bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional Analysis of Ground Squirrel Hepatitis Virus Enhancer II
Authors: Fourel Geneviève , Ringeisen François , Flajolet Marc , Tiollais Pierre , Buendia Marie Annick
Published: December 2019

We have characterized a major regulatory element of ground squirrel hepatitis virus (GSHV) located within a 90-nucleotide fragment of the core promoter upstream sequences and have compared its organization to that of woodchuck hepatitis virus (WHV) enhancer II (We2). The GSHV element (Ge2) stimulates transcription from the viral core promoter and heterologous promoters in an orientation-independent manner but displays a lower level of activity than We2 in transient transfection assays in human hepatoma cells. The general organization of Ge2 into binding sites for the liver-enriched HNF-1 and HNF-4 proteins and for ubiquitous factors of the NF1 and Oct families was found to be mostly conserved with respect to the homologous We2 region. Accordingly, transactivation by HNF-1 and HNF-4 plays an essential role in the liver-specific transcriptional activity of both the GSHV and WHV core promoters. Distinctive features of the GSHV enhancer consist of its ability to bind C/EBP family factors in a central motif that overlaps with one of the two HNF-4 sites and its differential binding affinities for HNF-4.
Visna Virus dUTPase Is Dispensable for Neuropathogenicity
Authors: Pétursson Gudmundur , Turelli Priscilla , Matthíasdóttir Sigrídur , Georgsson Gudmundur , Andrésson Ólafur S. , Torsteinsdóttir Sigurbjörg , Vigne Robert , Andrésdóttir Valgerdur , Gunnarsson Eggert , Agnarsdóttir Gudrún , Quérat Gilles
Published: December 2019

The major part of the dUTPase-encoding region of the visna virus genome was deleted. Intracerebral injection of the mutant virus resulted in a somewhat reduced viral load compared to that resulting from injection of the wild type, especially in the lungs, but the neuropathogenic effects were comparable. The dUTPase gene is dispensable for induction of lesions in the brain.
Enzymatic Characterization of Refolded Human Rhinovirus Type 14 2A Protease Expressed in
Escherichia coli
Authors: Wang Q. May , Johnson Robert B. , Cox Gregory A. , Villarreal Elcira C. , Churgay Lisa M. , Hale John E.
Published: December 2019

Reported here is the production of recombinant human rhinovirus 14 (HRV14) 2A protease from bacterial cells transformed with a heat-inducible plasmid containing the HRV14 2A cDNA sequence. Overexpressed 2A protein partitioned into the inclusion bodies was solubilized in urea and then refolded in the presence of Zn
2+
. Transition metals were required for the restoration of 2A protease activity as a structural component, but appeared to be inhibitory if added exogenously once the enzyme was refolded. Based on the cleavage specificity studies, a colorimetric assay was developed for the highly purified HRV14 2A protease. A peptide with the sequence RKGDIKSY–
p
-nitroanilide was found to be cleaved by the 2A protease with a
k
cat
/

K
m

ratio of ∼335 M
−1
s
−1
, which allows its activity to be measured continuously with a spectrophotometer or a microplate reader.
Cloning and Characterization of a Novel Hepatitis B Virus x Binding Protein That Inhibits Viral Replication
Authors: Melegari Margherita , Scaglioni Pier Paolo , Wands Jack R.
Published: December 2019

The hepatitis B virus and the mammalian hepadnavirus genomes encode for a short open reading frame called x. Expression of the protein product (HBx) appears necessary for establishment of natural infection. However, in vitro studies have suggested a multifunctional role for HBx as an indirect transcriptional transactivator of a variety of different viral and cellular promoters. Indeed, HBx has no known direct DNA binding properties but may interact with transcription factors as well as activate intracellular signaling pathways associated with cell growth. To further address the possible functional role of HBx in the life cycle of hepatitis B virus, we performed an analysis using the yeast two-hybrid system to screen a cDNA library derived from a hepatocellular carcinoma cell line with a HBx fusion bait in an attempt to identify cellular partners that may bind to and alter the biologic properties of HBx. A HBx-interacting protein that specifically complexes with the carboxy terminus of wild-type HBx was identified and designated XIP. This 9.6-kDa protein is capable of binding to HBx in vitro, and transient and stable expression in hepatocellular carcinoma cells abolishes the transactivation properties of HBx on luciferase constructs driven by AP-1 and endogenous hepatitis B virus enhancer/promoter elements. Investigation of the role of XIP in hepatitis B virus replication in differentiated hepatocellular carcinoma cells revealed that XIP expression reduces wild-type hepatitis B virus replication to levels observed following transfection with an HBx-minus virus. In contrast, the replication levels of the duck hepatitis B virus, a hepadnavirus that lacks the x open reading frame, were unchanged in the context of XIP expression. We propose that one of the physiologic functions of the cellular protein XIP is to negatively regulate HBx activity and thus to alter the replication life cycle of the virus.
Effect of Passive Immunization or Maternally Transferred Immunity on the Antibody Response to a Genetic Vaccine to Rabies Virus
Authors: Wang Yijie , Xiang Zhiquan , Pasquini Susanna , Ertl Hildegund C. J.
Published: December 2019

A plasmid vector, termed pSG5rab.gp, expressing the glycoprotein of rabies virus was tested in young adult or neonatal mice in the presence of maternally transferred immunity or passively administered antibodies to rabies virus for induction of an antibody response. Mice born to rabies virus-immune dams developed an impaired antibody response to genetic immunization at 6 weeks of age, as had been previously observed upon vaccination with an inactivated viral vaccine. Similarly, mice passively immunized with hyperimmune serum showed an inhibited B-cell response upon vaccination with the pSG5rab.gp vector, resulting in both cases in vaccine failures upon challenge with a virulent strain of rabies virus. In contrast, the immune responses of mice vaccinated as neonates in the presence of maternal immunity or upon passive immunization to rabies virus with the pSG5rab.gp construct were only marginally affected.
Polyadenylation of Vesicular Stomatitis Virus mRNA Dictates Efficient Transcription Termination at the Intercistronic Gene Junctions
Authors: Hwang Leroy N. , Englund Nathan , Pattnaik Asit K.
Published: December 2019

The intercistronic gene junctions of vesicular stomatitis virus (VSV) contain conserved sequence elements that are important for polyadenylation and transcription termination of upstream transcript as well as reinitiation of transcription of downstream transcript. To examine the role of the putative polyadenylation signal 3′AUACU
7
5′ at the gene junctions in polyadenylation and transcription termination, we constructed plasmids encoding antigenomic minireplicons containing one or two transcription units. In plasmid-transfected cells, analyses of the bicistronic minireplicon containing the wild-type or mutant intercistronic gene junctions for the ability to direct synthesis of polyadenylated upstream, downstream, and readthrough mRNAs showed that the AUACU
7
sequence element is required for polyadenylation of VSV mRNA. Deletion of AUAC or U
7
resulted in templates that did not support polyadenylation of upstream mRNA. Interestingly, we found that the loss of polyadenylation function led to antitermination of the upstream transcript and resulted in a readthrough transcript that contained the upstream and downstream mRNA sequences. Mutations that blocked polyadenylation also blocked transcription termination and generated mostly readthrough transcript. Reverse transcription-PCR of readthrough transcripts and subsequent nucleotide sequencing of the amplified product revealed no extra adenosine residues at the junction of the readthrough transcript. These results indicate that polyadenylation is required for transcription termination of VSV mRNA. The intergenic dinucleotide GA did not appear to be necessary for transcription termination. Furthermore, we found that insertion of the polyadenylation signal sequence AUACU
7
alone was sufficient to direct polyadenylation and efficient transcription termination at the inserted site. Taken together, the data presented here support the conclusion that polyadenylation is the major determinant of transcription termination at the intercistronic gene junctions of VSV.
Extended Minus-Strand DNA as Template for R-U5-Mediated Second-Strand Transfer in Recombinational Rescue of Primer Binding Site-Modified Retroviral Vectors
Authors: Mikkelsen Jacob Giehm , Lund Anders H. , Dybkær Karen , Duch Mogens , Pedersen Finn Skou
Published: December 2019

We have previously demonstrated recombinational rescue of primer binding site (PBS)-impaired Akv murine leukemia virus-based vectors involving initial priming on endogenous viral sequences and template switching during cDNA synthesis to obtain PBS complementarity in second-strand transfer of reverse transcription (Mikkelsen et al., J. Virol. 70:1439–1447, 1996). By use of the same forced recombination system, we have now found recombinant proviruses of different structures, suggesting that PBS knockout vectors may be rescued through initial priming on endogenous virus RNA, read-through of the mutated PBS during minus-strand synthesis, and subsequent second-strand transfer mediated by the R-U5 complementarity of the plus strand and the extended minus-strand DNA acceptor template. Mechanisms for R-U5-mediated second-strand transfer and its possible role in retrovirus replication and evolution are discussed.
Characterization of the DNA-Binding Domain of the Bovine Papillomavirus Replication Initiator E1
Authors: Chen Grace , Stenlund Arne
Published: December 2019

The bovine papillomavirus replication initiator protein E1 is an origin of replication (
ori
)-binding protein absolutely required for viral DNA replication. In the presence of the viral transcription factor E2, E1 binds to the
ori
and initiates DNA replication. To understand how the E1 initiator recognizes the
ori
and how E2 assists in this process, we have expressed and purified a 166-amino-acid fragment which corresponds to the minimal E1 DNA-binding domain (DBD). DNA binding studies using this protein demonstrate that the E1 DBD can bind to the palindromic E1 binding site in several forms but that binding of two monomers, each recognizing one half-site of the E1 palindrome, is the predominant form. This is reminiscent of the binding of the T-antigen DBD to the SV40
ori
, and interestingly, the arrangement of E1 binding sites shows striking similarities to the arrangement of T-antigen binding sites in the SV40
ori
even though the recognition sequences are unrelated. The E1 DBD is capable of interacting cooperatively with E2; however, the E2 DBD and not the E2 activation domain mediates this interaction. Furthermore, the E2 DBD stimulates binding of two monomers of the E1 DBD to the
ori
by binding cooperatively with one E1 monomer. Finally, we show that our results concerning the DNA-binding properties of the E1 DBD can be extended to full-length E1.
Role of Actin Microfilaments in Black Creek Canal Virus Morphogenesis
Authors: Ravkov Eugene V. , Nichol Stuart T. , Peters Clarence J. , Compans Richard W.
Published: December 2019

We have investigated the involvement of cytoskeletal proteins in the morphogenesis of Black Creek Canal virus (BCCV), a New World hantavirus. Immunofluorescent staining of BCCV-infected cells revealed a filamentous pattern of virus antigen, the appearance of which was sensitive to treatment with cytochalasin D, an actin microfilament-depolymerizing drug. Double immunofluorescence staining of BCCV-infected Vero cells with anti-BCCV nucleocapsid (N) monoclonal antibody and phalloidin revealed a colocalization of the BCCV N protein with actin microfilaments. A similar, though less prominent, filamentous pattern was observed in BHK21 cells transiently expressing the BCCV N protein alone but not in cells expressing the BCCV G1 and G2 glycoproteins. Moreover, the association of the N protein with actin microfilaments was confirmed by coimmunoprecipitation with β-actin-specific antibody. Treatment of the BCCV-infected Vero cells at 3 days postinfection with cytochalasin D decreased the yield of released BCCV by 94% relative to the yield from untreated cells. Pretreatment of Vero cells with cytochalasin D prior to and during BCCV adsorption and entry had no effect on the outcome of virus production. These results indicate that actin filaments may play an important role in hantavirus assembly and/or release.
Priming with Secreted Glycoprotein G of Respiratory Syncytial Virus (RSV) Augments Interleukin-5 Production and Tissue Eosinophilia after RSV Challenge
Authors: Johnson Teresa R. , Johnson Joyce E. , Roberts Sharon R. , Wertz Gail W. , Parker Robert A. , Graham Barney S.
Published: December 2019

The respiratory syncytial virus (RSV) G glycoprotein promotes differentiation of type 2 CD4+T lymphocytes and induces an eosinophilic response in lungs of RSV-infected mice. A unique feature of G is that a second initiation codon in the transmembrane region of the glycoprotein results in secretion of soluble protein from infected cells. Recombinant vaccinia viruses that express wild-type G (vvWT G), only secreted G (vvM48), or only membrane-anchored G (vvM48I) were used to define the influence of G priming on immunopathogenesis. Mice immunized with vvM48 had more severe illness following RSV challenge than did mice primed with vvWT G or vvM48I. Coadministration of purified G during priming with the construct expressing membrane-anchored G shifted immune responses following RSV challenge to a more Th2-like response. This was characterized by increased interleukin-5 in lung supernatants and an increase in G-specific immunoglobulin G1 antibodies. Eosinophils were present in the infiltrate of all mice primed with G-containing vectors but were greatest in mice primed with regimens including secreted G. These data suggest the form of G protein available for initial antigen processing and presentation is an important factor in promoting Th2-like immune responses, including the induction of lung eosinophilia. The ability of RSV to secrete G protein may therefore represent a viral strategy for immunomodulation and be a key determinant of disease pathogenesis.
Cytotoxic T-Lymphocyte Target Proteins and Their Major Histocompatibility Complex Class I Restriction in Response to Adenovirus Vectors Delivered to Mouse Liver
Authors: Jooss Karin , Ertl Hildegund C. J. , Wilson James M.
Published: December 2019

The activation of cytotoxic T lymphocytes (CTLs) to cells infected with adenovirus vectors contributes to problems of inflammation and transient gene expression that attend their use in gene therapy. The goal of this study was to identify in a murine model of liver gene therapy the proteins that provide targets to CTLs and to characterize the major histocompatibility complex (MHC) class I restricting elements. Mice of different MHC haplotypes were infected with an E1-deleted adenovirus expressing human alkaline phosphatase (ALP) or β-galactosidase as a reporter protein, and splenocytes were harvested for in vitro CTL assays to aid in the characterization of CTL epitopes. A library of vaccinia viruses was created to express individual viral open reading frames, as well as the ALP andlacZtransgenes. The MHC haplotype had a dramatic impact on the distribution of CTL targets: in C57BL/6 mice, the hexon protein presented by both H-2Kband H2Dbwas dominant, and in C3H mice, H-2Dk-restricted presentation of ALP was dominant. Adoptive transfer of CTLs specific for various adenovirus proteins or transgene products into either Rag-I or C3H-scid mice infected previously with an E1-deleted adenovirus verified the in vivo relevance of the adenovirus-specific CTL targets identified in vitro. The results of these experiments illustrate the impact of lr gene control on the response to gene therapy with adenovirus vectors and suggest that the efficacy of therapy with adenovirus vectors may exhibit considerable heterogeneity when applied in human populations.
Ubiquitin Is Covalently Attached to the p6
Gag
Proteins of Human Immunodeficiency Virus Type 1 and Simian Immunodeficiency Virus and to the p12
Gag
Protein of Moloney Murine Leukemia Virus
Authors: Ott David E. , Coren Lori V. , Copeland Terry D. , Kane Bradley P. , Johnson Donald G. , Sowder Raymond C. , Yoshinaka Yoshiyuki , Oroszlan Stephen , Arthur Larry O. , Henderson Louis E.
Published: December 2019

Host proteins are incorporated into retroviral virions during assembly and budding. We have examined three retroviruses, human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency virus (SIV), and Moloney murine leukemia virus (Mo-MuLV), for the presence of ubiquitin inside each of these virions. After a protease treatment to remove exterior viral as well as contaminating cellular proteins, the proteins remaining inside the virion were analyzed. The results presented here show that all three virions incorporate ubiquitin molecules at approximately 10% of the level of Gag found in virions. In addition to free ubiquitin, covalent ubiquitin-Gag complexes were detected, isolated, and characterized from all three viruses. Our immunoblot and protein sequencing results on treated virions showed that approximately 2% of either HIV-1 or SIV p6
Gag
was covalently attached to a single ubiquitin molecule inside the respective virions and that approximately 2 to 5% of the p12
Gag
in Mo-MuLV virions was monoubiquitinated. These results show that ubiquitination of Gag is conserved among these retroviruses and occurs in the p6
Gag
portion of the Gag polyprotein, a region that is likely to be involved in assembly and budding.
Adenovirus Type 5 E4 Open Reading Frame 4 Protein Induces Apoptosis in Transformed Cells
Authors: Shtrichman Ronit , Kleinberger Tamar
Published: December 2019

Adenovirus type 5 E4 open reading frame 4 (E4orf4) protein has been previously shown to counteract transactivation of the
junB
and c-
fos
genes by cyclic AMP plus E1A protein and to interact with protein phosphatase 2A (PP2A). Here, we show that the wild-type E4orf4 protein induces apoptosis in the E1A-expressing 293 cells, in NIH 3T3 cells transformed with v-Ras, and in the lung carcinoma cell line H1299. The induction of apoptosis is not accompanied by enhanced levels of p53 in 293 cells and occurs in the absence of p53 in H1299 cells, indicating involvement of a p53-independent pathway. A mutant E4orf4 protein that had lost the ability to induce apoptosis also lost its ability to bind PP2A. We suggest that E4orf4 antagonizes continuous signals to proliferate, like those given by E1A or v-Ras, and that the conflicting signals lead to the induction of cell death.
Viral Coat Protein Peptides with Limited Sequence Homology Bind Similar Domains of Alfalfa Mosaic Virus and Tobacco Streak Virus RNAs
Authors: Swanson Maud M. , Ansel-McKinney Patricia , Houser-Scott Felicia , Yusibov Vidadi , Loesch-Fries L. Sue , Gehrke Lee
Published: December 2019

An unusual and distinguishing feature of alfalfa mosaic virus (AMV) and ilarviruses such as tobacco streak virus (TSV) is that the viral coat protein is required to activate the early stages of viral RNA replication, a phenomenon known as genome activation. AMV-TSV coat protein homology is limited; however, they are functionally interchangeable in activating virus replication. For example, TSV coat protein will activate AMV RNA replication and vice versa. Although AMV and TSV coat proteins have little obvious amino acid homology, we recently reported that they share an N-terminal RNA binding consensus sequence (Ansel-McKinney et al., EMBO J. 15:5077–5084, 1996). Here, we biochemically compare the binding of chemically synthesized peptides that include the consensus RNA binding sequence and lysine-rich (AMV) or arginine-rich (TSV) environment to 3′-terminal TSV and AMV RNA fragments. The arginine-rich TSV coat protein peptide binds viral RNA with lower affinity than the lysine-rich AMV coat protein peptides; however, the ribose moieties protected from hydroxyl radical attack by the two different peptides are localized in the same area of the predicted RNA structures. When included in an infectious inoculum, both AMV and TSV 3′-terminal RNA fragments inhibited AMV RNA replication, while variant RNAs unable to bind coat protein did not affect replication significantly. The data suggest that RNA binding and genome activation functions may reside in the consensus RNA binding sequence that is apparently unique to AMV and ilarvirus coat proteins.
Synergistic Neutralization of Simian-Human Immunodeficiency Virus SHIV-vpu+by Triple and Quadruple Combinations of Human Monoclonal Antibodies and High-Titer Anti-Human Immunodeficiency Virus Type 1 Immunoglobulins
Authors: Li An , Katinger Hermann , Posner Marshall R. , Cavacini Lisa , Zolla-Pazner Susan , Gorny Miroslaw K. , Sodroski Joseph , Chou Ting-Chao , Baba Timothy W. , Ruprecht Ruth M.
Published: December 2019

We have tested triple and quadruple combinations of human monoclonal antibodies (MAbs), which are directed against various epitopes on human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins, and a high-titer anti-HIV-1 human immunoglobulin (HIVIG) preparation for their abilities to neutralize a chimeric simian-human immunodeficiency virus (SHIV-vpu+). This virus encodes the HIV-1 strain IIIBenv,tat,rev, andvpugenes. The quantitative nature of the Chou-Talalay method (Adv. Enzyme Regul. 22:27–55, 1984) allows ranking of various combinations under identical experimental conditions. Of all triple combinations tested, the most potent neutralization was seen with MAbs 694/98D plus 2F5 plus 2G12 (directed against domains on V3, gp41, and gp120, respectively) as measured by the total MAb concentration required to reach 90% neutralization (90% effective concentration [EC90], 2.0 μg/ml). All triple combinations involving MAbs and/or HIVIG that were tested yielded synergy with combination index values of <1; the dose reduction indices (DRIs) ranged from 3.1 to 26.2 at 90% neutralization. When four MAbs (the previous three plus MAb F105, directed against the CD4 binding site) were combined, higher neutralization potency (EC90, 1.8 μg/ml) and a higher degree of synergy compared to any triple combination were seen. The mean DRIs of the quadruple combination were approximately twice that of the most synergistic triple combination. We conclude that human MAbs targeting different HIV-1 envelope glycoprotein epitopes exhibit strong synergy when used in combination, a fact that could be exploited clinically for passive immunoprophylaxis against HIV-1.
Persistence and Expression of the Herpes Simplex Virus Genome in the Absence of Immediate-Early Proteins
Authors: Samaniego Lorna A. , Neiderhiser Lisa , DeLuca Neal A.
Published: December 2019

The immediate-early (IE) proteins of herpes simplex virus (HSV) function on input genomes and affect many aspects of host cell metabolism to ensure the efficient expression and regulation of the remainder of the genome and, subsequently, the production of progeny virions. Due to the many and varied effects of IE proteins on host cell metabolism, their expression is not conducive to normal cell function and viability. This presents a major impediment to the use of HSV as a vector system. In this study, we describe a series of ICP4 mutants that are defective in different subsets of the remaining IE genes. One mutant,
d
109, does not express any of the IE proteins and carries a green fluorescent protein (GFP) transgene under the control of the human cytomegalovirus IE promoter (HCMVIEp).
d
109 was nontoxic to Vero and human embryonic lung (HEL) cells at all multiplicities of infection tested and was capable of establishing persistent infections in both of these cell types. Paradoxically, the genetic manipulations that were required to eliminate toxicity and allow the genome to persist in cells for long periods of time also dramatically lowered the level of transgene expression. Efficient expression of the HCMVIEp-GFP transgene in the absence of ICP4 was dependent on the ICP0 protein. In
d
109-infected cells, the level of transgene expression was very low in most cells but abundant in a small subpopulation of cells. However, expression of the transgene could be induced in cells containing quiescent
d
109 genomes weeks after the initial infection, demonstrating the functionality of the persisting genomes.
Role of the β-Chemokine Receptors CCR3 and CCR5 in Human Immunodeficiency Virus Type 1 Infection of Monocytes and Microglia
Authors: Ghorpade Anuja , Xia Meng Qi , Hyman Bradley T. , Persidsky Yuri , Nukuna Adeline , Bock Paul , Che Myhanh , Limoges Jenae , Gendelman Howard E. , Mackay Charles R.
Published: December 2019

Human immunodeficiency virus type 1 (HIV-1) infection in mononuclear phagocyte lineage cells (monocytes, macrophages, and microglia) is a critical component in the pathogenesis of viral infection. Viral replication in macrophages serves as a reservoir, a site of dissemination, and an instigator for neurological sequelae during HIV-1 disease. Recent studies demonstrated that chemokine receptors are necessary coreceptors for HIV-1 entry which determine viral tropism for different cell types. To investigate the relative contribution of the β-chemokine receptors CCR3 and CCR5 to viral infection of mononuclear phagocytes we utilized a panel of macrophage-tropic HIV-1 strains (from blood and brain tissue) to infect highly purified populations of monocytes and microglia. Antibodies to CD4 (OKT4A) abrogated HIV-1 infection. The β chemokines and antibodies to CCR3 failed to affect viral infection of both macrophage cell types. Antibodies to CCR5 (3A9) prevented monocyte infection but only slowed HIV replication in microglia. Thus, CCR5, not CCR3, is an essential receptor for HIV-1 infection of monocytes. Microglia express both CCR5 and CCR3, but antibodies to them fail to inhibit viral entry, suggesting the presence of other chemokine receptors for infection of these cells. These studies demonstrate the importance of mononuclear phagocyte heterogeneity in establishing HIV-1 infection and persistence.
Rotavirus 2/6 Viruslike Particles Administered Intranasally with Cholera Toxin,
Escherichia coli
Heat-Labile Toxin (LT), and LT-R192G Induce Protection from Rotavirus Challenge
Authors: O’Neal Christine M. , Clements John D. , Estes Mary K. , Conner Margaret E.
Published: December 2019

We have shown that rotavirus 2/6 viruslike particles composed of proteins VP2 and VP6 (2/6-VLPs) administered to mice intranasally with cholera toxin (CT) induced protection from rotavirus challenge, as measured by virus shedding. Since it is unclear if CT will be approved for human use, we evaluated the adjuvanticity of
Escherichia coli
heat-labile toxin (LT) and LT-R192G. Mice were inoculated intranasally with 10 μg of 2/6-VLPs combined with CT, LT, or LT-R192G. All three adjuvants induced equivalent geometric mean titers of rotavirus-specific serum antibody and intestinal immunoglobulin G (IgG). Mice inoculated with 2/6-VLPs with LT produced significantly higher titers of intestinal IgA than mice given CT as the adjuvant. All mice inoculated with 2/6-VLPs mixed with LT and LT-R192G were totally protected (100%) from rotavirus challenge, while mice inoculated with 2/6-VLPs mixed with CT showed a mean 91% protection from challenge. The availability of a safe, effective mucosal adjuvant such as LT-R192G will increase the practicality of administering recombinant vaccines mucosally.
Virological and Molecular Demonstration of Human Immunodeficiency Virus Type 2 Vertical Transmission
Authors: Cavaco-Silva Patricia , Taveira Nuno C. , Rosado Lino , Lourenço Maria H. , Moniz-Pereira José , Douglas Nigel W. , Daniels Rod S. , Santos-Ferreira Maria O.
Published: December 2019

To demonstrate that human immunodeficiency virus type 2 (HIV-2) mother-to-child transmission exists, HIV-2 isolates were obtained from both an asymptomatic mother (HIV-2 strain ARM), and her child (HIV-2 strain SAR), who had a diagnosis of AIDS. To determine their biological phenotype, primary isolates were used to infect various primary mononuclear cells and cell lines. HIV-2 ARM replicates in primary cells and Jurkat-
tat
, while HIV-2 SAR infects these cells plus SupT1, which led us to classify HIV-2 ARM as a slow/low virus and HIV-2 SAR as having an intermediate (slow/low-3) phenotype. Molecular analysis of the
env
region corresponding to gp125 was performed. Viral DNA was cloned, sequenced, and used to construct phylogenetic trees. The DNA sequence analysis demonstrated an overall nucleotide diversity of 7.6%. The results present evidence that the child’s strain is more virulent than the mother’s strain, which is in agreement with the immunodeficiency of the child. The phylogenetic trees that were constructed demonstrate that the two isolates cluster together, being closer to each other than to any other isolate described until now.
The Entire Nucleotide Sequence of Friend-Related and Paralysis-Inducing PVC-441 Murine Leukemia Virus (MuLV) and Its Comparison with Those of PVC-211 MuLV and Friend MuLV
Authors: Tanaka Atsushi , Oka Kiyomasa , Tanaka Keiji , Jinno Atsushi , Ruscetti Sandra K. , Kai Kazushige
Published: December 2019

PVC-441 murine leukemia virus (MuLV) is a member of the PVC group of Friend MuLV (F-MuLV)-derived neuropathogenic retroviruses. In order to determine the molecular basis for the difference in neuropathogenicity between PVC-441 and the previously characterized PVC-211 MuLVs, the entire nucleotide sequence of PVC-441 MuLV was determined and compared with those of PVC-211 and F-MuLV. The results suggest that PVC-441 and PVC-211 MuLVs were formed as a result of random mutations of F-MuLV and developed differently. The distinct pathogenicities of PVC-441 and PVC-211 MuLVs were maintained in the viruses regenerated from their molecular clones, and the sequences responsible for the pathological differences observed can be localized to the
env
gene. The amino acid sequence of PVC-441 deduced from its nucleotide sequence revealed a number of differences from PVC-211, the most striking of which was a difference at position 129 of the SU proteins in the two viruses. Host range studies with a brain capillary endothelial cell line (RTEC-6) and Chinese hamster ovary cells (CHO-K1) revealed that PVC-441, like PVC-211, could infect these cells but its efficiency of infection was lower than that of PVC-211. These results may account for the difference in neuropathogenicity between PVC-441 and PVC-211.
The U3 Promoter and the
nef
Gene of Simian Immunodeficiency Virus (SIV) smmPBj1.9 Do Not Confer Acute Pathogenicity upon SIVagm
Authors: Wagener Stefan , Dittmar Matthias T. , Beer Brigitte , König Renate , Plesker Roland , Norley Stephen , Kurth Reinhard , Cichutek Klaus
Published: December 2019

Two chimeric proviruses comprising the U3 promoter and the
nef
gene of simian immunodeficiency virus (SIV) smmPBj1.9 in addition to other genomic regions of SIVagm3mc from African green monkeys (
Cercopithecus aethiops
) were constructed. The derived chimeric viruses (SIVagm3mc/SIVsmmPBj1.9) were both able to replicate in nonstimulated peripheral blood leukocytes from pig-tailed macaques (
Macaca nemestrina
), a biological property often correlated with acute pathogenicity. However, only one of the chimeric viruses was acutely pathogenic, inducing a rapid depletion of the peripheral CD4
+
T cells in two infected pig-tailed macaques within 10 days after infection in a manner similar to infection with SIVsmmPBj1.9 itself. The other chimeric virus actively replicated during the first 8 weeks after experimental infection of two pig-tailed macaques but induced neither acute disease nor CD4
+
T-cell depletion for 113 weeks after infection. Thus, the U3 promoter and the
nef
gene of SIVsmmPBj1.9 alone appear to be insufficient to confer acute pathogenicity to SIVagm3mc.
Cleavage of Human Immunodeficiency Virus Type 1 Proteinase from the N-Terminally Adjacent p6* Protein Is Essential for Efficient Gag Polyprotein Processing and Viral Infectivity
Authors: Tessmer Uwe , Kräusslich Hans-Georg
Published: December 2019

Maturation of infectious human immunodeficiency virus (HIV) particles requires proteolytic cleavage of the structural polyproteins by the viral proteinase (PR), which is itself encoded as part of the Gag-Pol polyprotein. Expression of truncated PR-containing sequences in heterologous systems has mostly led to the autocatalytic release of an 11-kDa species of PR which is capable of processing all known cleavage sites on the viral precursor proteins. Relatively little is known about cleavages within the nascent virus particle, on the other hand, and controversial results concerning the active PR species inside the virion and the relative activities of extended PR species have been reported. Here, we report that HIV type 1 (HIV-1) particles of four different strains obtained from different cell lines contain an 11-kDa PR, with no extended PR proteins detectable. Furthermore, mutation of the N-terminal PR cleavage site leading to production of an N-terminally extended 17-kDa PR species caused a severe defect in Gag polyprotein processing and a complete loss of viral infectivity. We conclude that N-terminal release of PR from the HIV-1 polyprotein is essential for viral replication and suggest that extended versions of PR may have a transient function in the proteolytic cascade.
Pneumococcal Bacteriophage Cp-1 Encodes Its Own Protease Essential for Phage Maturation
Authors: Martín Ana C. , López Rubens , García Pedro
Published: December 2019

The major capsid protein of the pneumococcal phage Cp-1 that accounts for 90% of the total protein found in the purified virions is synthesized by posttranslational processing of the product of the open reading frame (ORF)
orf9
. Cloning of different ORFs of the Cp-1 genome in
Escherichia coli
and
Streptococcus pneumoniae
combined with Western blot analysis of the expressed products led to the conclusion that the product of
orf13
is an endoprotease that cleaves off the first 48 amino acid residues of the major head protein. This protease appears to be a key enzyme in the morphopoietic pathway of the Cp-1 phage head. To our knowledge, this is the first case of a bacteriophage infecting gram-positive bacteria that encodes a protease involved in phage maturation.
The TAATGARAT Motif in the Herpes Simplex Virus Immediate-Early Gene Promoters Can Confer both Positive and Negative Responses to Cellular Octamer-Binding Proteins When It Is Located within the Viral Genome
Authors: Thomas Suzanne , Coffin Robert S. , Watts Paul , Gough Gerald , Latchman David S.
Published: December 2019

The TAATGARAT motif in the herpes simplex virus (HSV) immediate-early (IE) gene promoters plays a key role in their activation by the Oct-1–Vmw65 complex, but its role in mediating inhibitory effects of cellular octamer-binding proteins is less clear. We have used indicator viruses containing reporter constructs with different IE promoters driving a reporter β-galactosidase gene within the viral genome to investigate this. We showed that deletion of the upstream IE promoter region containing the TAATGARAT motifs abolishes the inhibitory effect of the cellular octamer-binding proteins Oct-2.4 and Oct-2.5 on the viral IE promoter. This inhibitory effect can be restored by addition of a single TAATGARAT motif to the minimal promoter within the viral genome. Hence, the TAATGARAT motif can indeed mediate both positive and negative effects of cellular transcription factors when it is located within the viral genome.
Human Immunodeficiency Virus Type 1 Subtype F Reverse Transcriptase Sequence and Drug Susceptibility
Authors: Apetrei Cristian , Descamps Diane , Collin Gilles , Loussert-Ajaka Ibtisam , Damond Florence , Duca Mihai , Simon François , Brun-Vézinet Françoise
Published: December 2019

We sequenced and phylogenetically analyzed the reverse transcriptase (RT) regions of the
pol
genes of 14 human immunodeficiency virus type 1 (HIV-1) isolates from Romanian patients, which were classified as subtype F on the basis of
env
gene structure. The RT sequences showed that the strains clustered phylogenetically and were equidistant from other HIV-1 subtypes as shown by the neighbor-joining and maximum-likelihood methods, allowing us to define HIV-1 subtype F according to the
pol
classification. The subtype F RT sequences differed from reported group M RT sequences by 10.94% (for nucleotides) and 7.6% (for amino acids). Phenotypic analysis of subtype F susceptibility to three classes of antiretroviral compounds showed an increase in the 50% inhibitory concentration of the tetrahydroimidazo[4,5,1-
jk
][1,4]-benzodiazepin-2-(1
H
)-one and -thione (TIBO) derivate R82913 for one strain which was naturally resistant to this compound. This first report of subtype F
pol
sequences confirms the perfect correlation between the phylogenetic positions determined by
env
and
pol
analyses and suggests that virus variability might influence the efficacy of antiretroviral treatments. This finding warrants a global evaluation of the phenotypic and genotypic susceptibility of HIV-1 subtypes to antiretroviral drugs.
Cross-Reactions between the Cytotoxic T-Lymphocyte Responses of Human Immunodeficiency Virus-Infected African and European Patients
Authors: Durali Deniz , Morvan Jacques , Letourneur Franck , Schmitt Doris , Guegan Nelly , Dalod Marc , Saragosti Sentob , Sicard Didier , Levy Jean-Paul , Gomard Elisabeth
Published: December 2019

The great variability of protein sequences from human immunodeficiency virus (HIV) type 1 (HIV-1) isolates represents a major obstacle to the development of an effective vaccine against this virus. The surface protein (Env), which is the predominant target of neutralizing antibodies, is particularly variable. Here we examine the impact of variability among different HIV-1 subtypes (clades) on cytotoxic T-lymphocyte (CTL) activities, the other major component of the antiviral immune response. CTLs are produced not only against Env but also against other structural proteins, as well as some regulatory proteins. The genetic subtypes of HIV-1 were determined for Env and Gag from several patients infected either in France or in Africa. The cross-reactivities of the CTLs were tested with target cells expressing selected proteins from HIV-1 isolates of clade A or B or from HIV type 2 isolates. All African patients were infected with viruses belonging to clade A for Env and for Gag, except for one patient who was infected with a clade A Env-clade G Gag recombinant virus. All patients infected in France were infected with clade B viruses. The CTL responses obtained from all the African and all the French individuals tested showed frequent cross-reactions with proteins of the heterologous clade. Epitopes conserved between the viruses of clades A and B appeared especially frequent in Gag p24, Gag p18, integrase, and the central region of Nef. Cross-reactivity also existed among Gag epitopes of clades A, B, and G, as shown by the results for the patient infected with the clade A Env-clade G Gag recombinant virus. These results show that CTLs raised against viral antigens from different clades are able to cross-react, emphasizing the possibility of obtaining cross-immunizations for this part of the immune response in vaccinated individuals.
Identification of Regions of Poliovirus 2BC Protein That Are Involved in Cytotoxicity
Authors: Barco Angel , Carrasco Luis
Published: December 2019

The expression of poliovirus 2BC protein in yeast and mammalian cells leads to a number of metabolic and morphological alterations, such as growth inhibition, intracellular membrane proliferation, blockade of the exocytic pathway, and enhanced membrane permeability. Yeast cells that express poliovirus 2BC in an inducible manner were used to identify the regions of 2BC implicated in the modifications of these cellular functions. Several 2BC deletion mutants were generated to define the minimal portion of 2BC required to alter these activities. Additional deletion mutants that were obtained by random mutagenesis followed by selection in yeast cells provided new insights into the structure and mechanism of action of 2BC. The activity responsible for membrane proliferation is located in 2C, while the activities responsible for membrane permeabilization and inhibition of the exocytic pathway are located in 2B. Several regions of 2B and 2C required for the different functions of 2BC were identified. Thus, the integrity of the N termini of both 2B and 2C is necessary for 2BC-induced cytotoxicity. It is also possible to separate the different cellular alterations provoked by 2BC by the use of several 2BC variants. Deletion of amino acids 52 to 65 in 2B generates a 2BC deletion variant, 2bCΔAvrII, that still blocks yeast growth but is unable to enhance membrane permeability or to inhibit the exocytic pathway. On the other hand, 2Bc128*.32b and 2Bc128*.3c, which contain only 73 and 77 amino acids of 2B, interfere with yeast division and enhance membrane permeability but affect the exocytic pathway only weakly and do not induce membrane proliferation. Our findings indicate that
Saccharomyces cerevisiae
represents a useful model system to analyze the functions of poliovirus 2BC and show the feasibility of separating the activities assigned to this protein.
Human Immunodeficiency Virus Type 1 Attachment to HeLa CD4 Cells Is CD4 Independent and gp120 Dependent and Requires Cell Surface Heparans
Authors: Mondor Isabelle , Ugolini Sophie , Sattentau Quentin J.
Published: December 2019

The binding of human immunodeficiency virus type 1 (HIV-1) (Hx10) virions to two different cell lines was analyzed by using a novel assay based on the detection, by anti-HLA-DR-specific antibodies, of HLA-DR+virus binding to HLA-DR−cells. Virion attachment to the CD4+-T-cell line A3.01 was highly CD4 dependent in that it was potently inhibited by CD4 monoclonal antibodies (MAbs), and little virus binding to the CD4−sister A2.01 line was observed. By contrast, virion binding to HeLa cells expressing moderate or high levels of CD4 was equivalent to, or lower than, binding to wild-type CD4−HeLa cells. Moreover, several CD4 MAbs did not reduce, but enhanced, HIV-1 attachment to HeLa-CD4 cells. CD4 was required for infection of HeLa cells, however, demonstrating a postattachment role for this receptor. MAbs specific for the V2 and V3 loops and the CD4i epitope of gp120 strongly inhibited virion binding to HeLa-CD4 cells, whereas MAbs specific for the CD4bs and the 2G12 epitopes enhanced attachment. Despite this, all gp120- and gp41-specific MAbs tested neutralized infectivity on HeLa-CD4 cells. HIV-1 attachment to HeLa cells was only partially inhibited by MAbs specific for adhesion molecules present on the virus or target cells but was completely blocked by polyanions such as heparin, dextran sulfate, and pentosan sulfate. Treatment of HeLa-CD4 cells with heparinases completely eliminated HIV attachment and infection, strongly implicating cell surface heparans in the attachment process. CD4 dependence for HIV-1 attachment to target cells is thus highly cell line specific and may be replaced by other ligand-receptor interactions.
Hepatitis Delta Antigen Mediates the Nuclear Import of Hepatitis Delta Virus RNA
Authors: Chou Huei-Chi , Hsieh Tsai-Yuan , Sheu Gwo-Tarng , Lai Michael M. C.
Published: December 2019

Hepatitis delta virus (HDV) RNA replicates in the nuclei of virus-infected cells. The mechanism of nuclear import of HDV RNA is so far unknown. Using a fluorescein-labeled HDV RNA introduced into partially permeabilized HeLa cells, we found that HDV RNA accumulated only in the cytoplasm. However, in the presence of hepatitis delta antigen (HDAg), which is the only protein encoded by HDV RNA, the HDV RNA was translocated into the nucleus, suggesting that nuclear import of HDV RNA is mediated by HDAg. Deletion of the nuclear localization signal (NLS) or RNA-binding motifs of HDAg resulted in the failure of nuclear import of HDV RNA, indicating that both the NLS and an RNA-binding motif of HDAg are required for the RNA-transporting activity of HDAg. Surprisingly, any one of the three previously identified RNA-binding motifs was sufficient to confer the RNA-transporting activity. We have further shown that HDAg, via its NLS, interacts with karyopherin α2 in vitro, suggesting that nuclear import of the HDAg-HDV RNA complex is mediated by the karyopherin α2β heterodimer. The nuclear import of HDV RNA may be the first biological function of HDAg in the HDV life cycle.
Two RING Finger Proteins, the Oncoprotein PML and the Arenavirus Z Protein, Colocalize with the Nuclear Fraction of the Ribosomal P Proteins
Authors: Borden Katherine L. B. , CampbellDwyer Elizabeth J. , Carlile Graeme W. , Djavani Mahmoud , Salvato Maria S.
Published: December 2019

The promyelocytic leukemia (PML) protein forms nuclear bodies which are relocated to the cytoplasm by the RNA virus lymphocytic choriomeningitis virus (LCMV). The viral Z protein directly binds to PML and can relocate the nuclear bodies. Others have observed that LCMV virions may contain ribosomes; hence, we investigated the effects of infection on the distribution of ribosomal P proteins (P0, P1, and P2) with PML as a reference point. We demonstrate an association of PML bodies with P proteins by indirect immunofluorescence and coimmunoprecipitation experiments, providing the first evidence of nucleic acid-binding proteins associated with PML bodies. We show that unlike PML, the P proteins are not redistributed upon infection. Immunofluorescence and coimmunoprecipitation studies indicate that the viral Z protein binds the nuclear, but not the cytoplasmic, fraction of P0. The nuclear fraction of P0 has been associated with translationally coupled DNA excision repair and with nonspecific endonuclease activity; thus, P0 may be involved in nucleic acid processing activities necessary for LCMV replication. During the infection process, PML, P1, and P2 are downregulated but P0 remains unchanged. Further, P0 is present in virions while PML is not, indicating some selectivity in the assembly of LCMV.
Expression Kinetics and Mapping of the Thymidine Kinase Transcript and an Immediate-Early Transcript from Channel Catfish Virus
Authors: Silverstein Peter S. , van Santen Vicky L. , Nusbaum Kenneth E. , Bird R. Curtis
Published: December 2019

Three transcripts from the terminal repeat of the channel catfish virus (CCV; also known as ictalurid herpesvirus 1) genome were mapped by S1 nuclease and primer extension analyses as well as by cDNA sequencing. These transcripts,
TR3
,
TR5/6
, and
TR6
, are encoded by open reading frame (ORF) 3, ORFs 5 and 6, and ORF 6, respectively, and correspond to those previously identified by sequence analysis (A. J. Davison, Virology 186:9–14, 1992). ORF 5 has previously been determined to encode thymidine kinase, but ORF 3 and ORF 6 encode proteins of unknown function. Although all three transcripts accumulate to high levels in cells infected in the presence of cycloheximide, kinetic analysis demonstrates that
TR5/6
and
TR6
are either early or late transcripts that leak through the cycloheximide block. In addition, two transcripts from the terminal repeat of the CCV genome that were mapped previously and were thought to be immediate-early in character,
TR8a/9
and
TR9
, exhibit kinetics characteristic of early or late transcripts.
TR3
is an immediate-early transcript that appears to have a very short half-life. In the 3′ untranslated region of
TR3
, there are three copies of an AU-rich element which has previously been shown to be involved in destabilization of the oncogene c-
fos
and granulocyte/macrophage colony-stimulating factor mRNAs. mRNA destabilization may represent another mechanism by which herpesviruses regulate the rapid switch in expression from immediate-early genes to early genes during the transition to the early phase of infection.