bluuebunny/crossref_metadata_2025 huggingface dataset:
Structures of Endogenous Nonecotropic Murine Leukemia Virus (MLV) Long Terminal Repeats in Wild Mice: Implication for Evolution of MLVs
Authors: Tomonaga Keizo , Coffin John M.
Published: December 2019

To develop a better understanding of the interaction between retroviruses and their hosts, we have investigated the polymorphism in endogenous murine leukemia proviruses (MLVs). We used genomic libraries of wild mouse DNAs and PCR to analyze genetic variation in the proviruses found in wild mouse species, including
Mus musculus
(
M. m. castaneus
,
M. m. musculus
,
M. m. molossinus
, and
M. m. domesticus
),
Mus spretus
, and
Mus spicelegus
, as well as some inbred laboratory strains. In this analysis, we detected several unique forms of sequence organization in the U3 regions of the long terminal repeats of these proviruses. The distribution of the proviruses with unique U3 structures demonstrated that xenotropic MLV-related proviruses were present only in
M. musculus
subspecies, while polytropic MLV-related proviruses were found in both
M. musculus
and
M. spretus
. Furthermore, one unique provirus from
M. spicelegus
was found to be equidistant from ecotropic provirus and nonecotropic provirus by phylogenetic analysis. This provirus, termed HEMV, was thus likely to be related to the common ancestor of these MLVs. Moreover, an ancestral type of polytropic MLV-related provirus was detected in
M. spretus
species. Despite their “ancestral” phylogenetic position, proviruses of these types are not widespread in mice, implying more-recent spread by infection rather than inheritance. These results imply that recent evolution of these proviruses involved alternating periods of replication as virus and residence in the germ line.
Mapping EBNA-1 Domains Involved in Binding to Metaphase Chromosomes
Authors: Marechal Vincent , Dehee Axelle , Chikhi-Brachet Roxane , Piolot Tristan , Coppey-Moisan Maité , Nicolas Jean-Claude
Published: December 2019

The Epstein-Barr virus (EBV) genome can persist in dividing human B cells as multicopy circular episomes. Viral episomes replicate in synchrony with host cell DNA and are maintained at a relatively constant copy number for a long time. Only two viral elements, the replication origin OriP and the EBNA-1 protein, are required for the persistence of viral genomes during latency. EBNA-1 activates OriP during the S phase and may also contribute to the partition and/or retention of viral genomes during mitosis. Indeed, EBNA-1 has been shown to interact with mitotic chromatin. Moreover, viral genomes are noncovalently associated with metaphase chromosomes. This suggests that EBNA-1 may facilitate the anchorage of viral genomes on cellular chromosomes, thus ensuring proper partition and retention. In the present paper, we have investigated the chromosome-binding activity of EBV EBNA-1, herpesvirus papio (HVP) EBNA-1, and various derivatives of EBV EBNA-1, fused to a variant of the green fluorescent protein. The results show that binding to metaphase chromosomes is a common property of EBV and HVP EBNA-1. Further studies indicated that at least three independent domains (CBS-1, -2, and -3) mediate EBNA-1 binding to metaphase chromosomes. In agreement with the anchorage model, two of these domains mapped to a region that has been previously demonstrated to be required for the long-term persistence of OriP-containing plasmids.
Subtypes of Human Immunodeficiency Virus Type 1 and Disease Stage among Women in Nairobi, Kenya
Authors: Neilson Joel R. , John Grace C. , Carr Jean K. , Lewis Paul , Kreiss Joan K. , Jackson Stephanie , Nduati Ruth W. , Mbori-Ngacha Dorothy , Panteleeff Dana D. , Bodrug Sharon , Giachetti Christina , Bott Martha A. , Richardson Barbra A. , Bwayo Job , Ndinya-Achola Jeckoniah , Overbaugh Julie
Published: December 2019

In sub-Saharan Africa, where the effects of human immunodeficiency virus type 1 (HIV-1) have been most devastating, there are multiple subtypes of this virus. The distribution of different subtypes within African populations is generally not linked to particular risk behaviors. Thus, Africa is an ideal setting in which to examine the diversity and mixing of viruses from different subtypes on a population basis. In this setting, it is also possible to address whether infection with a particular subtype is associated with differences in disease stage. To address these questions, we analyzed the HIV-1 subtype, plasma viral loads, and CD4 lymphocyte levels in 320 women from Nairobi, Kenya. Subtype was determined by a combination of heteroduplex mobility assays and sequence analyses of envelope genes, using geographically diverse subtype reference sequences as well as envelope sequences of known subtype from Kenya. The distribution of subtypes in this population was as follows: subtype A, 225 (70.3%); subtype D, 65 (20.5%); subtype C, 22 (6.9%); and subtype G, 1 (0.3%). Intersubtype recombinant envelope genes were detected in 2.2% of the sequences analyzed. Given that the sequences analyzed represented only a small fraction of the proviral genome, this suggests that intersubtype recombinant viral genomes may be very common in Kenya and in other parts of Africa where there are multiple subtypes. The plasma viral RNA levels were highest in women infected with subtype C virus, and women infected with subtype C virus had significantly lower CD4 lymphocyte levels than women infected with the other subtypes. Together, these data suggest that women in Kenya who are infected with subtype C viruses are at more advanced stages of immunosuppression than women infected with subtype A or D. There are at least two models to explain the data from this cross-sectional study; one is that infection with subtype C is associated with a more rapid disease progression, and the second is that subtype C represents an older epidemic in Kenya. Discriminating between these possibilities in a longitudinal study will be important for increasing our understanding of the role of specific subtypes in the transmission and pathogenesis of HIV-1.
Comparative Analysis of Evolutionary Mechanisms of the Hemagglutinin and Three Internal Protein Genes of Influenza B Virus: Multiple Cocirculating Lineages and Frequent Reassortment of the NP, M, and NS Genes
Authors: Lindstrom Stephen E. , Hiromoto Yasuaki , Nishimura Hidekazu , Saito Takehiko , Nerome Reiko , Nerome Kuniaki
Published: December 2019

Phylogenetic profiles of the genes coding for the hemagglutinin (HA) protein, nucleoprotein (NP), matrix (M) protein, and nonstructural (NS) proteins of influenza B viruses isolated from 1940 to 1998 were analyzed in a parallel manner in order to understand the evolutionary mechanisms of these viruses. Unlike human influenza A (H3N2) viruses, the evolutionary pathways of all four genes of recent influenza B viruses revealed similar patterns of genetic divergence into two major lineages. Although evolutionary rates of the HA, NP, M, and NS genes of influenza B viruses were estimated to be generally lower than those of human influenza A viruses, genes of influenza B viruses demonstrated complex phylogenetic patterns, indicating alternative mechanisms for generation of virus variability. Topologies of the evolutionary trees of each gene were determined to be quite distinct from one another, showing that these genes were evolving in an independent manner. Furthermore, variable topologies were apparently the result of frequent genetic exchange among cocirculating epidemic viruses. Evolutionary analysis done in the present study provided further evidence for cocirculation of multiple lineages as well as sequestering and reemergence of phylogenetic lineages of the internal genes. In addition, comparison of deduced amino acid sequences revealed a novel amino acid deletion in the HA1 domain of the HA protein of recent isolates from 1998 belonging to the B/Yamagata/16/88-like lineage. It thus became apparent that, despite lower evolutionary rates, influenza B viruses were able to generate genetic diversity among circulating viruses through a combination of evolutionary mechanisms involving cocirculating lineages and genetic reassortment by which new variants with distinct gene constellations emerged.
Immunization with a Live, Attenuated Simian Immunodeficiency Virus Vaccine Leads to Restriction of Viral Diversity in Rhesus Macaques Not Protected from Pathogenic Challenge
Authors: Sodora Donald L. , Sheridan Kristine E. , Marx Preston A. , Connor Ruth I.
Published: December 2019

Rhesus macaques immunized with simian immunodeficiency virus SIVmac239Δnef but not protected from SIVmac251 challenge were studied to determine the genetic and biological characteristics of the breakthrough viruses. Assessment of SIV genetic diversity (
env
V1-V2) revealed a reduction in the number of viral species in the immunized, unprotected macaques, compared to the number in nonimmunized controls. However, no evidence for selection of a specific V1-V2 genotype was observed, and biologically cloned isolates from the animals with breakthrough virus were similar with respect to replication kinetics and coreceptor use in vitro.
Molecular Cloning and Expression of Major Structural Protein VP1 of the Human Polyomavirus JC Virus: Formation of Virus-Like Particles Useful for Immunological and Therapeutic Studies
Authors: Goldmann Claudia , Petry Harald , Frye Stephan , Ast Oliver , Ebitsch Susanne , Jentsch Klaus-Dieter , Kaup Franz-Josef , Weber Frank , Trebst Corinna , Nisslein Thomas , Hunsmann Gerhard , Weber Thomas , Lüke Wolfgang
Published: December 2019

The major structural viral protein, VP1, of the human polyomavirus JC virus (JCV), the causative agent of progressive multifocal leukoencephalopathy (PML), was expressed by using recombinant baculoviruses. Recombinant VP1 formed virus-like particles (VLP) with the typical morphology of empty JCV capsids. Purified VP1 VLP bind to SVG, B, and T cells, as well as to monkey kidney cells. After binding, VP1 VLP were also internalized with high efficiency and transported to the nucleus. Immunization studies revealed these particles as highly immunogenic when administered with adjuvant, while immunization without adjuvant induced no immune response. VP1 VLP hyperimmune serum inhibits binding to SVG cells and neutralizes natural JCV. Furthermore, the potential of VP1 VLP as an efficient transporter system for gene therapy was demonstrated. Exogenous DNA could be efficiently packaged into VP1 VLP, and the packaged DNA was transferred into COS-7 cells as shown by the expression of a marker gene. Thus, VP1 VLP are useful for PML vaccine development and represent a potential new transporter system for human gene therapy.
A Sodium-Dependent Neutral-Amino-Acid Transporter Mediates Infections of Feline and Baboon Endogenous Retroviruses and Simian Type D Retroviruses
Authors: Tailor Chetankumar S. , Nouri Ali , Zhao Yuan , Takeuchi Yasuhiro , Kabat David
Published: December 2019

The type D simian retroviruses cause immunosuppression in macaques and have been reported as a presumptive opportunistic infection in a patient with AIDS. Previous evidence based on viral interference has strongly suggested that the type D simian viruses share a common but unknown cell surface receptor with three type C viruses: feline endogenous virus (RD114), baboon endogenous virus, and avian reticuloendotheliosis virus. Furthermore, the receptor gene for these viruses has been mapped to human chromosome 19q13.1–13.2. We now report the isolation and characterization of a cell surface receptor for this group of retroviruses by using a human T-lymphocyte cDNA library in a retroviral vector. Swiss mouse fibroblasts (NIH 3T3), which are naturally resistant to RD114, were transduced with the retroviral library and then challenged with an RD114-pseudotyped virus containing a dominant selectable gene for puromycin resistance. Puromycin selection yielded 12 cellular clones that were highly susceptible to a β-galactosidase-encoding lacZ(RD114) pseudotype virus. Using PCR primers specific for vector sequences, we amplified a common 2.9-kb product from 10 positive clones. Expression of the 2.9-kb cDNA in Chinese hamster ovary cells conferred susceptibility to RD114, baboon endogenous virus, and the type D simian retroviruses. The 2.9-kb cDNA predicted a protein of 541 amino acids that had 98% identity with the previously cloned human Na
+
-dependent neutral-amino-acid transporter B
o
. Accordingly, expression of the RD114 receptor in NIH 3T3 cells resulted in enhanced cellular uptake of
l
-[
3
H]alanine and
l
-[
3
H]glutamine. RNA blot (Northern) analysis suggested that the RD114 receptor is widely expressed in human tissues and cell lines, including hematopoietic cells. The human B
o
transporter gene has been previously mapped to 19q13.3, which is closely linked to the gene locus of the RD114 receptor.
Monoclonal Antibodies against the Minimal DNA-Binding Domain in the Carboxyl-Terminal Region of Human Immunodeficiency Virus Type 1 Integrase
Authors: Ishikawa Tetsuya , Okui Nobuo , Kobayashi Noriko , Sakuma Ryuta , Kitamura Tadaichi , Kitamura Yoshihiro
Published: December 2019

Integrase of human immunodeficiency virus type 1 (HIVIN) consists of 288 amino acids, and its minimum DNA-binding domain (MDBD) (amino acids [aa] 220 to 270) is required for the integration reaction. We produced and characterized four murine monoclonal antibodies (MAbs) to the MDBD of HIVIN (strain LAI). Immunoblot and enzyme-linked immunosorbent assays with truncated HIVINs showed that those MAbs recognized sequential epitopes within the MDBD (aa 228 to 236, 237 to 252, 253 to 261, and 262 to 270). Their binding to HIVIN inhibited terminal cleavage and strand transfer activities but not disintegration activity in vitro. This collection of MAbs is useful for studying the structure and function of the MDBD by complementing mutational analyses and other biochemical studies.
The Proto-Oncogene c-
myc
Is a Direct Target Gene of Epstein-Barr Virus Nuclear Antigen 2
Authors: Kaiser Carmen , Laux Gerhard , Eick Dirk , Jochner Nicola , Bornkamm Georg W. , Kempkes Bettina
Published: December 2019

Epstein-Barr virus (EBV) infects and transforms primary B lymphocytes in vitro. Viral infection initiates the cell cycle entry of the resting B lymphocytes. The maintenance of proliferation in the infected cells is strictly dependent on functional EBNA2. We have recently developed a conditional immortalization system for EBV by rendering the function of EBNA2, and thus proliferation of the immortalized cells, dependent on estrogen. This cellular system was used to identify early events preceding induction of proliferation. We show that LMP1 and c-
myc
are directly activated by EBNA2, indicating that all cellular factors essential for induction of these genes by EBNA2 are present in the resting cells. In contrast, induction of the cell cycle regulators cyclin D2 and cdk4 are secondary events, which require de novo protein synthesis.
Novel Role for E4 Region Genes in Protection of Adenovirus Vectors from Lysis by Cytotoxic T Lymphocytes
Authors: Kaplan Johanne M. , Armentano Donna , Scaria Abraham , Woodworth Lisa A. , Pennington Sarah E. , Wadsworth Samuel C. , Smith Alan E. , Gregory Richard J.
Published: December 2019

Target cells infected with adenovirus (Ad) vectors containing intact E3 and E4 regions were found to be relatively resistant to lysis by Ad-specific cytotoxic T lymphocytes. Elements from both the E3 and the E4 regions were required for this effect, leading to the identification of a previously undescribed role for E4 gene products in resistance to cytolysis.
An Arginine-Faced Amphipathic Alpha Helix Is Required for Adenovirus Type 5 E4orf6 Protein Function
Authors: Orlando Joseph S. , Ornelles David A.
Published: December 2019

A region in the carboxy terminus of the protein encoded by open reading frame 6 in early region 4 (E4orf6) of adenovirus type 5 was determined to be required for directing nuclear localization of the E1B 55-kDa protein and for efficient virus replication. A peptide encompassing this region, corresponding to amino acids 239 through 255 of the E4orf6 protein, was analyzed by circular dichroism spectroscopy. The peptide showed evidence of self-interaction and displayed the characteristic spectra of an amphipathic α helix in the helix-stabilizing solvent trifluoroethanol. Disrupting the integrity of this α helix in the E4orf6 protein by proline substitutions or by removing amino acids 241 through 250 abolished its ability to direct the E1B 55-kDa protein to the nucleus when both proteins were transiently expressed in HeLa cells. Expression of E4orf6 variants that failed to direct nuclear localization of the E1B 55-kDa protein failed to enhance replication of the E4 mutant virus,
dl
1014, whereas expression of the wild-type E4orf6 protein restored growth of
dl
1014 to near-wild-type levels. These results suggest that the E4orf6 protein contains an arginine-faced, amphipathic α helix that is critical for a functional interaction with the E1B 55-kDa protein in the cell and for the function of the E4orf6 protein during a lytic infection.
Mutational Analysis, Using a Full-Length Rubella Virus cDNA Clone, of Rubella Virus E1 Transmembrane and Cytoplasmic Domains Required for Virus Release
Authors: Yao Jiansheng , Gillam Shirley
Published: December 2019

We report on the construction of a full-length cDNA clone, pBRM33, derived from wild-type rubella virus M33 strain. The RNA transcripts synthesized in vitro from pBRM33 are highly infectious, and the viruses produced retain the phenotypic characteristics of the parental M33 virus in growth rate and plaque size. This cDNA clone was used to study the role of E1 transmembrane and cytoplasmic domains in virus assembly by site-directed mutagenesis. Three different alanine substitutions were introduced in the transmembrane domain of E1. These included substitution of leucine 464, cysteine 466, cysteine 467, and both cysteines 466 and 467 to alanine. In the E1 cytoplasmic domain, cysteine 470 and leucine 471 were altered to alanine. We found that these mutations did not significantly affect viral RNA replication, viral structural protein synthesis and transport, or E2/E1 heterodimer formation. Except for the substitution of cysteine 470, these mutations did, however, lead to a reduction in virus release. Substitution of cysteine 467 in the transmembrane region and of leucine 471 in the cytoplasmic domain dramatically reduced virus yield, resulting in the production of only 1 and 10% of the parental virus yield, respectively, in a parallel infection. These data show that E1 transmembrane and cytoplasmic domains play an important role in late stages of virus assembly, possibly during virus budding, consistent with earlier studies indicating that the E1 cytoplasmic domain may interact with nucleocapsids and that this interaction drives virus budding.
Novel Endogenous Type D Retroviral Particles Expressed at High Levels in a SCID Mouse Thymic Lymphoma
Authors: Ristevski Sika , Purcell Damian F. J. , Marshall John , Campagna Daniella , Nouri Sara , Fenton Simon P. , McPhee Dale A. , Kannourakis George
Published: December 2019

A xenograft model of the human disease Langerhans cell histiocytosis (LCH) was investigated with severe combined immunodeficiency (SCID) mice. Transplantation of human LCH biopsy material into SCID mice resulted in the generation of mouse tumors resembling lymphomas. A thymoma cell line (ThyE1M6) was generated from one of these mice and found to display significant levels of Mg2+-dependent reverse transcriptase activity. Electron microscopy revealed particles with type D retroviral morphology budding from ThyE1M6 cells at a high frequency, whereas control cultures were negative. Reverse transcription-PCR of virion RNA with degenerate primers for conserved regions of various mouse, human, and primate retroviruses amplified novel sequences related to primate type D retroviruses, murine intracisternal A particles, Jaagsiekte sheep retrovirus, and murine long interspersed nuclear elements but not other retroviral classes. We demonstrate that these sequences represent a novel group of endogenous retroviruses expressed at low levels in mice but expressed at high levels in the ThyE1M6 cell line. Furthermore, we propose that the activation of endogenous retroviral elements may be associated with a high incidence of thymomas in SCID mice.
Proline Residues in Human Immunodeficiency Virus Type 1 p6
Gag
Exert a Cell Type-Dependent Effect on Viral Replication and Virion Incorporation of Pol Proteins
Authors: Dettenhofer Markus , Yu Xiao-Fang
Published: December 2019

The C terminus of the HIV-1 Gag protein contains a proline-rich domain termed p6
Gag
. This domain has been shown to play a role in efficient virus release and incorporation of Vpr into virions. In a previous study (X. F. Yu, L. Dawson, C. J. Tian, C. Flexner, and M. Dettenhofer, J. Virol. 72:3412–3417, 1998), we observed that the removal of the p6 domain of Gag as well as drastic mutations in the PTAP motif resulted in reduced virion-associated Pol proteins from transfected COS cells. In the present study, amino acid substitutions at residues 5 and 7 of p6
Gag
resulted in a cell type-dependent replication of the mutant virus in CD4
+
T cells; the virus was replication competent in Jurkat cells but restricted in H9 cells and primary blood-derived monocytes. Established Jurkat and H9 cell lines expressing p6
Gag
mutant and parental virus were used to further understand this defect. Mutant virions produced from H9 cells, which displayed no defect in extracellular virion production, showed an ∼16-fold reduction in Pol protein levels, whereas the levels of Pol proteins were only marginally reduced in mutant virions produced from Jurkat cells. The reduction in the virion-associated Pol proteins could not be accounted for by differences in the levels of intracellular p160
Gag-Pol
or in the interaction between p55
Gag
and p160
Gag-Pol
precursors. Electron microscopic analysis of the p6
Gag
mutant virions showed a predominately immature morphology in the absence of significant defects in Gag proteolytic cleavage. Taken together, these data suggest that the proline-rich motif of p6
Gag
is involved in the late stages of virus maturation, which include the packaging of cleaved Pol proteins in viral particles, a process which may involve cell-type-specific factors.
Phenotypic Consequences of Rearranging the P, M, and G Genes of Vesicular Stomatitis Virus
Authors: Ball L. Andrew , Pringle Craig R. , Flanagan Brian , Perepelitsa Victoria P. , Wertz Gail W.
Published: December 2019

The nonsegmented negative-strand RNA viruses (order
Mononegavirales
) include many important human pathogens. The order of their genes, which is highly conserved, is the major determinant of the relative levels of gene expression, since genes that are close to the single promoter site at the 3′ end of the viral genome are transcribed at higher levels than those that occupy more distal positions. We manipulated an infectious cDNA clone of the prototypic vesicular stomatitis virus (VSV) to rearrange three of the five viral genes, using an approach which left the viral nucleotide sequence otherwise unaltered. The central three genes in the gene order, which encode the phosphoprotein P, the matrix protein M, and the glycoprotein G, were rearranged into all six possible orders. Viable viruses were recovered from each of the rearranged cDNAs. The recovered viruses were examined for their levels of gene expression, growth potential in cell culture, and virulence in mice. Gene rearrangement changed the expression levels of the encoded proteins in concordance with their distance from the 3′ promoter. Some of the viruses with rearranged genomes replicated as well or slightly better than wild-type virus in cultured cells, while others showed decreased replication. All of the viruses were lethal for mice, although the time to symptoms and death following inoculation varied. These data show that despite the highly conserved gene order of the
Mononegavirales
, gene rearrangement is not lethal or necessarily even detrimental to the virus. These findings suggest that the conservation of the gene order observed among the
Mononegavirales
may result from immobilization of the ancestral gene order due to the lack of a mechanism for homologous recombination in this group of viruses. As a consequence, gene rearrangement should be irreversible and provide an approach for constructing viruses with novel phenotypes.
Hepatitis C Virus Core Protein Inhibits Fas- and Tumor Necrosis Factor Alpha-Mediated Apoptosis via NF-κB Activation
Authors: Marusawa Hiroyuki , Hijikata Makoto , Chiba Tsutomu , Shimotohno Kunitada
Published: December 2019

The effects of hepatitis C virus (HCV) proteins on anti-Fas (CD95/APO-1) antibody- and tumor necrosis factor alpha (TNF-α)-mediated apoptosis in different human cell lines were investigated by magnetic concentration of cells which transiently produced the exogenous protein. HepG2 cells, which produced whole HCV proteins, became resistant to anti-Fas-induced apoptotic cell death. Furthermore, the core protein among HCV proteins had a key role in protecting the various cells from apoptosis mediated by not only anti-Fas but also TNF-α. We also found that the core functioned in the activation of nuclear factor κB (NF-κB) in all cells examined. Deletion analysis of the core revealed that the region required for NF-κB activation was closely correlated with that for its antiapoptotic function. In addition, we revealed in some cases that the antiapoptotic effect of the core was restrained by coproduction of the inhibitor of NF-κB, IκB-α protein. These results demonstrated that the core inhibits Fas- and TNF-α-mediated apoptotic cell death via a mechanism dependent on the activation of NF-κB in particular cell lines.
Role of the Individual Interferon Systems and Specific Immunity in Mice in Controlling Systemic Dissemination of Attenuated Pseudorabies Virus Infection
Authors: Grob Philipp , Schijns Virgil E. C. J. , van den Broek Maries F. , Cox Silvie P. J. , Ackermann Mathias , Suter Mark
Published: December 2019

The importance of each of the two interferon (IFN) systems in impeding herpesvirus replication and in stimulating virus-specific lymphocytes to control an acute systemic infection is not completely understood. To further our knowledge, pseudorabies virus, attenuated by deletion of the glycoprotein E gene to impair its neurovirulence and by deletion of the thymidine kinase gene (gE−TK−PRV), was used to infect wild-type 129Sv/Ev and congenic mice with immune system-associated genetic deficiencies. Mice with mature B and T lymphocytes but lacking either one or both functional receptors for members of each of the two IFN families were infected with gE−TK−PRV. At 3 and 7 but not 14 days after infection, replicating gE−TK−PRV could be isolated only from livers or spleens of mice lacking the receptors for both IFN families, and these mice survived the infection. Therefore, functional IFN receptors were not required to induce a protective immune response against an acute infection with gE−TK−PRV. Furthermore, PRV-specific antibodies of all immunoglobulin G isotypes were produced in these mice. Mice without mature B and T lymphocytes and lacking either one or both functional receptors for members of each of the two IFN families were also infected with gE−TK−PRV. Three days after infection, replicating virus could be isolated only from mice lacking both mature B and T lymphocytes and functional IFN receptors, and these mice were not able to clear the virus. We present evidence that mice with an intact gamma IFN system but without mature B and T cells were able to prevent systemic dissemination of gE−TK−PRV.
The 5′ RNA Terminus of Spleen Necrosis Virus Contains a Novel Posttranscriptional Control Element That Facilitates Human Immunodeficiency Virus Rev/RRE-Independent Gag Production
Authors: Butsch Melinda , Hull Stacey , Wang Yalai , Roberts Tiffiney M. , Boris-Lawrie Kathleen
Published: December 2019

Previous work has shown that spleen necrosis virus (SNV) long terminal repeats (LTRs) are associated with Rex/Rex-responsive element-independent expression of bovine leukemia virus RNA and supports the hypothesis that SNV RNA contains a
cis
-acting element that interacts with cellular Rex-like proteins. To test this hypothesis, the human immunodeficiency virus type 1 (HIV) Rev/RRE-dependent
gag
gene was used as a reporter to analyze various SNV sequences. Gag enzyme-linked immunosorbent assay and Western blot analyses reveal that HIV Gag production is enhanced at least 20,000-fold by the 5′ SNV LTR in COS, D17, and 293 cells. Furthermore, SNV RU5 in the sense but not the antisense orientation is sufficient to confer Rev/RRE-independent expression onto a cytomegalovirus-
gag
plasmid. In contrast, the SNV 3′ LTR and 3′ untranslated sequence between
env
and the LTR did not support Rev-independent
gag
expression. Quantitative RNase protection assays indicate that the SNV 5′ RNA terminus enhances cytoplasmic accumulation and polysome association of HIV unspliced and spliced transcripts. However, comparison of the absolute amounts of polysomal RNA indicates that polysome association is not sufficient to account for the significant increase in Gag production by the SNV sequences. Our analysis reveals that the SNV 5′ RNA terminus contains a unique
cis
-acting posttranscriptional control element that interacts with hypothetical cellular Rev-like proteins to facilitate HIV RNA transport and efficient translation.
Immortalization of CD4+and CD8+T Lymphocytes by Human T-Cell Leukemia Virus Type 1 Tax Mutants Expressed in a Functional Molecular Clone
Authors: Robek Michael D. , Ratner Lee
Published: December 2019

The human T-cell leukemia virus type 1 (HTLV-1) transcriptionaltrans-activator Tax has been demonstrated to have transforming activity in multiple cell culture and transgenic-mouse models. In addition to activating transcription from the viral long terminal repeat (LTR) through the cyclic AMP response element binding protein/activating transcription factor (CREB/ATF) family of transcription factors, Tax activates the expression of multiple cellular promoters through the NF-κB pathway of transcriptional activation. The Tax mutants M22 and M47 have previously been demonstrated to selectively abrogate the ability of Tax to activate transcription through the NF-κB or CREB/ATF pathway, respectively. These mutations were introduced in thetaxgene of the ACH functional molecular clone of HTLV-1, and virus produced from the mutant ACH clones was examined for the ability to replicate and immortalize primary human lymphocytes. While virus derived from the clone containing the M47 mutation retained the ability to immortalize T lymphocytes, the M22 mutant lost the ability to immortalize infected cells. These results indicate that activation of the CREB/ATF pathway by Tax is dispensable for the immortalization of T cells by HTLV-1, whereas activation of the NF-κB pathway may be critical.
Identification of the Murine
Mx2
Gene: Interferon-Induced Expression of the Mx2 Protein from the Feral Mouse Gene Confers Resistance to Vesicular Stomatitis Virus
Authors: Jin Hee Kyung , Takada Ayato , Kon Yasuhiro , Haller Otto , Watanabe Tomomasa
Published: December 2019

The mouse genome contains two related interferon-regulated genes,
Mx1
and
Mx2
. Whereas
Mx1
codes for the nuclear 72-kDa protein that interferes with influenza virus replication after interferon treatment, the
Mx2
gene is nonfunctional in all laboratory mouse strains examined, since its open reading frame (ORF) is interrupted by an insertional mutation and a subsequent frameshift mutation. In the present study, we demonstrate that
Mx2
mRNA of cells from feral mouse strains NJL (
Mus musculus musculus
) and SPR (
Mus spretus
) differs from that of the laboratory mouse strains tested. The
Mx2
mRNA of the feral strains contains a single long ORF consisting of 656 amino acids. We further show that Mx2 protein in the feral strains is expressed upon interferon treatment and localizes to the cytoplasm much like the rat Mx2 protein, which inhibits vesicular stomatitis virus replication. Furthermore, transfected 3T3 cell lines of laboratory mouse origin expressing
Mx2
from feral strains acquire slight resistance to vesicular stomatitis virus.
Natural Variation in Translational Activities of the 5′ Nontranslated RNAs of Hepatitis C Virus Genotypes 1a and 1b: Evidence for a Long-Range RNA-RNA Interaction outside of the Internal Ribosomal Entry Site
Authors: Honda Masao , Rijnbrand Rene , Abell Geoff , Kim Desok , Lemon Stanley M.
Published: December 2019

The 5′ nontranslated RNA (5′NTR) of a genotype 1b hepatitis C virus (HCV-N) directs cap-independent translation of the HCV-N polyprotein with about twofold less efficiency than the 5′NTR of a genotype 1a virus under physiologic conditions (Hutchinson strain, or HCV-H) (M. Honda et al., Virology 222:31–42, 1996). Here, we show by mutational analysis that substitution of the AG dinucleotide sequence at nucleotides (nt) 34 and 35 of HCV-N with GA (present in HCV-H) restores the translational activity to that of the HCV-H 5′NTR both in vitro and in vivo. These nucleotides are located upstream of the minimal essential internal ribosome entry site (IRES), as a 6-nt deletion spanning nt 32 to 37 also increased the translational activity of the HCV-N 5′NTR to that of HCV-H. Thus, the upstream AG dinucleotide sequence has an inhibitory effect on IRES-directed translation. Surprisingly, however, this inhibitory effect was observed only when the translated, downstream RNA sequence contained nt 408 to 929 of HCV (capsid-coding RNA). Further analysis of RNA transcripts containing frameshift mutations demonstrated that the nucleotide sequence of the transcript, and not the amino acid sequence of the expressed capsid protein, determines this difference in translation efficiency. The difference between the translational activities of the HCV-N and HCV-H transcripts was increased when translation was carried out in reticulocyte lysates containing high K
+
concentrations, with a sevenfold difference evident at 130 to 150 mM K
+
. These results suggest that there is an RNA-RNA interaction involving 5′NTR and capsid-coding sequences flanking the IRES and that this is responsible for the reduced IRES activity of the genotype 1b virus, HCV-N.
Oligomerization within Virions and Subcellular Localization of Human Immunodeficiency Virus Type 1 Integrase
Authors: Petit Caroline , Schwartz Olivier , Mammano Fabrizio
Published: December 2019

Previous biochemical and genetic evidence indicated that the functional form of retroviral integrase protein (IN) is a multimer. A direct demonstration of IN oligomerization during the infectious cycle was, however, missing, due to the absence of a sensitive detection method. We describe here the generation of infectious human immunodeficiency virus type 1 (HIV-1) viral clones carrying IN protein tagged with highly antigenic epitopes. In this setting, we could readily visualize IN both in producer cells and in viral particles. More interestingly, we detected IN oligomers, the formation of which was dependent on disulfide bridges and took place inside virions. Additionally, expression of a tagged HIV-1 IN in the absence of other viral components resulted in almost exclusive nuclear accumulation of the protein. Mutation of a conserved cysteine in the proposed dimer interface determined the loss of viral infectivity, associated with a reduction of IN oligomer formation and the redistribution of the mutated protein in the nucleus and cytoplasm. Epitope tagging of HIV-1 IN expressed alone or in the context of a replication-competent viral clone provides powerful tools to validate debated issues on the implication of this enzyme in different steps of the viral cycle.
Inhibition of Tumor Necrosis Factor Alpha by an Adenovirus-Encoded Soluble Fusion Protein Extends Transgene Expression in the Liver and Lung
Authors: Peng YuFeng , Trevejo Jose , Zhou JunLiang , Marino Michael W. , Crystal Ronald G. , Falck-Pedersen Erik , Elkon Keith B.
Published: December 2019

The cellular and humoral immune responses to adenovirus (Ad) remain a major barrier to Ad-mediated gene therapy. We recently reported that mice deficient in tumor necrosis factor alpha (TNF-α) or Fas (APO-1, CD95) have prolonged expression of an Ad transgene expressing a foreign protein in the liver. To determine whether blockade of TNF-α or Fas would have the same effect in normal mice, we created transgenes that expressed soluble murine CD8 or CD8 fused to the extracellular regions of TNF receptor 1 (TNFR) or Fas and inserted into the left-end region of first-generation (E1/E3−) Ad vectors. Consistent with the results observed in TNF-deficient mice, expression of the TNFR-CD8 fusion protein was prolonged in vivo compared to that of control proteins. Not only did expression of TNFR-CD8 persist in the liver and the lung, but when coadministered with another first-generation vector, the protein provided “transprotection” for the companion vector and transgene. In addition, TNFR-CD8 attenuated the humoral immune response to the Ad. Together, these findings demonstrate that blockade of TNF-α is likely to be useful in extending the expression of an Ad-encoded transgene in a gene therapy application.
Viral Immediate-Early Proteins Abrogate the Modification by SUMO-1 of PML and Sp100 Proteins, Correlating with Nuclear Body Disruption
Authors: Müller Stefan , Dejean Anne
Published: December 2019

PML nuclear bodies (NBs) are subnuclear structures whose integrity is compromised in certain human diseases, including leukemia and neurodegenerative disorders. Infection by a number of DNA viruses similarly triggers the reorganization of these structures, suggesting an important role for the NBs in the viral infection process. While expression of the adenovirus E4 ORF3 protein leads to only a moderate redistribution of PML to filamentous structures, the herpes simplex virus (HSV) ICP0 protein and the cytomegalovirus (CMV) IE1 protein both induce a complete disruption of the NB structure. Recently, we and others have shown that the NB proteins PML and Sp100 are posttranslationally modified by covalent linkage with the ubiquitin-related SUMO-1 protein and that this modification may promote the assembly of these structures. Here we show that the HSV ICP0 and CMV IE1 proteins specifically abrogate the SUMO-1 modification of PML and Sp100, whereas the adenovirus E4 ORF3 protein does not affect this process. The potential of ICP0 and IE1 to alter SUMO-1 modification is directly linked to their capacity to disassemble NBs, thus strengthening the role for SUMO-1 conjugation in maintenance of the structural integrity of the NBs. This observation supports a model in which ICP0 and IE1 disrupt the NBs either by preventing the formation or by degrading of the SUMO-1-modified PML and Sp100 protein species. Finally, we show that the IE1 protein itself is a substrate for SUMO-1 modification, thus representing the first viral protein found to undergo this new type of posttranslational modification.
A Single Amino Acid Substitution in the Phosphoprotein of Respiratory Syncytial Virus Confers Thermosensitivity in a Reconstituted RNA Polymerase System
Authors: Marriott Anthony C. , Wilson Steven D. , Randhawa Jaspal S. , Easton Andrew J.
Published: December 2019

The single amino acid change Gly172 to Ser in the phosphoprotein (P) of respiratory syncytial virus (RSV) has previously been shown to be responsible for the thermosensitivity and protein-negative phenotype of
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N19, a mutant of the B subgroup RSN-2 strain. This single change was inserted into the P gene of the A subgroup virus RSS-2, and the resulting phenotype was observed in a plasmid-driven reconstituted RSV RNA polymerase system. Expression from a genome analogue containing two reporter genes was thermosensitive when directed by plasmids containing the N, L, M2, and mutant P genes cloned under the control of T7 promoters. Analysis of RNA synthesis showed that mutant P protein was unable to produce genome, antigenome, or mRNA at the restrictive temperature. At a semipermissive temperature, genome, antigenome, and mRNA synthesis were all reduced, 6- to 30-fold, relative to synthesis directed by a wild-type P plasmid. Binding of the mutant P protein to N protein in the absence of other viral proteins was unaffected by temperature, indicating that the lesion did not produce a large enough structural change to disrupt this binding. These data suggest that the plasmid rescue system is suitable for investigation of the role of thermosensitive mutations in RSV polymerase components in RNA synthesis.
Human Immunodeficiency Virus Type 2 Produces a Defect in CD3-γ Gene Transcripts Similar to That Observed for Human Immunodeficiency Virus Type 1
Authors: Segura Iris , Delmelle-Wibaut Christine , Janssens Michel , Cleuter Yvette , van den Broeke Anne , Kettmann Richard , Willard-Gallo Karen E.
Published: December 2019

T cells are central players in the immune response to infectious disease, with the specificity of their responses controlled by the T-cell receptor (TCR)/CD3 complex on the cell surface. Impairment of TCR/CD3-directed CD4+T-cell immune responses is frequently observed in individuals infected with human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2). Virus replication is also regulated by T-cell activation factors, with HIV-1 and HIV-2 responding to different TCR/CD3-directed cellular pathways. We previously demonstrated that HIV-1 infection of the human interleukin-2-dependent CD4+T-cell line WE17/10 abrogates TCR/CD3 function and surface expression by a specific loss of CD3-γ gene transcripts. In this study, we show that HIV-2 provokes the same molecular defect in CD3-γ gene transcripts, resulting in a similar but delayed progressive loss of TCR/CD3 surface expression after infection.
Epstein-Barr Virus (EBV) Nuclear Protein 2-Induced Disruption of EBV Latency in the Burkitt’s Lymphoma Cell Line Akata: Analysis by Tetracycline-Regulated Expression
Authors: Fujiwara Shigeyoshi , Nitadori Yoshikazu , Nakamura Hiroyuki , Nagaishi Takashi , Ono Yasushi
Published: December 2019

The Burkitt’s lymphoma (BL) cell line Akata retains the latency I program of Epstein-Barr virus (EBV) gene expression and cross-linking of its surface immunoglobulin G (IgG) by antibodies results in activation of viral replication. When EBV nuclear antigen 2 (EBNA2) was artificially expressed by a constitutive expression vector, the Cp EBNA promoter remained inactive and accordingly the latency III program was not induced. In contrast, expression of LMP2A and activity of the Fp lytic promoter were activated. Consistent with this Fp activity, the rate of spontaneous activation of the EBV replicative cycle was increased significantly, suggesting the possibility that EBNA2 can induce EBV replication. The efficiency of anti-IgG-induced activation of the viral replication was reduced in Akata cells expressing EBNA2. To obtain more direct evidence for EBNA2-induced activation of the EBV replicative cycle, this protein was next expressed by a tetracycline-regulated expression system. EBNA2 was undetectable with low doses (<0.5 μg/ml) of tetracycline, while its expression was rapidly induced after removal of the antibiotic. This induced expression of EBNA2 was immediately followed by expression of EBV replicative cycle proteins in up to 50% of the cells, as shown by indirect immunofluorescence and immunoblot analysis. These results suggest an unexpected potential of EBNA2 to disrupt EBV latency and to activate viral replication.
Infection of Chicken Embryonic Fibroblasts by Measles Virus: Adaptation at the Virus Entry Level
Authors: Escoffier Carine , Gerlier Denis
Published: December 2019

Measles virus (MV) has a tropism restricted to humans and primates and uses the human CD46 molecule as a cellular receptor. MV has been adapted to grow in chicken embryonic fibroblasts (CEF) and gave rise to an attenuated live vaccine. Hallé and Schwarz MV strains were compared in their ability to infect both simian Vero cells and CEF. Whereas both strains infected Vero cells, only the CEF-adapted Schwarz strain was able to efficiently infect CEF. Since the expression of the human MV receptor CD46 rendered the chicken embryonic cell line TCF more permissive to the infection by the Hallé MV strain, the MV entry into CEF appeared to be a limiting step in the absence of prior MV adaptation. CEF lacked reactivity with anti-CD46 antibodies but were found to express another protein allowing MV binding as an alternative receptor to CD46.
Primary Virus Envelope Cross-Reactivity of the Broadening Neutralizing Antibody Response during Early Chronic Human Immunodeficiency Virus Type 1 Infection
Authors: Zhang Peng Fei , Chen Xi , Fu Da Wei , Margolick Joseph B. , Quinnan Gerald V.
Published: December 2019

To test the hypothesis that changing neutralizing antibody responses against human immunodeficiency virus type 1 (HIV-1) during chronic infection were a response to emergence of neutralization escape mutants, we cloned expressed and characterized envelope clones from patients in the Multicenter AIDS Cohort Study (MACS). Pseudotyped HIV-1 envelope clones obtained from differing time points were assessed for sensitivity to neutralization by using sera from different times from the same and different patients. Clones from early and late time points during chronic infection had similar neutralization sensitivity, and neutralizing antibody responses cross-reacted with early, late, and heterologous envelopes. The potential for broadly effective HIV-1 immunization is supported.
An Orphan G Protein-Coupled Receptor, GPR1, Acts as a Coreceptor To Allow Replication of Human Immunodeficiency Virus Types 1 and 2 in Brain-Derived Cells
Authors: Shimizu Nobuaki , Soda Yasushi , Kanbe Katsuaki , Liu Hui-Yu , Jinno Atsushi , Kitamura Toshio , Hoshino Hiroo
Published: December 2019

Twelve G protein-coupled receptors, including chemokine receptors, act as coreceptors and determinants for the cell tropisms of human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus (SIV). We isolated HIV-1 variants from T-cell-line (T)- and macrophage (M)-tropic (i.e., dualtropic) (R5-R3-X4) HIV-1 strains and also produced six HIV-1 mutants carrying single-point amino acid substitutions at the tip of the V3 region of the Env protein of HIV-1. These variants and three mutants infected brain-derived CD4-positive cells that are resistant to M-, T-, or dualtropic (R5, X4, or R5-X4) HIV-1 strains. However, a factor that determines this cell tropism has not been identified. This study shows that primary brain-derived fibroblast-like cell strains, BT-3 and BT-20/N, as well as a CD4-transduced glioma cell line, U87/CD4, which were susceptible to these HIV-1 variants and mutants and the HIV-2RODstrain, expressed mRNA of an orphan G protein-coupled receptor (GPCR), GPR1. When a CD4-positive cell line which was strictly resistant to infection with diverse HIV-1 and HIV-2 strains was transduced with GPR1, the cell line became susceptible to these HIV-1 variants and mutants and to an HIV-2 strain but not to T- or dualtropic HIV-1 strains, and numerous syncytia formed after infection. These results indicate that GPR1 functions as a coreceptor for the HIV-1 variants and mutants and for the HIV-2RODstrain in vitro.