bluuebunny/crossref_metadata_2025 huggingface dataset:
Two Widely Spaced Initiator Binding Sites Create an HMG1-Dependent Parvovirus Rolling-Hairpin Replication Origin
Authors: Cotmore Susan F. , Christensen Jesper , Tattersall Peter
Published: July 2002

Minute virus of mice (MVM) replicates via a linearized form of rolling-circle replication in which the viral nickase, NS1, initiates DNA synthesis by introducing a site-specific nick into either of two distinct origin sequences. In vitro nicking and replication assays with substrates that had deletions or mutations were used to explore the sequences and structural elements essential for activity of one of these origins, located in the right-end (5′) viral telomere. This structure contains 248 nucleotides, most-favorably arranged as a simple hairpin with six unpaired bases. However, a pair of opposing NS1 binding sites, located near its outboard end, create a 33-bp palindrome that could potentially assume an alternate cruciform configuration and hence directly bind HMG1, the essential cofactor for this origin. The palindromic nature of this sequence, and thus its ability to fold into a cruciform, was dispensable for origin function, as was the NS1 binding site occupying the inboard arm of the palindrome. In contrast, the NS1 site in the outboard arm was essential for initiation, even though positioned 120 bp from the nick site. The specific sequence of the nick site and an additional NS1 binding site which directly orients NS1 over the initiation site were also essential and delimited the inboard border of the minimal right-end origin. DNase I and hydroxyl radical footprints defined sequences protected by NS1 and suggest that HMG1 allows the NS1 molecules positioned at each end of the origin to interact, creating a distortion characteristic of a double helical loop.
Productive Replication of Human Adenoviruses in Mouse Epidermal Cells
Authors: Ganly Ian , Mautner Vivien , Balmain Allan
Published: July 2002

In contrast to most cells of mouse origin, cell lines derived from mouse epidermis are permissive for replication of human adenovirus type 5. The extent of epidermal cell differentiation correlated with the level of E1A expression and virus replication. Mouse epidermal cells may provide useful models for cancer therapy using replication-competent human adenoviruses.
Characterization of the R572T Point Mutant of a Putative Cleavage Site in Human Foamy Virus Env
Authors: Bansal Anju , Shaw Kit L. , Edwards Bradley H. , Goepfert Paul A. , Mulligan Mark J.
Published: July 2002

A putative cleavage site of the human foamy virus (HFV) envelope glycoprotein (Env) was altered. Transientenvexpression revealed that the R572T mutant Env was normally expressed and modified by asparagine-linked oligosaccharide chains. However, this single-amino-acid substitution was sufficient to abolish all detectable cleavage of the gp130 precursor polyprotein. Cell surface biotinylation demonstrated that the uncleaved mutant gp130 was transported to the plasma membrane. The uncleaved mutant protein was incapable of syncytium formation. Glycoprotein-driven virion budding, a unique aspect of HFV assembly, occurred despite the absence of Env cleavage. We then substituted the R572T mutantenvinto a replication-competent HFV molecular clone. Transfection of the mutant viral DNA into BHK-21 cells followed by viral titration with the FAB (foamy virus-activated β-galactosidase expression) assay revealed that proteolysis of the HFV Env was essential for viral infectivity. Wild-type HFV Env partially complemented the defective virus phenotype. Taken together, these experimental results established the location of the HFV Env proteolytic site; the effects of cleavage on Env transport, processing, and function; and the importance of Env proteolysis for virus maturation and infectivity.
Assembly and Processing of Human Immunodeficiency Virus Gag Mutants Containing a Partial Replacement of the Matrix Domain by the Viral Protease Domain
Authors: Wang Chin-Tien , Chou Yen-Chiou , Chiang Chien-Cheng
Published: July 2002

We constructed human immunodeficiency virus (HIV) mutants by replacing the matrix domain with sequences encoding the viral protease or p6* and protease. The chimeras retaining matrix myristylation and processing signals underwent efficient autoprocessing with severely defective particle budding. The budding defects of the chimeras were rescued by suppressing the chimera protease activity either through addition of an HIV protease inhibitor or through inactivating the chimera protease via a substitution mutation of the catalytic aspartic acid residue. This resulted in the release of chimeric virus-like particles with the density of a wild-type retrovirus particle. In addition, the assembly-competent but processing-defective chimeras produced proteolytically processed particles with significant reverse transcriptase activity when a downstream native
pol
gene was present. These results suggest that HIV has the potential to adapt heterologous sequences in place of the matrix sequence without major effects on virus-like particle budding. In addition, the positions of the protease and substrate accessibility may contribute significantly toward avoiding a premature Gag or Gag-Pol process, which leads to severe defects in both particle budding and incorporation.
Mutations in the 2C Region of Poliovirus Responsible for Altered Sensitivity to Benzimidazole Derivatives
Authors: Shimizu Hiroyuki , Agoh Masanobu , Agoh Yumi , Yoshida Hiromu , Yoshii Kumiko , Yoneyama Tetsuo , Hagiwara Akio , Miyamura Tatsuo
Published: July 2002

MRL-1237, [1-(4-fluorophenyl)-2-(4-imino-1,4-dihydropyridin-1-yl) methylbenzimidazole hydrochloride], is a potent and selective inhibitor of the replication of enteroviruses. To reveal the target molecule of MRL-1237 in viral replication, we selected spontaneous MRL-1237-resistant poliovirus mutants. Of 15 MRL-1237-resistant mutants obtained, 14 were cross-resistant to guanidine hydrochloride (
mrgr
), while 1 was susceptible (
mrgs
). Sequence analysis of the 2C region revealed that the 14
mrgr
mutants contained a single nucleotide substitution that altered an amino acid residue from Phe-164 to Tyr. The
mrgs
mutant, on the other hand, contained a substitution of Ile-120 to Val. Through the construction of a cDNA-derived mutant, we confirmed that the single mutation at Phe-164 was really responsible for the reduced susceptibility to MRL-1237. MRL-1237 inhibited poliovirus-specific RNA synthesis in HeLa cells infected with a wild strain but not with an F164Y mutant. We furthermore examined the effect of mutations of the 2C region on the drug sensitivity of cDNA-derived guanidine-resistant and -dependent mutants. Two guanidine-resistant mutants were cross-resistant to MRL-1237 but remained susceptible to another benzimidazole, enviroxime. Either MRL-1237 or guanidine stimulated the viral replication of two guanidine-dependent mutants, but enviroxime did not. These results indicate that MRL-1237, like guanidine, targets the 2C protein of poliovirus for its antiviral effect.
Differences in Affinity of Binding of Lymphocytic Choriomeningitis Virus Strains to the Cellular Receptor α-Dystroglycan Correlate with Viral Tropism and Disease Kinetics
Authors: Smelt Sara C. , Borrow Persephone , Kunz Stefan , Cao Wei , Tishon Antoinette , Lewicki Hanna , Campbell Kevin P. , Oldstone Michael B. A.
Published: July 2002

α-Dystroglycan (α-DG) was recently identified as a receptor for lymphocytic choriomeningitis virus (LCMV) and several other arenaviruses, including Lassa fever virus (W. Cao, M. D. Henry, P. Borrow, H. Yamada, J. H. Elder, E. V. Ravkov, S. T. Nichol, R. W. Compans, K. P. Campbell, and M. B. A. Oldstone, Science 282:2079–2081, 1998). Data presented in this paper indicate that the affinity of binding of LCMV to α-DG determines viral tropism and the outcome of infection in mice. To characterize this relationship, we evaluated the interaction between α-DG and several LCMV strains, variants, and reassortants. These viruses could be divided into two groups with respect to affinity of binding to α-DG, dependence on this protein for cell entry, viral tropism, and disease course. Viruses that exhibited high-affinity binding to α-DG displayed a marked dependence on α-DG for cell entry and were blocked from infecting mouse 3T6 fibroblasts by 1 to 4 nM soluble α-DG. In addition, high-affinity binding to α-DG correlated with an ability to infiltrate the white pulp (T-dependent) area of the spleen, cause ablation of the cytotoxic T-lymphocyte (CTL) response by day 7 postinfection, and establish a persistent infection. In contrast, viruses with a lower affinity of binding to α-DG were only partially inhibited from infecting α-DG−/−embryonic stem cells and required a concentration of soluble α-DG higher than 100 nM to prevent infection of mouse 3T6 fibroblasts. These viruses that bound at low affinity were mainly restricted to the splenic red pulp, and the host generated an effective CTL response that rapidly cleared the infection. Reassortants of viruses that bound to α-DG at high and low affinities were used to map genes responsible for the differences described to the S RNA, containing the virus attachment protein glycoprotein 1.
Identification and Characterization of a Peptide That Specifically Binds the Human, Broadly Neutralizing Anti-Human Immunodeficiency Virus Type 1 Antibody b12
Authors: Zwick Michael B. , Bonnycastle Lori L. C. , Menendez Alfredo , Irving Melita B. , Barbas Carlos F. , Parren Paul W. H. I. , Burton Dennis R. , Scott Jamie K.
Published: July 2002

Human monoclonal antibody (MAb) b12 recognizes a conformational epitope that overlaps the CD-4-binding site of the human immunodeficiency virus type 1 (HIV-1) envelope. MAb b12 neutralizes a broad range of HIV-1 primary isolates and protects against primary virus challenge in animal models. We report here the discovery and characterization of B2.1, a peptide that binds specifically to MAb b12. B2.1 was selected from a phage-displayed peptide library by using immunoglobulin G1 b12 as the selecting agent. The peptide is a homodimer whose activity depends on an intact disulfide bridge joining its polypeptide chains. Competition studies with gp120 indicate that B2.1 occupies the b12 antigen-binding site. The affinity of b12 for B2.1 depends on the form in which the peptide is presented; b12 binds best to the homodimer as a recombinant polypeptide fused to the phage coat. Originally, b12 was isolated from a phage-displayed Fab library constructed from the bone marrow of an HIV-1-infected donor. The B2.1 peptide is highly specific for b12 since it selected only phage bearing b12 Fab from this large and diverse antibody library.
African Swine Fever Virus Structural Protein pE120R Is Essential for Virus Transport from Assembly Sites to Plasma Membrane but Not for Infectivity
Authors: Andrés Germán , Garcı́a-Escudero Ramón , Viñuela Eladio , Salas Marı́a L. , Rodrı́guez Javier M.
Published: July 2002

This report examines the role of African swine fever virus (ASFV) structural protein pE120R in virus replication. Immunoelectron microscopy revealed that protein pE120R localizes at the surface of the intracellular virions. Consistent with this, coimmunoprecipitation assays showed that protein pE120R binds to the major capsid protein p72. Moreover, it was found that, in cells infected with an ASFV recombinant that inducibly expresses protein p72, the incorporation of pE120R into the virus particle is dependent on p72 expression. Protein pE120R was also studied using an ASFV recombinant in which E120R gene expression is regulated by the
Escherichia coli lac
repressor-operator system. In the absence of inducer, pE120R expression was reduced about 100-fold compared to that obtained with the parental virus or the recombinant virus grown under permissive conditions. One-step virus growth curves showed that, under conditions that repress pE120R expression, the titer of intracellular progeny was similar to the total virus yield obtained under permissive conditions, whereas the extracellular virus yield was about 100-fold lower than in control infections. Immunofluorescence and electron microscopy demonstrated that, under restrictive conditions, intracellular mature virions are properly assembled but remain confined to the replication areas. Altogether, these results indicate that pE120R is necessary for virus dissemination but not for virus infectivity. The data also suggest that protein pE120R might be involved in the microtubule-mediated transport of ASFV particles from the viral factories to the plasma membrane.
The Number of a U3 Repeat Box Acting as an Enhancer in Long Terminal Repeats of Polytropic Replication-Competent Porcine Endogenous Retroviruses Dynamically Fluctuates during Serial Virus Passages in Human Cells
Authors: Scheef Gregor , Fischer Nicole , Krach Ulrich , Tönjes Ralf R.
Published: July 2002

The organization and transcriptional regulation of porcine endogenous retrovirus (PERV) long terminal repeats (LTRs) are unknown. We have studied the activity of LTRs from replication-competent molecular clones by performing luciferase reporter assays. The LTRs differ in the presence and number of 39-bp repeats located in U3 that confer strong promoter activity in human, simian, canine, feline, and porcine cell lines, whereas for LTRs devoid of the repeats, the promoter strength was significantly reduced. As the activity of a heterologous simian virus 40 promoter and a homologous repeat-deficient LTR was elevated by four 39-bp repeats independently of its orientation and location, the repeat box complies with the definition of an enhancer. During serial virus passaging of molecular PERV clones on human 293 cells, proviral LTRs demonstrated adaptation of transcriptional activity by dynamic changes of the number of 39-bp repeats in the course of up to 12 passaging cycles.
Wild
Mandrillus sphinx
Are Carriers of Two Types of Lentivirus
Authors: Souquière Sandrine , Bibollet-Ruche Frédéric , Robertson David L. , Makuwa Maria , Apetrei Cristian , Onanga Richard , Kornfeld Christopher , Plantier Jean-Christophe , Gao Feng , Abernethy Katharine , White Lee J. T. , Karesh William , Telfer Paul , Wickings E. Jean , Mauclère Philippe , Marx Preston A. , Barré-Sinoussi Françoise , Hahn Beatrice H. , Müller-Trutwin Michaela C. , Simon François
Published: July 2002

Mandrillus sphinx
, a large primate living in Cameroon and Gabon and belonging to the Papionini tribe, was reported to be infected by a simian immunodeficiency virus (SIV) (SIVmndGB1) as early as 1988. Here, we have identified a second, highly divergent SIVmnd (designated SIVmnd-2). Genomic organization differs between the two viral types; SIVmnd-2 has the additional
vpx
gene, like other SIVs naturally infecting the Papionini tribe (SIVsm and SIVrcm) and in contrast to the other SIVmnd type (here designated SIVmnd-1), which is more closely related to SIVs infecting l'hoest (
Cercopithecus lhoesti lhoesti
) and sun-tailed (
Cercopithecus lhoesti solatus
) monkeys. Importantly, our epidemiological studies indicate a high prevalence of both types of SIVmnd; all 10 sexually mature wild-living monkeys and 3 out of 17 wild-born juveniles tested were infected. The geographic distribution of SIVmnd seems to be distinct for the two types: SIVmnd-1 viruses were exclusively identified in mandrills from central and southern Gabon, whereas SIVmnd-2 viruses were identified in monkeys from northern and western Gabon, as well as in Cameroon. SIVmnd-2 full-length sequence analysis, together with analysis of partial sequences from SIVmnd-1 and SIVmnd-2 from wild-born or wild-living mandrills, shows that the
gag
and
pol
regions of SIVmnd-2 are closest to those of SIVrcm, isolated from red-capped mangabeys (
Cercocebus torquatus
), while the
env
gene is closest to that of SIVmnd-1.
pol
and
env
sequence analyses of SIV from a related Papionini species, the drill (
Mandrillus leucophaeus
), shows a closer relationship of SIVdrl to SIVmnd-2 than to SIVmnd-1. Epidemiological surveys of human immunodeficiency virus revealed a case in Cameroon of a human infected by a virus serologically related to SIVmnd, raising the possibility that mandrills represent a viral reservoir for humans similar to sooty mangabeys in Western Africa and chimpanzees in Central Africa.
Functional Analysis of Adenovirus Protein IX Identifies Domains Involved in Capsid Stability, Transcriptional Activity, and Nuclear Reorganization
Authors: Rosa-Calatrava Manuel , Grave Linda , Puvion-Dutilleul Francine , Chatton Bruno , Kedinger Claude
Published: July 2002

The product of adenovirus (Ad) type 5 gene IX (pIX) is known to actively participate in the stability of the viral icosahedron, acting as a capsid cement. We have previously demonstrated that pIX is also a transcriptional activator of several viral and cellular TATA-containing promoters, likely contributing to the transactivation of the Ad expression program. By extensive mutagenesis, we have now delineated the functional domains involved in each of the pIX properties: residues 22 to 26 of the highly conserved N-terminal domain are crucial for incorporation of the protein into the virion; specific residues of the C-terminal leucine repeat are responsible for pIX interactions with itself and possibly other proteins, a property that is critical for pIX transcriptional activity. We also show that pIX takes part in the virus-induced nuclear reorganization of late infected cells: the protein induces, most likely through self-assembly, the formation of specific nuclear structures which appear as dispersed nuclear globules by immunofluorescence staining and as clear amorphous spherical inclusions by electron microscopy. The integrity of the leucine repeat appears to be essential for the formation and nuclear retention of these inclusions. Together, our results demonstrate the multifunctional nature of pIX and provide new insights into Ad biology.
Multimerization of Human Immunodeficiency Virus Type 1 Gag Promotes Its Localization to Barges, Raft-Like Membrane Microdomains
Authors: Lindwasser O. Wolf , Resh Marilyn D.
Published: July 2002

The Gag polyprotein of human immunodeficiency virus type 1 (HIV-1) organizes the assembly of nascent virions at the plasma membrane of infected cells. Here we demonstrate that a population of Gag is present in distinct raft-like membrane microdomains that we have termed “barges.” Barges have a higher density than standard rafts, most likely due to the presence of oligomeric Gag-Gag assembly complexes. The regions of the Gag protein responsible for barge targeting were mapped by examining the flotation behavior of wild-type and mutant proteins on Optiprep density gradients. N-myristoylation of Gag was necessary for association with barges. Removal of the NC and p6 domains shifted much of the Gag from barges into typical raft fractions. These data are consistent with a model in which multimerization of myristoylated Gag proteins drives association of Gag oligomers into raft-like barges. The functional significance of barge association was revealed by several lines of evidence. First, Gag isolated from virus-like particles was almost entirely localized in barges. Moreover, a comparison of wild-type Gag with Fyn(10)Gag, a chimeric protein containing the N-terminal sequence of Fyn, revealed that Fyn(10)Gag exhibited increased affinity for barges and a two- to fourfold increase in particle production. These results imply that association of Gag with raft-like barge membrane microdomains plays an important role in the HIV-1 assembly process.
Nuclear Export of Human Immunodeficiency Virus Type 1 Vpr Is Not Required for Virion Packaging
Authors: Jenkins Yonchu , Sanchez Patricia V. , Meyer Barbara E. , Malim Michael H.
Published: July 2002

The human immunodeficiency virus type 1 Vpr protein is both packaged into virions and efficiently localized to the nucleus. In this report, we show that a significant fraction of Vpr also accumulates in the cytoplasm of virus-producing cells. Although Vpr shuttles between the nucleus and the cytoplasm, studies with an export-deficient Vpr mutant reveal that nuclear export is not required for virion incorporation.
Antiretroviral Agents Restore Mycobacterium-Specific T-Cell Immune Responses and Facilitate Controlling a Fatal Tuberculosis-Like Disease in Macaques Coinfected with Simian Immunodeficiency Virus andMycobacterium bovisBCG
Authors: Shen Yun , Shen Ling , Sehgal Prabhat , Zhou Dejiang , Simon Meredith , Miller Michael , Enimi Emilio A. , Henckler Bill , Chalifoux Laura , Sehgal Nitu , Gastron Michael , Letvin Norman L. , Chen Zheng W.
Published: July 2002

The contribution of immune reconstitution following antiretroviral treatment to the prevention or treatment of human immunodeficiency virus-related primary or reactivation tuberculosis remains unknown. Macaque models of simian immunodeficiency virus-Mycobacterium bovisBCG (SIV/BCG) coinfection were employed to determine the extent to which anti-Mycobacterium tuberculosisimmunity can be restored by antiretroviral therapy. Both SIV-infected macaques with active BCG reinfection and naive animals with simultaneous SIV/BCG coinfection were evaluated. The suppression of SIV replication by antiretroviral treatment resulted in control of the active BCG infection and blocked development of the fatal SIV-related tuberculosis-like disease. The resolution of this disease coincided with the restoration of BCG purified protein derivative (PPD)-specific T-cell immune responses. In contrast, macaques similarly coinfected with SIV/BCG but not receiving antiretroviral therapy had depressed PPD-specific primary and memory T-cell immune responses and died from tuberculosis-like disease. These results provide in vivo evidence that the restoration of anti-mycobacterial immunity by antiretroviral agents can improve the clinical outcome of an AIDS virus-related tuberculosis-like disease.
Borna Disease Virus Phosphoprotein Binds a Neurite Outgrowth Factor, Amphoterin/HMG-1
Authors: Kamitani Wataru , Shoya Yuko , Kobayashi Takeshi , Watanabe Makiko , Lee Byeong-Jae , Zhang Guoqi , Tomonaga Keizo , Ikuta Kazuyoshi
Published: July 2002

The Borna disease virus (BDV) p24 phosphoprotein is an abundant protein in BDV-infected cultured cells and animal brains. Therefore, there is a possibility that binding of the p24 protein to cellular factor(s) induces functional alterations of infected neural cells in the brain. To identify a cellular protein(s) that interacts with BDV p24 protein, we performed far-Western blotting with extracts from various cell lines. Using recombinant p24 protein as a probe, we detected a 30-kDa protein in all cell lines examined. Binding between the 30-kDa and BDV p24 proteins was also demonstrated using BDV p24 affinity and ion-exchange chromatography columns. Microsequence analysis of the purified 30-kDa protein revealed that its N terminus showed complete homology with rat amphoterin protein, which is a neurite outgrowth factor abundant in the brain during development. Mammalian two-hybrid and immunoprecipitation analyses also confirmed that amphoterin is a specific target for the p24 protein in vivo. Furthermore, we showed that infection by BDV, as well as purified p24 protein in the medium, significantly decreased cell process outgrowth of cells grown on laminin, indicating the functional inhibition of amphoterin by interaction with the p24 protein. Immunohistochemical analysis revealed decreased levels of amphoterin protein at the leading edges of BDV-infected cells. Moreover, the expression of the receptor for advanced glycation end products, of which the extracellular moiety is a receptor for amphoterin, was not significantly activated in BDV-infected cells during the process of extension, suggesting that the secretion of amphoterin from the cell surface is inhibited by the binding of the p24 protein. These results suggested that BDV infection may cause direct damage in the developing brain by inhibiting the function of amphoterin due to binding by the p24 phosphoprotein.
Syndecans Serve as Attachment Receptors for Human Immunodeficiency Virus Type 1 on Macrophages
Authors: Saphire Andrew C. S. , Bobardt Michael D. , Zhang Zhe , David Guido , Gallay Philippe A.
Published: July 2002

Macrophages are thought to represent one of the first cell types in the body to be infected during the early stage of human immunodeficiency virus type 1 (HIV-1) transmission and represent a potential viral reservoir in vivo. Thus, an understanding of HIV-1 attachment to these cells is fundamental to the development of novel anti-HIV-1 therapies. Although one of the major targets of HIV-1 in vivo—CD4+T lymphocytes—express high CD4 levels, other major targets such as macrophages do not. We asked in this study whether this low CD4 level on macrophages is sufficient to support HIV-1 attachment to these cells or whether cell surface proteins other than CD4 are required for this process. We show that CD4 alone is not sufficient to support the initial adsorption of HIV-1 to macrophages. Importantly, we find that heparan sulfate proteoglycans (HSPGs) serve as the main class of attachment receptors for HIV-1 on macrophages. Most importantly, we demonstrate that a single family of HSPGs, the syndecans, efficiently mediates HIV-1 attachment and represents an abundant class of attachment receptors on macrophages.
Identification of Human Immunodeficiency Virus Type 1 Subtype C Gag-, Tat-, Rev-, and Nef-Specific Elispot-Based Cytotoxic T-Lymphocyte Responses for AIDS Vaccine Design
Authors: Novitsky V. , Rybak N. , McLane M. F. , Gilbert P. , Chigwedere P. , Klein I. , Gaolekwe S. , Chang S. Y. , Peter T. , Thior I. , Ndung'u T. , Vannberg F. , Foley B. T. , Marlink R. , Lee T. H. , Essex M.
Published: July 2002

The most severe human immunodeficiency virus type 1 (HIV-1) epidemic is occurring in southern Africa. It is caused by HIV-1 subtype C (HIV-1C). In this study we present the identification and analysis of cumulative cytotoxic T-lymphocyte (CTL) responses in the southern African country of Botswana. CTLs were shown to be an important component of the immune response to control HIV-1 infection. The definition of optimal and dominant epitopes across the HIV-1C genome that are targeted by CTL is critical for vaccine design. The characteristics of the predominant virus that causes the HIV-1 epidemic in a certain geographic area and also the genetic background of the population, through the distribution of common HLA class I alleles, might impact dominant CTL responses in the vaccinee and in the general population. The enzyme-linked immunospot (Elispot) gamma interferon assay has recently been shown to be a reliable tool to map optimal CTL epitopes, correlating well with other methods, such as intracellular staining, tetramer staining, and the classical chromium release assay. Using Elispot with overlapping synthetic peptides across Gag, Tat, Rev, and Nef, we analyzed HIV-1C-specific CTL responses of HIV-1-infected blood donors. Profiles of cumulative Elispot-based CTL responses combined with diversity and sequence consensus data provide an additional characterization of immunodominant regions across the HIV-1C genome. Results of the study suggest that the construction of a poly-epitope subtype-specific HIV-1 vaccine that includes multiple copies of immunodominant CTL epitopes across the viral genome, derived from predominant HIV-1 viruses, might be a logical approach to the design of a vaccine against AIDS.
The vhs1 Mutant Form of Herpes Simplex Virus Virion Host Shutoff Protein Retains Significant Internal Ribosome Entry Site-Directed RNA Cleavage Activity
Authors: Lu Patricia , Saffran Holly A. , Smiley James R.
Published: July 2002

The virion host shutoff (vhs) protein of herpes simplex virus (HSV) triggers global shutoff of host protein synthesis and accelerated turnover of host and viral mRNAs during HSV infection. As well, it induces endoribonucleolytic cleavage of RNA substrates when produced in a rabbit reticulocyte lysate (RRL) in vitro translation system. The vhs1 point mutation (Thr 214→Ile) eliminates vhs function during virus infection and in transiently transfected mammalian cells and was therefore previously considered to abolish vhs activity. Here we demonstrate that the vhs1 mutant protein induces readily detectable endoribonuclease activity on RNA substrates bearing the internal ribosome entry site of encephalomyocarditis virus in the RRL assay system. These data document that the vhs1 mutation does not eliminate catalytic activity and raise the possibility that the vhs-dependent endoribonuclease employs more than one mode of substrate recognition.
Molecular Characterization of
Bombyx mori
Cytoplasmic Polyhedrosis Virus Genome Segment 4
Authors: Ikeda Keiko , Nagaoka Sumiharu , Winkler Stefan , Kotani Kumiko , Yagi Hiroaki , Nakanishi Kae , Miyajima Shigetoshi , Kobayashi Jun , Mori Hajime
Published: July 2002

The complete nucleotide sequence of the genome segment 4 (S4) of
Bombyx mori
cytoplasmic polyhedrosis virus (BmCPV) was determined. The 3,259-nucleotide sequence contains a single long open reading frame which spans nucleotides 14 to 3187 and which is predicted to encode a protein with a molecular mass of about 130 kDa. Western blot analysis showed that S4 encodes BmCPV protein VP3, which is one of the outer components of the BmCPV virion. Sequence analysis of the deduced amino acid sequence of BmCPV VP3 revealed possible sequence homology with proteins from rice ragged stunt virus (RRSV) S2,
Nilaparvata lugens
reovirus S4, and Fiji disease fijivirus S4. This may suggest that plant reoviruses originated from insect viruses and that RRSV emerged more recently than other plant reoviruses. A chimeric protein consisting of BmCPV VP3 and green fluorescent protein (GFP) was constructed and expressed with BmCPV polyhedrin using a baculovirus expression vector. The VP3-GFP chimera was incorporated into BmCPV polyhedra and released under alkaline conditions. The results indicate that specific interactions occur between BmCPV polyhedrin and VP3 which might facilitate BmCPV virion occlusion into the polyhedra.
Yaba-Like Disease Virus: an Alternative Replicating Poxvirus Vector for Cancer Gene Therapy
Authors: Hu Yun , Lee John , McCart J. Andrea , Xu Hui , Moss Bernard , Alexander H. Richard , Bartlett David L.
Published: July 2002

Vaccinia virus is being investigated as a replicating vector for tumor-directed gene therapy. However, the majority of cancer patients have preformed immunologic reactivity against vaccinia virus, as a result of smallpox vaccination, which may limit its use as a vector. The Yaba-like disease (YLD) virus was investigated here as an alternative, replicating poxvirus for cancer gene therapy. We have demonstrated that the YLD virus does not cross-react with vaccinia virus antibodies, and it replicates efficiently in human tumor cells. YLD virus can be expanded and purified to high titer in CV-1 cells under conditions utilized for vaccinia virus. The YLD virus RNA polymerase was able to express genes regulated by a synthetic promoter designed for use in orthopoxviruses. We sequenced the YLD virus TK gene and created a shuttle plasmid, which allowed the recombination of the green fluorescent protein (GFP) gene into the YLD virus. In a murine model of ovarian cancer, up to 38% of cells in the tumor expressed the GFP transgene 12 days after intraperitoneal virus delivery. YLD virus has favorable characteristics as a vector for cancer gene therapy, and this potential should be explored further.
Transgenically Expressed T-Rep of Tomato Yellow Leaf Curl Sardinia Virus Acts as a
trans
-Dominant-Negative Mutant, Inhibiting Viral Transcription and Replication
Authors: Brunetti Angela , Tavazza Raffaela , Noris Emanuela , Lucioli Alessandra , Accotto Gian Paolo , Tavazza Mario
Published: July 2002

We have previously shown that transgenic expression of a truncated C1 gene of
Tomato yellow leaf curl Sardinia virus
(TYLCSV), expressing the first 210 amino acids of the replication-associated protein (T-Rep) and potentially coexpressing the C4 protein, confers resistance to the homologous virus in
Nicotiana benthamiana
plants. In the present study we have investigated the role of T-Rep and C4 proteins in the resistance mechanism, analyzing changes in virus transcription and replication. Transgenic plants and protoplasts were challenged with TYLCSV and the related TYLCSV Murcia strain (TYLCSV-ES[1]). TYLCSV-resistant plants were susceptible to TYLCSV-ES[1]; moreover, TYLCSV but not TYLCSV-ES[1] replication was strongly inhibited in transgenic protoplasts as well as in wild-type (wt) protoplasts transiently expressing T-Rep but not the C4 protein. Viral circular single-stranded DNA (cssDNA) was usually undetectable in transgenically and transiently T-Rep-expressing protoplasts, while viral DNAs migrating more slowly than the cssDNA were observed. Biochemical studies showed that these DNAs were partial duplexes with the minus strand incomplete. Interestingly, similar viral DNA forms were also found at early stages of TYLCSV replication in wt
N. benthamiana
protoplasts. Transgenically expressed T-Rep repressed the transcription of the GUS reporter gene up to 300-fold when fused to the homologous (TYLCSV) but not to the heterologous (TYLCSV-ES[1]) C1 promoter. Similarly, transiently expressed T-Rep but not C4 protein strongly repressed GUS transcription when fused to the C1 promoter of TYLCSV. A model of T-Rep interference with TYLCSV transcription-replication is proposed.
Exogenous Interleukin-2 Administration Corrects the Cell Cycle Perturbation of Lymphocytes from Human Immunodeficiency Virus-Infected Individuals
Authors: Paiardini Mirko , Galati Domenico , Cervasi Barbara , Cannavo Giuseppe , Galluzzi Luca , Montroni Maria , Guetard Denise , Magnani Mauro , Piedimonte Giuseppe , Silvestri Guido
Published: July 2002

Human immunodeficiency virus (HIV)-induced immunodeficiency is characterized by progressive loss of CD4+T cells associated with functional abnormalities of the surviving lymphocytes. Increased susceptibility to apoptosis and loss of proper cell cycle control can be observed in lymphocytes from HIV-infected individuals and may contribute to the lymphocyte dysfunction of AIDS patients. To better understand the relation between T-cell activation, apoptosis, and cell cycle perturbation, we studied the effect of exogenous interleukin-2 (IL-2) administration on the intracellular turnover of phase-dependent proteins. Circulating T cells from HIV-infected patients display a marked discrepancy between a metabolic profile typical of G0and a pattern of expression of phase-dependent proteins that indicates a more-advanced position within the cell cycle. This discrepancy is enhanced by in vitro activation with ConA and ultimately results in a marked increase of apoptotic events. Conversely, treatment of lymphocytes with IL-2 alone restores the phase-specific pattern of expression of cell cycle-dependent proteins and is associated with low levels of apoptosis. Interestingly, exogenous IL-2 administration normalizes the overall intracellular protein turnover, as measured by protein synthesis, half-life of newly synthesised proteins, and total protein ubiquitination, thus providing a possible explanation for the effect of IL-2 on the intracellular kinetics of cell cycle-dependent proteins. The beneficial effect of IL-2 administration is consistent with the possibility of defective IL-2 function in vivo, which is confirmed by the observation that lymphocytes from HIV-infected patients show abnormal endogenous IL-2 paracrine/autocrine function upon in vitro mitogen stimulation. Overall these results confirm that perturbation of cell cycle control contributes to HIV-related lymphocyte dysfunction and, by showing that IL-2 administration can revert this perturbation, suggest a new mechanism of action of IL-2 therapy in HIV-infected patients.
Genome Replication and Postencapsidation Functions Mapping to the Nonstructural Gene Restrict the Host Range of a Murine Parvovirus in Human Cells
Authors: Rubio Mari-Paz , Guerra Susana , Almendral José M.
Published: July 2002

The infection outcome of theParvoviridaelargely relies on poorly characterized intracellular factors modulated by proliferation, differentiation, and transformation of host cells. We have studied the interactions displayed by the highly homologous p and i strains of the murine parvovirus minute virus of mice (MVM), with a series of transformed cells of rat (C6) and human (U373, U87, SW1088, SK-N-SH) nervous system origin, seeking for molecular mechanisms governing parvovirus host range. The MVMp infection of C6 and U373 cells was cytotoxic and productive, whereas the other nervous cells behaved essentially as resistant to this virus. In contrast, MVMi did not complete its life cycle in any of the human nervous cells, though it efficiently killed the astrocytic tumor cells by two types of nonproductive infections: (i) normal synthesis of all viral macromolecules with a late defect in infectious virion maturation and release to the medium in U373; and (ii) high levels of accumulation of the full set of viral messenger RNAs and of both nonstructural (NS-1) and structural (VP-1 and VP-2) proteins, under a very low viral DNA amplification, in U87 and SW1088 cells. Further analyses showed that U87 was permissive for nuclear transport of MVMi proteins, leading to efficient assembly of empty viral capsids with a normal phosphorylation and VP1-to-VP2 ratio. The DNA amplification blockade in U87 occurred after conversion of the incoming MVMi genome to the monomeric replicative form, and it operated independently of the delivery pathway used by the viral particle, since it could not be overcome by transfection with cloned infectious viral DNA. Significantly, a chimeric MVMi virus harboring the coding region of the nonstructural (NS) gene replaced with that of MVMp showed a similar pattern of restriction in U87 cells as the parental MVMi virus, and it attained in U373 cultures an infectious titer above 100-fold higher under equal levels of DNA amplification and genome encapsidation. The results suggest that the activity of complexes formed by the NS polypeptides and recruited cellular factors restrict parvovirus DNA amplification in a cell type-dependent manner and that NS functions may in addition determine MVM host range acting at postencapsidation steps of viral maturation. These data are relevant for understanding the increased multiplication of autonomous parvovirus in some transformed cells and the transduction efficacy of nonreplicative parvoviral vectors, as well as a general remark on the mechanisms by which NS genes may regulate viral tropism and pathogenesis.
The Membrane M Protein Carboxy Terminus Binds to Transmissible Gastroenteritis Coronavirus Core and Contributes to Core Stability
Authors: Escors David , Ortego Javier , Laude Hubert , Enjuanes Luis
Published: July 2002

The architecture of transmissible gastroenteritis coronavirus includes three different structural levels, the envelope, an internal core, and the nucleocapsid that is released when the core is disrupted. Starting from purified virions, core structures have been reproducibly isolated as independent entities. The cores were stabilized at basic pH and by the presence of divalent cations, with Mg
2+
ions more effectively contributing to core stability. Core structures showed high resistance to different concentrations of detergents, reducing agents, and urea and low concentrations of monovalent ions (<200 mM). Cores were composed of the nucleoprotein, RNA, and the C domain of the membrane (M) protein. At high salt concentrations (200 to 300 mM), the M protein was no longer associated with the nucleocapsid, which resulted in destruction of the core structure. A specific ionic interaction between the M protein carboxy terminus and the nucleocapsid was demonstrated using three complementary approaches: (i) a binding assay performed between a collection of M protein amino acid substitution or deletion mutants and purified nucleocapsids that led to the identification of a 16-amino-acid (aa) domain (aa 237 to 252) as being responsible for binding the M protein to the nucleocapsid; (ii) the specific inhibition of this binding by monoclonal antibodies (MAbs) binding to a carboxy-terminal M protein domain close to the indicated peptide but not by MAbs specific for the M protein amino terminus; and (iii) a 26-residue peptide, including the predicted sequence (aa 237 to 252), which specifically inhibited the binding. Direct binding of the M protein to the nucleoprotein was predicted, since degradation of the exposed RNA by RNase treatment did not affect the binding. It is proposed that the M protein is embedded within the virus membrane and that the C region, exposed to the interior face of the virion in a population of these molecules, interacts with the nucleocapsid to which it is anchored, forming the core. Only the C region of the M protein is part of the core.
Infectivity-Enhancing Antibodies to Ebola Virus Glycoprotein
Authors: Takada Ayato , Watanabe Shinji , Okazaki Katsunori , Kida Hiroshi , Kawaoka Yoshihiro
Published: July 2002

Ebola virus causes severe hemorrhagic fever in primates, resulting in mortality rates of up to 100%, yet there are no satisfactory biologic explanations for this extreme virulence. Here we show that antisera produced by DNA immunization with a plasmid encoding the surface glycoprotein (GP) of the Zaire strain of Ebola virus enhances the infectivity of vesicular stomatitis virus pseudotyped with the GP. Substantially weaker enhancement was observed with antiserum to the GP of the Reston strain, which is much less pathogenic in humans than the Ebola Zaire and Sudan viruses. The enhancing activity was abolished by heat but was increased in the presence of complement system inhibitors, suggesting that heat-labile factors other than the complement system are required for this effect. We also generated an anti-Zaire GP monoclonal antibody that enhanced viral infectivity and another that neutralized it, indicating the presence of distinct epitopes for these properties. Our findings suggest that antibody-dependent enhancement of infectivity may account for the extreme virulence of the virus. They also raise issues about the development of Ebola virus vaccines and the use of passive prophylaxis or therapy with Ebola virus GP antibodies.
Human Papillomavirus Type 16 Minor Capsid Protein L2 N-Terminal Region Containing a Common Neutralization Epitope Binds to the Cell Surface and Enters the Cytoplasm
Authors: Kawana Yukiko , Kawana Kei , Yoshikawa Hiroyuki , Taketani Yuji , Yoshiike Kunito , Kanda Tadahito
Published: July 2002

The first step of papillomavirus infection is believed to be binding of major capsid protein L1 to the cell surface without involvement of minor capsid protein L2, but the viral infectivity can be neutralized either by anti-L1 or anti-L2 antibody. To understand the role of L2 in human papillomavirus (HPV) infection, we examined a segment of HPV type 16 (HPV16) L2, which contains a neutralization epitope common to HPV6, for its involvement in adsorption and penetration of the capsids. Preincubation of monkey COS-1 cells with a synthetic peptide having amino acids (aa) 108 to 120 of HPV16 L2 reduced the susceptibility of COS-1 cells to infection with HPV16 pseudovirions. Confocal microscopy showed that the green fluorescence protein (GFP) fused with the L2 peptide was found to bind to the surface of a HeLa cell, an HPV18-positive human cancer cell line, at 4°C and to enter the cytoplasm after subsequent incubation at 37°C. Flow cytometry showed that fused GFP did not bind to HeLa cells that had been treated with trypsin. Besides COS-1 and HeLa cells, some human and rodent cell lines were detected by flow cytometry to be susceptible to binding with fused GFP, showing a tendency of epithelial cells toward higher susceptibility. Substitutions at aa 108 to 111 inhibited fused GFP from binding to HeLa cells and reduced the infectivity in COS-1 cells of the in vitro-constructed pseudovirions. The results suggest that L2 plays an important role in enhancing HPV infection through interaction between the N-terminal region and a cellular surface protein, facilitating penetration of the virions and determining part of the tropism of HPVs.
Requirements for the Nuclear-Cytoplasmic Translocation of Infected-Cell Protein 0 of Herpes Simplex Virus 1
Authors: Lopez Pascal , Van Sant Charles , Roizman Bernard
Published: July 2002

Earlier studies have shown that wild-type infected-cell protein 0 (ICP0), a key herpes simplex virus regulatory protein, translocates from the nucleus to the cytoplasm of human embryonic lung (HEL) fibroblasts within several hours after infection (Y. Kawaguchi, R. Bruni, and B. Roizman, J. Virol. 71:1019–1024, 1997). Translocation of ICP0 was also observed in cells infected with the
d
120 mutant, in which both copies of the gene encoding ICP4, the major regulatory protein, had been deleted (V. Galvan, R. Brandimarti, J. Munger, and B. Roizman, J. Virol. 74:1931–1938, 2000). Furthermore, a mutant (R7914) carrying the D199A substitution in ICP0 does not bind or stabilize cyclin D3 and is retained in the nucleus (C. Van Sant, P. Lopez, S. J. Advani, and B. Roizman, J. Virol. 75:1888–1898, 2001). Studies designed to elucidate the requirements for the translocation of ICP0 between cellular compartments revealed the following. (i) Translocation of ICP0 to the cytoplasm in productive infection maps to the D199 amino acid, inasmuch as wild-type ICP0 delivered in
trans
to cells infected with an ICP0 null mutant was translocated to the cytoplasm whereas the D199A-substituted mutant ICP0 was not. (ii) Translocation of wild-type ICP0 requires a function expressed late in infection, inasmuch as phosphonoacetate blocked the translocation of ICP0 in wild-type virus-infected cells but not in
d
120 mutant-infected cells. Moreover, whereas in
d
120 mutant-infected cells ICP0 was translocated rapidly from the cytoplasm to the nucleus at approximately 5 h after infection, the translocation of ICP0 in wild-type virus-infected cells extended from 5 to at least 9 h after infection. (iii) In wild-type virus-infected cells, the MG132 proteasomal inhibitor blocked the translocation of ICP0 to the cytoplasm early in infection, but when added late in infection, it caused ICP0 to be relocated back to the nucleus from the cytoplasm. (iv) MG132 blocked the translocation of ICP0 in
d
120 mutant-infected cells early in infection but had no effect on the ICP0 aggregated in vesicle-like structures late in infection. However, in
d
120 mutant-infected cells treated with MG132 at late times, proteasomes formed a shell-like structure around the aggregated ICP0. These structures were not seen in wild-type virus or R7914 mutant-infected cells. The results indicate the following. (i) In the absence of β or γ protein synthesis, ICP0 dynamically associates with proteasomes and is translocated to the cytoplasm. (ii) In cells productively infected beyond α gene expression, ICP0 is retained in the nucleus until after the onset of viral DNA synthesis and the synthesis of γ
2
proteins. (iii) Late in infection, ICP0 is actively sequestered in the cytoplasm by a process mediated by proteasomes, inasmuch as interference with proteasomal function causes rapid relocation of ICP0 to the nucleus.