in vitro data showing neutralization synergy by antibody combinations, we evaluated HIV immune globulin (HIVIG), and human monoclonal antibodies (MAbs) 2F5 and 2G12 given alone, compared with the double combination 2F5/2G12 and the triple combination HIVIG/2F5/2G12. Antibodies were administered 24 h prior to intravenous challenge with the pathogenic SHIV-89.6PD. Six control monkeys displayed high plasma viremia, rapid CD4+-cell decline, and clinical AIDS within 14 weeks. Of six animals given HIVIG/2F5/2G12, three were completely protected; the remaining three animals became SHIV infected but displayed reduced plasma viremia and near normal CD4+-cell counts. One of three monkeys given 2F5/2G12 exhibited only transient evidence of infection; the other two had marked reductions in viral load. All monkeys that received HIVIG, 2F5, or 2G12 alone became infected and developed high-level plasma viremia. However, compared to controls, monkeys that received HIVIG or MAb 2G12 displayed a less profound drop in CD4+T cells and a more benign clinical course. These data indicate a general correlation between in vitro neutralization and protection and suggest that a vaccine that elicits neutralizing antibody should have a protective effect against HIV-1 infection or disease.
The C-Terminal Region but Not the Arg-X-Pro Repeat of Epstein-Barr Virus Protein EB2 Is Required for Its Effect on RNA Splicing and Transport
Authors: Buisson Monique , Hans Fabienne , Kusters Inca , Duran Nathalie , Sergeant Alain
Published: December 2019

The Epstein-Barr virus BMLF1 gene product EB2 has been shown to efficiently transform immortalized Rat1 and NIH 3T3 cells, to bind RNA, and to shuttle from the nucleus to the cytoplasm. In transient-expression assays EB2 seems to affect mRNA nuclear export of intronless RNAs and pre-mRNA 3′ processing, but no direct proof of EB2 being involved in RNA processing and transport has been provided, and no specific functional domain of EB2 has been mapped. Here we significantly extend these findings and directly demonstrate that (i) EB2 inhibits the cytoplasmic accumulation of mRNAs, but only if they are generated from precursors containing weak (cryptic) 5′ splice sites, (ii) EB2 has no effect on the cytoplasmic accumulation of mRNA generated from precursors containing constitutive splice sites, and (iii) EB2 has no effect on the 3′ processing of precursor RNAs containing canonical and noncanonical cleavage-polyadenylation signals. We also show that in the presence of EB2, intron-containing and intronless RNAs accumulate in the cytoplasm. EB2 contains an Arg-X-Pro tripeptide repeated eight times, similar to that described as an RNA-binding domain in the herpes simplex virus type 1 protein US11. As glutathione
S
-transferase fusion proteins, both EB2 and the Arg-X-Pro repeat bound RNA in vitro. However, by using EB2 deletion mutants, we demonstrated that the effect of EB2 on splicing and RNA transport requires the C-terminal half of the protein but not the Arg-X-Pro repeat.
Binding of Human Immunodeficiency Virus Type 1 Gag to Membrane: Role of the Matrix Amino Terminus
Authors: Ono Akira , Freed Eric O.
Published: December 2019

Binding of the human immunodeficiency virus type 1 (HIV-1) Gag protein precursor, Pr55
Gag
, to membrane is an indispensable step in virus assembly. Previously, we reported that a matrix (MA) residue 6 substitution (6VR) imposed a virus assembly defect similar to that observed with myristylation-defective mutants, suggesting that the 6VR change impaired membrane binding. Intriguingly, the 6VR mutation had no effect on Gag myristylation. The defective phenotype imposed by 6VR was reversed by changes at other positions in MA, including residue 97. In this study, we use several biochemical methods to demonstrate that the residue 6 mutation, as well as additional substitutions in MA amino acids 7 and 8, reduce membrane binding without affecting N-terminal myristylation. This effect is observed in the context of Pr55
Gag
, a truncated Gag containing only MA and CA, and in MA itself. The membrane binding defect imposed by the 6VR mutation is reversed by second-site changes in MA residues 20 and 97, both of which, when present alone, increase membrane binding to levels greater than those for the wild type. Both reduced and enhanced membrane binding imposed by the MA substitutions depend upon the presence of the N-terminal myristate. The results support the myristyl switch model recently proposed for the regulation of Gag membrane binding, according to which membrane binding is determined by the degree of exposure or sequestration of the N-terminal myristate moiety. Alternatively, insertion of the myristate into the lipid bilayer might be a prerequisite event for the function of other distinct MA-encoded membrane binding domains.
Characterization of Varicella-Zoster Virus Glycoprotein K (Open Reading Frame 5) and Its Role in Virus Growth
Authors: Mo Chengjun , Suen Jeffrey , Sommer Marvin , Arvin Ann
Published: December 2019

Varicella-zoster virus (VZV) is an alphaherpesvirus that is the causative agent of chickenpox and herpes zoster. VZV open reading frame 5 (ORF5) encodes glycoprotein K (gK), which is conserved among alphaherpesviruses. While VZV gK has not been characterized, and its role in viral replication is unknown, homologs of VZV gK in herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PRV) have been well studied. To identify the VZV ORF5 gene product, we raised a polyclonal antibody against a fusion protein of ORF5 codons 25 to 122 with glutathione
S
-transferase and used it to study the protein in infected cells. A 40,000-molecular-weight protein was detected in cell-free virus by Western blotting. In immunogold electron microscopic studies, VZV gK was in enveloped virions and was evenly distributed in the cytoplasm in infected cells. To determine the function of VZV gK in virus growth, a series of gK deletion mutants were constructed with VZV cosmid DNA derived from the Oka strain. Full and partial deletions in gK prevented viral replication when the gK mutant cosmids were transfected into melanoma cells. Insertion of the HSV-1 (KOS) gK gene into the endogenous VZV gK site did not compensate for the deletion of VZV gK. The replacement of VZV gK at a nonnative
Avr
II site in the VZV genome restored the phenotypic characteristics of intact recombinant Oka (rOka) virus. Moreover, gK complementing cells transfected with a full gK deletion mutant exhibited viral plaques indistinguishable from those of rOka. Our results are consistent with the studies of gK proteins of HSV-1 and PRV showing that gK is indispensable for viral replication.
CCAAT Displacement Protein Binds to and Negatively Regulates Human Papillomavirus Type 6 E6, E7, and E1 Promoters
Authors: Ai Wandong , Toussaint Esra , Roman Ann
Published: December 2019

Expression of human papillomavirus genes increases as the target cell, the keratinocyte, differentiates. CCAAT displacement protein (CDP) is a cellular protein which has been shown in other cell types to negatively regulate gene expression in undifferentiated cells but not in differentiated cells. We have previously shown that a 66-bp purine-thymidine-rich sequence (the 66-mer) binds CDP and negatively regulates the human papillomavirus type 6 (HPV-6) E6 promoter (S. Pattison, D. G. Skalnik, and A. Roman, J. Virol. 71:2013–2022, 1997). Cotransfection experiments with a plasmid expressing luciferase from the HPV-6 E6, E7, or E1 regulatory region and a plasmid carrying the CDP gene indicate that CDP represses transcription from all three HPV-6 promoters. Using electrophoretic mobility shift assays (EMSAs), we have shown that CDP binds HPV-6 both upstream and downstream of the E6, E7, and E1 transcription initiation start sites. Furthermore, when keratinocytes were induced to differentiate, all three promoter activities increased. Consistent with this, immunoblotting and EMSAs revealed that endogenous nucleus CDP and, correspondingly, DNA binding activity decreased when keratinocytes were induced to differentiate. The elevated promoter activities were abrogated by exogenously transfected CDP. Our data demonstrate that CDP fulfills the requirement of a differentiation-dependent negative regulator that could tie the HPV life cycle to keratinocyte differentiation.