bluuebunny/crossref_metadata_2025 huggingface dataset:
The Vaccinia Virus Soluble Alpha/Beta Interferon (IFN) Receptor Binds to the Cell Surface and Protects Cells from the Antiviral Effects of IFN
Authors: Alcamı́ Antonio , Symons Julian A. , Smith Geoffrey L.
Published: July 2002

Poxviruses encode a broad range of proteins that interfere with host immune functions, such as soluble versions of receptors for the cytokines tumor necrosis factor, interleukin-1β, gamma interferon (IFN-γ), IFN-α/β, and chemokines. These virus-encoded cytokine receptors have a profound effect on virus pathogenesis and enable the study of the role of cytokines in virus infections. The vaccinia virus (VV) Western Reserve gene B18R encodes a secreted protein with 3 immunoglobulin domains that functions as a soluble receptor for IFN-α/β. We have found that after secretion B18R binds to both uninfected and infected cells. The B18R protein present at the cell surface maintains the properties of the soluble receptor, binding IFN-α/β with high affinity and with broad species specificity, and protects cells from the antiviral state induced by IFN-α/β. VV strain Wyeth expressed a truncated B18R protein lacking the C-terminal immunoglobulin domain. This protein binds IFN with lower affinity and retains its ability to bind to cells, indicating that the C-terminal region of B18R contributes to IFN binding. The replication of a VV B18R deletion mutant in tissue culture was restricted in the presence of IFN-α, whereas the wild-type virus replicated normally. Binding of soluble recombinant B18R to cells protected the cultures from IFN and allowed VV replication. This represents a novel strategy of virus immune evasion in which secreted IFN-α/β receptors not only bind the soluble cytokine but also bind to uninfected cells and protect them from the antiviral effects of IFN-α/β, maintaining the cells' susceptibility to virus infections. The adaptation of this soluble receptor to block IFN-α/β activity locally will help VV to replicate in the host and spread in tissues. This emphasizes the importance of local effects of IFN-α/β against virus infections.
Expression and Characterization of a Single-Chain Polypeptide Analogue of the Human Immunodeficiency Virus Type 1 gp120-CD4 Receptor Complex
Authors: Fouts Timothy R. , Tuskan Robert , Godfrey Karla , Reitz Marvin , Hone David , Lewis George K. , DeVico Anthony L.
Published: July 2002

The infection of CD4
+
host cells by human immunodeficiency virus type 1 (HIV-1) is initiated by a temporal progression of interactions between specific cell surface receptors and the viral envelope protein, gp120. These interactions produce a number of intermediate structures with distinct conformational, functional, and antigenic features that may provide important targets for therapeutic and vaccination strategies against HIV infection. One such intermediate, the gp120-CD4 complex, arises from the interaction of gp120 with the CD4 receptor and enables interactions with specific coreceptors needed for viral entry. gp120-CD4 complexes are thus promising targets for anti-HIV vaccines and therapies. The development of such strategies would be greatly facilitated by a means to produce the gp120-CD4 complexes in a wide variety of contexts. Accordingly, we have developed single-chain polypeptide analogues that accurately replicate structural, functional, and antigenic features of the gp120-CD4 complex. One analogue (FLSC) consists of full-length HIV-1BaL gp120 and the D1D2 domains of CD4 joined by a 20-amino-acid linker. The second analogue (TcSC) contains a truncated form of the gp120 lacking portions of the C1, C5, V1, and V2 domains. Both molecules exhibited increased exposure of epitopes in the gp120 coreceptor-binding site but did not present epitopes of either gp120 or CD4 responsible for complex formation. Further, the FLSC and TcSC analogues bound specifically to CCR5 (R5) and blocked R5 virus infection. Thus, these single-chain chimeric molecules represent the first generation of soluble recombinant proteins that mimic the gp120-CD4 complex intermediate that arises during HIV replication.
The Membrane-Proximal Stem Region of Vesicular Stomatitis Virus G Protein Confers Efficient Virus Assembly
Authors: Robison Clinton S. , Whitt Michael A.
Published: July 2002

In this report, we show that the glycoprotein of vesicular stomatitis virus (VSV G) contains within its extracellular membrane-proximal stem (GS) a domain that is required for efficient VSV budding. To determine a minimal sequence in GS that provides for high-level virus assembly, we have generated a series of recombinant ΔG-VSVs which express chimeric glycoproteins having truncated stem sequences. The recombinant viruses having chimeras with 12 or more membrane-proximal residues of the G stem, and including the G protein transmembrane-cytoplasmic tail domains, produced near-wild-type levels of particles. In contrast, viruses encoding chimeras with shorter or no G-stem sequences produced ∼10- to 20-fold less. This budding domain when present in chimeric glycoproteins also promoted their incorporation into the VSV envelope. We suggest that the G-stem budding domain promotes virus release by inducing membrane curvature at sites where virus budding occurs or by recruiting condensed nucleocapsids to sites on the plasma membrane which are competent for efficient virus budding.
Disulfide Bonds and Membrane Topology of the Vaccinia Virus A17L Envelope Protein
Authors: Betakova Tatiana , Moss Bernard
Published: July 2002

The envelope protein encoded by the vaccinia virus A17L open reading frame is essential for virion assembly. Our mutagenesis studies indicated that cysteines 101 and 121 form an intramolecular disulfide bond and that cysteine 178 forms an intermolecular disulfide linking two A17L molecules. This arrangement of disulfide bonds has important implications for the topology of the A17L protein and supports a two-transmembrane model in which cysteines 101 and 121 are intraluminal and cysteine 178 is cytoplasmic. The structure of the A17L protein, however, was not dependent on these disulfide bonds, as a recombinant vaccinia virus with all three cysteine codons mutated to serines retained infectivity.
Human T-Cell Leukemia Virus Type 2 Tax Mutants That Selectively Abrogate NFκB or CREB/ATF Activation Fail To Transform Primary Human T Cells
Authors: Ross Ted M. , Narayan Murli , Fang Zhi-Yu , Minella Alex C. , Green Patrick L.
Published: July 2002

Human T-cell leukemia virus (HTLV) Tax protein has been implicated in the HTLV oncogenic process, primarily due to its pleiotropic effects on cellular genes involved in growth regulation and cell cycle control. To date, several approaches attempting to correlate Tax activation of the CREB/activating transcription factor (ATF) or NFκB/Rel transcriptional activation pathway to cellular transformation have yielded conflicting results. In this study, we use a unique HTLV-2 provirus (HTLV
c-enh
) that replicates by a Tax-independent mechanism to directly assess the role of Tax transactivation in HTLV-mediated T-lymphocyte transformation. A panel of well-characterized
tax-2
mutations is utilized to correlate the respective roles of the CREB/ATF or NFκB/Rel signaling pathway. Our results demonstrate that viruses expressing
tax-2
mutations that selectively abrogate NFκB/Rel or CREB/ATF activation display distinct phenotypes but ultimately fail to transform primary human T lymphocytes. One conclusion consistent with our results is that the activation of NFκB/Rel provides a critical proliferative signal early in the cellular transformation process, whereas CREB/ATF activation is required to promote the fully transformed state. However, complete understanding will require correlation of Tax domains important in cellular transformation to those Tax domains important in the modulation of gene transcription, cell cycle control, induction of DNA damage, and other undefined activities.
Recombinant Modified Vaccinia Virus Ankara Expressing the Surface gp120 of Simian Immunodeficiency Virus (SIV) Primes for a Rapid Neutralizing Antibody Response to SIV Infection in Macaques
Authors: Ourmanov Ilnour , Bilska Miroslawa , Hirsch Vanessa M. , Montefiori David C.
Published: July 2002

Neutralizing antibodies were assessed before and after intravenous challenge with pathogenic SIVsmE660 in rhesus macaques that had been immunized with recombinant modified vaccinia virus Ankara expressing one or more simian immunodeficiency virus gene products (MVA-SIV). Animals received either MVA-gag-pol, MVA-env, MVA-gag-pol-env, or nonrecombinant MVA. Although no animals were completely protected from infection with SIV, animals immunized with recombinant MVA-SIV vaccines had lower virus loads and prolonged survival relative to control animals that received nonrecombinant MVA (I. Ourmanov et al., J. Virol. 74:2740–2751, 2000). Titers of neutralizing antibodies measured with the vaccine strain SIVsmH-4 were low in the MVA-envand MVA-gag-pol-envgroups of animals and were undetectable in the MVA-gag-poland nonrecombinant MVA groups of animals on the day of challenge (4 weeks after final immunization). Titers of SIVsmH-4-neutralizing antibodies remained unchanged 1 week later but increased approximately 100-fold 2 weeks postchallenge in the MVA-envand MVA-gag-pol-envgroups while the titers remained low or undetectable in the MVA-gag-poland nonrecombinant MVA groups. This anamnestic neutralizing antibody response was also detected with T-cell-line-adapted stocks of SIVmac251 and SIV/DeltaB670 but not with SIVmac239, as this latter virus resisted neutralization. Most animals in each group had high titers of SIVsmH-4-neutralizing antibodies 8 weeks postchallenge. Titers of neutralizing antibodies were low or undetectable until about 12 weeks of infection in all groups of animals and showed little or no evidence of an anamnestic response when measured with SIVsmE660. The results indicate that recombinant MVA is a promising vector to use to prime for an anamnestic neutralizing antibody response following infection with primate lentiviruses that cause AIDS. However, the Env component of the present vaccine needs improvement in order to target a broad spectrum of viral variants, including those that resemble primary isolates.
Nonrandom Transduction of Recombinant Adeno-Associated Virus Vectors in Mouse Hepatocytes In Vivo: Cell Cycling Does Not Influence Hepatocyte Transduction
Authors: Miao Carol H. , Nakai Hiroyuki , Thompson Arthur R. , Storm Theresa A. , Chiu Winnie , Snyder Richard O. , Kay Mark A.
Published: July 2002

Recombinant adeno-associated virus vectors (rAAV) show promise in preclinical trials for the treatment of genetic diseases including hemophilia. Liver-directed gene transfer results in a slow rise in transgene expression, reaching steady-state levels over a period of 5 weeks concomitant with the conversion of the single-stranded rAAV molecules into high-molecular-weight concatemers in about 5% of hepatocytes. Immunohistochemistry and RNA in situ hybridization show that the transgene product is made in about ∼5% of hepatocytes, suggesting that most rAAV-mediated gene expression occurs in hepatocytes containing the double-stranded concatemers. In this study, the mechanism(s) involved in stable transduction in vivo was evaluated. While only ∼5% of hepatocytes are stably transduced, in situ hybridization experiments demonstrated that the vast majority of the hepatocytes take up AAV-DNA genomes after portal vein infusion of the vector. Two different vectors were infused together or staggered by 1, 3, or 5 weeks, and two-color fluorescent in situ hybridization and molecular analyses were performed 5 weeks after the infusion of the second vector. These experiments revealed that a small but changing subpopulation of hepatocytes were permissive to stable transduction. Furthermore, in animals that received a single infusion of two vectors, about one-third of the transduced cells contained heteroconcatemers, suggesting that dimer formation was a critical event in the process of concatemer formation. To determine if the progression through the cell cycle was important for rAAV transduction, animals were continuously infused with 5′-bromo-2′-deoxyuridine (BrdU), starting at the time of administration of a rAAV vector that expressed cytoplasmic β-galactosidase. Colabeling for β-galactosidase and BrdU revealed that there was no preference for transduction of cycling cells. This was further confirmed by demonstrating no increase in rAAV transduction efficiencies in animals whose livers were induced to cycle at the time of or after vector administration. Taken together, our studies suggest that while virtually all hepatocytes take up vector, unknown cellular factors are required for stable transduction, and that dimer formation is a critical event in the transduction pathway. These studies have important implications for understanding the mechanism of integration and may be useful for improving liver gene transfer in vivo.
Mus cervicolor
Murine Leukemia Virus Isolate M813 Belongs to a Unique Receptor Interference Group
Authors: Prassolov Vladimir , Hein Sibyll , Ziegler Marion , Ivanov Dmitry , Münk Carsten , Löhler Jürgen , Stocking Carol
Published: July 2002

Murine leukemia virus (MuLV) M813 was originally isolated from the Southeast Asian rodent
Mus cervicolor
. As with the ecotropic MuLVs derived from
Mus musculus
, its host range is limited to rodent cells. Earlier studies have mapped its receptor to chromosome 2, but it has not been established whether M813 shares a common receptor with any other MuLVs. In this study, we have performed interference assays with M813 and viruses from four interference groups of MuLV. The infection efficiency of M813 was not compromised in cells expressing any one of the other MuLVs, demonstrating that M813 must use a distinct receptor for cell entry. The entire M813
env
coding region was molecularly cloned. Sequence analysis revealed high similarity with other MuLVs but with a unique receptor-binding domain. Substitution of M813
env
sequences in Moloney MuLV resulted in a replication-competent virus with a host range and interference profile similar to those of the biological clone M813. M813 thus defines a novel receptor interference group of type C MuLVs.
MEK-Specific Inhibitor U0126 Blocks Spread of Borna Disease Virus in Cultured Cells
Authors: Planz Oliver , Pleschka Stephan , Ludwig Stephan
Published: July 2002

Borna disease virus (BDV) is a highly neurotropic virus that causes Borna disease, a virus-induced immune-mediated encephalomyelitis, in a variety of warm-blooded animals. Recent studies reported that BDV can be detected in patients with psychiatric disorders. BDV is noncytopathic, replicates in the nucleus of infected cells, and spreads intraaxonally in vivo. Upon infection of susceptible cultured cells, virus can be detected in foci. Little is known about the cellular components required for BDV replication. Here, we show that the cellular Raf/MEK/ERK signaling cascade is activated upon infection with BDV. In the presence of the MEK-specific inhibitor U0126, cells get infected with BDV; however, there is a block in virus spread to neighboring cells. The effect of the inhibitor on virus spread was still observed when the compound was added 2 h postinfection but not if treatment was initiated as late as 4 h after infection. Our results provide new insights into the BDV-host cell interaction and show that virus infection can be controlled with drugs interfering with a cellular signaling pathway. Since concentrations of the MEK inhibitor required to block BDV focus formation are not toxic for the host cells, our finding may be important with respect to antiviral drug development.
Patient-Specific Cytotoxic T-Lymphocyte Cross-Recognition of Naturally Occurring Variants of a Human Immunodeficiency Virus Type 1 (HIV-1) p24gagEpitope by HIV-1-Infected Children
Authors: Buseyne F. , Chaix M.-L. , Rouzioux C. , Blanche S. , Rivière Y.
Published: July 2002

We tested seven human immunodeficiency virus-infected children for their cytotoxic T-lymphocyte (CTL) activities towards the p24gagQASQEVKNW epitope and its nine variant sequences. Our data confirm that most, but not all, CTL responses are broadly cross-specific. For the first time, we show the high interpatient variability in cross-recognition of mutant CTL epitopes. These interindividual variations in the CTL response to the same epitope should be taken into account in the design and the evaluation of vaccines.
Virological Properties and Nucleotide Sequences of Cas-E-Type Endogenous Ecotropic Murine Leukemia Viruses in South Asian Wild Mice,
Mus musculus castaneus
Authors: Ikeda Hidetoshi , Kato Kanako , Kitani Hiroshi , Suzuki Takako , Yoshida Takamasa , Inaguma Yutaka , Yamamoto Naoyuki , Suh Jun-Gyo , Hyun Byung-Hwa , Yamagata Takahiro , Namikawa Takao , Tomita Takeshi
Published: July 2002

Two types of endogenous ecotropic murine leukemia viruses (MuLVs), termed AKV- and Cas-E-type MuLVs, differ in nucleotide sequence and distribution in wild mouse subspecies. In contrast to AKV-type MuLV, Cas-E-type MuLV is not carried by common laboratory mice. Wild mice of
Mus musculus (M. m.) castaneus
carry multiple copies of Cas-E-type endogenous MuLV, including the

Fv-4
r

gene that is a truncated form of integrated MuLV and functions as a host's resistance gene against ecotropic MuLV infection. Our genetic cross experiments showed that only the

Fv-4
r

gene was associated with resistance to ecotropic F-MuLV infection. Because the spontaneous expression of infectious virus was not detected in
M. m. castaneus
, we generated mice that did not carry the

Fv-4
r

gene but did carry a single or a few endogenous MuLV loci. In mice not carrying the

Fv-4
r

gene, infectious MuLVs were isolated in association with three of six Cas-E-type endogenous MuLV loci. The isolated viruses showed a weak syncytium-forming activity for XC cells, an interfering property of ecotropic MuLV, and a slight antigenic variation. Two genomic DNAs containing endogenous Cas-E-type MuLV were cloned and partially sequenced. All of the Cas-E-type endogenous MuLVs were closely related, hybrid-type viruses with an ecotropic
env
gene and a xenotropic long terminal repeat. Duplications and a deletion were found in a restricted region of the hypervariable proline-rich region of Env glycoprotein.
Marek's Disease Virus (MDV) Encodes an Interleukin-8 Homolog (vIL-8): Characterization of the vIL-8 Protein and a vIL-8 Deletion Mutant MDV
Authors: Parcells Mark S. , Lin Su-Fang , Dienglewicz Robert L. , Majerciak Vladimir , Robinson Dan R. , Chen Hua-Chien , Wu Zining , Dubyak George R. , Brunovskis Peter , Hunt Henry D. , Lee Lucy F. , Kung Hsing-Jien
Published: July 2002

Chemokines induce chemotaxis, cell migration, and inflammatory responses. We report the identification of an interleukin-8 (IL-8) homolog, termed vIL-8, encoded within the genome of Marek's disease virus (MDV). The 134-amino-acid vIL-8 shares closest homology to mammalian and avian IL-8, molecules representing the prototype CXC chemokine. The gene for vIL-8 consists of three exons which map to theBamHI-L fragment within the repeats flanking the unique long region of the MDV genome. A 0.7-kb transcript encoding vIL-8 was detected in ann-butyrate-treated, MDV-transformed T-lymphoblastoid cell line, MSB-1. This induction is essentially abolished by cycloheximide and herpesvirus DNA polymerase inhibitor phosphonoacetate, indicating that vIL-8 is expressed with true late (γ2) kinetics. Baculovirus-expressed vIL-8 was found to be secreted into the medium and shown to be functional as a chemoattractant for chicken peripheral blood mononuclear cells but not for heterophils. To characterize the function of vIL-8 with respect to MDV infection in vivo, a recombinant MDV was constructed with a deletion of all three exons and a soluble-modified green fluorescent protein (smGFP) expression cassette inserted at the site of deletion. In two in vivo experiments, the vIL-8 deletion mutant (RB1BvIL-8ΔsmGFP) showed a decreased level of lytic infection in comparison to its parent virus, an equal-passage-level parent virus, and to another recombinant MDV containing the insertion of a GFP expression cassette at the nonessential US2 gene. RB1BvIL-8ΔsmGFP retained oncogenicity, albeit at a greatly reduced level. Nonetheless, we have been able to establish a lymphoblastoid cell line from an RB1BvIL-8ΔsmGFP-induced ovarian lymphoma (MDCC-UA20) and verify the presence of a latent MDV genome lacking vIL-8. Taken together, these data describe the identification and characterization of a chemokine homolog encoded within the MDV genome that is dispensable for transformation but may affect the level of MDV in vivo lytic infection.
Hepatitis C Virus Nonstructural 5A Protein Induces Interleukin-8, Leading to Partial Inhibition of the Interferon-Induced Antiviral Response
Authors: Polyak Stephen J. , Khabar Khalid S. A. , Paschal Denise M. , Ezelle Heather J. , Duverlie Gilles , Barber Glen N. , Levy David E. , Mukaida Naofumi , Gretch David R.
Published: July 2002

Hepatitis C virus (HCV), a major cause of liver disease worldwide, is frequently resistant to the antiviral alpha interferon (IFN). The HCV nonstructural 5A (NS5A) protein has been implicated in HCV antiviral resistance in many studies. NS5A antagonizes the IFN antiviral response in vitro, and one mechanism is via inhibition of a key IFN-induced enzyme, the double-stranded-RNA-activated protein kinase (PKR). In the present study we determined if NS5A uses other strategies to subvert the IFN system. Expression of full-length NS5A proteins from patients who exhibited a complete response (FL-NS5A-CR) or were nonresponsive (FL-NS5A-NR) to IFN therapy in HeLa cells had no effect on IFN induction of IFN-stimulated gene factor 3 (ISGF-3). Expression of mutant NS5A proteins lacking 110 (NS5A-ΔN110), 222 (NS5A-ΔN222), and 334 amino-terminal amino acids and mutants lacking 117 and 230 carboxy-terminal amino acids also had no effect on ISGF-3 induction by IFN. Expression of FL-NS5A-CR and FL-NS5A-NR did not affect IFN-induced STAT-1 tyrosine phosphorylation or upregulation of PKR and major histocompatibility complex class I antigens. However, NS5A expression in human cells induced interleukin 8 (IL-8) mRNA and protein, and this effect correlated with inhibition of the antiviral effects of IFN in an in vitro bioassay. NS5A induced transcription of a reporter gene driven by the IL-8 promoter, and the first 133 bp of the IL-8 promoter made up the minimal domain required for NS5A transactivation. NS5A-ΔN110 and NS5A-ΔN222 stimulated the IL-8 promoter to higher levels than did the full-length NS5A protein, and this correlated with increased nuclear localization of the proteins. Additional mutagenesis of the IL-8 promoter suggested that NF-κB and AP-1 were important in NS5A-ΔN222 transactivation in the presence of tumor necrosis factor alpha and that NF–IL-6 was inhibitory to this process. This study suggests that NS5A inhibits the antiviral actions of IFN by at least two mechanisms and provides the first evidence for a biological effect of the transcriptional activity of the NS5A protein. During HCV infection, viral proteins may induce chemokines that contribute to HCV antiviral resistance and pathogenesis.
ICP0, ICP4, or VP16 Expressed from Adenovirus Vectors Induces Reactivation of Latent Herpes Simplex Virus Type 1 in Primary Cultures of Latently Infected Trigeminal Ganglion Cells
Authors: Halford William P. , Kemp Clinton D. , Isler Jennifer A. , Davido David J. , Schaffer Priscilla A.
Published: July 2002

In a previous study, we demonstrated that infected-cell polypeptide 0 (ICP0) is necessary for the efficient reactivation of herpes simplex virus type 1 (HSV-1) in primary cultures of latently infected trigeminal ganglion (TG) cells (W. P. Halford and P. A. Schaffer, J. Virol. 75:3240–3249, 2001). The present study was undertaken to determine whether ICP0 is sufficient to trigger HSV-1 reactivation in latently infected TG cells. To test this hypothesis, replication-defective adenovirus vectors that express wild-type and mutant forms of ICP0 under the control of a tetracycline response element (TRE) promoter were constructed. Similar adenovirus vectors encoding wild-type ICP4, wild-type and mutant forms of the HSV-1 origin-binding protein (OBP), and wild-type and mutant forms of VP16 were also constructed. The TRE promoter was induced by coinfection of Vero cells with the test vector and an adenovirus vector that expresses the reverse tetracycline-regulated transactivator in the presence of doxycycline. Northern blot analysis demonstrated that transcription of the OBP gene in the adenovirus expression vector increased as a function of doxycycline concentration over a range of 0.1 to 10 μM. Likewise, Western blot analysis demonstrated that addition of 3 μM doxycycline to adenovirus vector-infected Vero cells resulted in a 100-fold increase in OBP expression. Wild-type forms of ICP0, ICP4, OBP, and VP16 expressed from adenovirus vectors were functional based on their ability to complement plaque formation in Vero cells by replication-defective HSV-1 strains with mutations in these genes. Adenovirus vectors that express wild-type forms of ICP0, ICP4, or VP16 induced reactivation of HSV-1 in 86% ± 5%, 86% ± 5%, and 97% ± 5% of TG cell cultures, respectively (means ± standard deviations). In contrast, vectors that express wild-type OBP or mutant forms of ICP0, OBP, or VP16 induced reactivation in 5% ± 5%, 8% ± 0%, 0% ± 0%, and 13% ± 6% of TG cell cultures, respectively. In control infections, an adenovirus vector expressed green fluorescent protein efficiently in TG neurons but did not induce HSV-1 reactivation. Therefore, expression of ICP0, ICP4, or VP16 is sufficient to induce HSV-1 reactivation in latently infected TG cell cultures. We conclude that this system provides a powerful tool for determining which cellular and viral proteins are sufficient to induce HSV-1 reactivation from neuronal latency.
Requirement for Uracil-DNA Glycosylase during the Transition to Late-Phase Cytomegalovirus DNA Replication
Authors: Courcelle Charmain Tan , Courcelle Justin , Prichard Mark N. , Mocarski Edward S.
Published: July 2002

Cytomegalovirus gene UL114, a homolog of mammalian uracil-DNA glycosylase (UNG), is required for efficient viral DNA replication. In quiescent fibroblasts, UNG mutant virus replication is delayed for 48 h and follows the virus-induced expression of cellular UNG. In contrast, mutant virus replication proceeds without delay in actively growing fibroblasts that express host cell UNG. In the absence of viral or host cell UNG expression, mutant virus fails to proceed to late-phase DNA replication, characterized by rapid DNA amplification. The data suggest that uracil incorporated early during wild-type viral DNA replication must be removed by virus or host UNG prior to late-phase amplification and encapsidation into progeny virions. The process of uracil incorporation and excision may introduce strand breaks to facilitate the transition from early-phase replication to late-phase amplification.
Identification of Aleutian Mink Disease Parvovirus Capsid Sequences Mediating Antibody-Dependent Enhancement of Infection, Virus Neutralization, and Immune Complex Formation
Authors: Bloom Marshall E. , Best Sonja M. , Hayes Stanley F. , Wells Richard D. , Wolfinbarger James B. , McKenna Robert , Agbandje-McKenna Mavis
Published: July 2002

Aleutian mink disease parvovirus (ADV) causes a persistent infection associated with circulating immune complexes, immune complex disease, hypergammaglobulinemia, and high levels of antiviral antibody. Although antibody can neutralize ADV infectivity in Crandell feline kidney cells in vitro, virus is not cleared in vivo, and capsid-based vaccines have proven uniformly ineffective. Antiviral antibody also enables ADV to infect macrophages, the target cells for persistent infection, by Fc-receptor-mediated antibody-dependent enhancement (ADE). The antibodies involved in these unique aspects of ADV pathogenesis may have specific targets on the ADV capsid. Prominent differences exist between the structure of ADV and other, more-typical parvoviruses, which can be accounted for by short peptide sequences in the flexible loop regions of the capsid proteins. In order to determine whether these short sequences are targets for antibodies involved in ADV pathogenesis, we studied heterologous antibodies against several peptides present in the major capsid protein, VP2. Of these antibodies, a polyclonal rabbit antibody to peptide VP2:428-446 was the most interesting. The anti-VP2:428-446 antibody aggregated virus particles into immune complexes, mediated ADE, and neutralized virus infectivity in vitro. Thus, antibody against this short peptide can be implicated in key facets of ADV pathogenesis. Structural modeling suggested that surface-exposed residues of VP2:428-446 are readily accessible for antibody binding. The observation that antibodies against a single target peptide in the ADV capsid can mediate both neutralization and ADE may explain the failure of capsid-based vaccines.
Infectious Simian/Human Immunodeficiency Virus with Human Immunodeficiency Virus Type 1 Subtype C from an African Isolate: Rhesus Macaque Model
Authors: Ndung'u Thumbi , Lu Yichen , Renjifo Boris , Touzjian Neal , Kushner Nicholas , Pena-Cruz Victor , Novitsky Vladimir A. , Lee Tun-Hou , Essex Max
Published: July 2002

Human immunodeficiency virus type 1 (HIV-1) subtype C is responsible for more than 56% of all infections in the HIV and AIDS pandemic. It is the predominant subtype in the rapidly expanding epidemic in southern Africa. To develop a relevant model that would facilitate studies of transmission, pathogenesis, and vaccine development for this subtype, we generated SHIV
MJ4
, a simian/human immunodeficiency virus (SHIV) chimera based on HIV-1 subtype C. SHIV
MJ4
contains the majority of
env
, the entire second exon of
tat
, and a partial sequence of the second exon of
rev
, all derived from a CCR5-tropic, primary isolate envelope clone from southern Africa. SHIV
MJ4
replicated efficiently in human, rhesus, and pig-tailed macaque peripheral blood mononuclear cells (PBMCs) in vitro but not in CEMx174 cells. To assess in vivo infectivity, SHIV
MJ4
was intravenously inoculated into four rhesus macaques (
Macaca mulatta
). All four animals became infected as determined through virus isolation, PCR analysis, and viral loads of 10
7
to 10
8
copies of viral RNA per ml of plasma during the primary infection phase. We have established a CCR5-tropic SHIV
MJ4
/rhesus macaque model that may be useful in the studies of HIV-1 subtype C immunology and biology and may also facilitate the evaluation of vaccines to control the spread of HIV-1 subtype C in southern Africa and elsewhere.
Rat CRM1 Is Responsible for the Poor Activity of Human T-Cell Leukemia Virus Type 1 Rex Protein in Rat Cells
Authors: Hakata Yoshiyuki , Yamada Masami , Shida Hisatoshi
Published: July 2002

Rat models of human T-cell leukemia virus type 1 (HTLV-1)-related diseases such as adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis have been reported. However, these models do not completely reproduce human diseases partly because HTLV-1 replicates poorly in rats. We investigated here the possible reason for this. We found that the activity of Rex in rat cells is quite low compared to that in human cells. As Rex function depends largely on the CRM1 protein, whose human type (human CRM1 [hCRM1]) directly binds to Rex and exports it from the nucleus to the cytoplasm, we assessed whether rat CRM1 (rCRM1) could act as well as hCRM1 as a cofactor for Rex activity. We first cloned a cDNA encoding rCRM1 and found that both rCRM1 and hCRM1 could bind to and export Rex protein to the cytoplasm with similar efficiencies. However, unlike hCRM1, rCRM1 could hardly support Rex function because of its poor ability in inducing the Rex-Rex interaction required for RNA export into the cytoplasm. These observations suggest that the poor ability of rCRM1 to act as a cofactor for Rex function may be responsible for the poor replication of HTLV-1 in rats.
Functional Specialization and Evolution of Leader Proteinases in the Family
Closteroviridae
Authors: Peng Chih-Wen , Peremyslov Valera V. , Mushegian Arcady R. , Dawson William O. , Dolja Valerian V.
Published: July 2002

Members of the
Closteroviridae
and
Potyviridae
families of the plant positive-strand RNA viruses encode one or two papain-like leader proteinases. In addition to a C-terminal proteolytic domain, each of these proteinases possesses a nonproteolytic N-terminal domain. We compared functions of the several leader proteinases using a gene swapping approach. The leader proteinase (L-Pro) of
Beet yellows virus
(BYV; a closterovirus) was replaced with L1 or L2 proteinases of
Citrus tristeza virus
(CTV; another closterovirus), P-Pro proteinase of
Lettuce infectious yellows virus
(LIYV; a crinivirus), and HC-Pro proteinase of
Tobacco etch virus
(a potyvirus). Each foreign proteinase efficiently processed the chimeric BYV polyprotein in vitro. However, only L1 and P-Pro, not L2 and HC-Pro, were able to rescue the amplification of the chimeric BYV variants. The combined expression of L1 and L2 resulted in an increased RNA accumulation compared to that of the parental BYV. Remarkably, this L1-L2 chimera exhibited reduced invasiveness and inability to move from cell to cell. Similar analyses of the BYV hybrids, in which only the papain-like domain of L-Pro was replaced with those derived from L1, L2, P-Pro, and HC-Pro, also revealed functional specialization of these domains. In subcellular-localization experiments, distinct patterns were observed for the leader proteinases of BYV, CTV, and LIYV. Taken together, these results demonstrated that, in addition to a common proteolytic activity, the leader proteinases of closteroviruses possess specialized functions in virus RNA amplification, virus invasion, and cell-to-cell movement. The phylogenetic analysis suggested that functionally distinct L1 and L2 of CTV originated by a gene duplication event.
Hantavirus Nucleocapsid Protein Is Expressed as a Membrane-Associated Protein in the Perinuclear Region
Authors: Ravkov Eugene V. , Compans Richard W.
Published: July 2002

Black Creek Canal virus (BCCV) is a New World hantavirus which is associated with hantavirus pulmonary syndrome. We have examined the site of expression of the BCCV nucleocapsid protein (NBCCV) in the absence of BCCV glycoproteins and found that the majority of the protein is localized to the Golgi region. Immunofluorescence analysis of BHK21 cells expressing the NBCCV and La Crosse virus nucleocapsid protein (NLACV) showed different intracellular localization patterns of these proteins within the same cell: NLACV is cytoplasmic, whereas NBCCV is perinuclear. NBCCV was found to be colocalized with α-mannosidase II, a marker for the Golgi complex. Also, NBCCV was found to be associated with microsomal membranes following cell fractionation. Sedimentation analysis in density gradients revealed that the membrane association of NBCCV is sensitive to treatments with high-salt and high-pH solutions, which indicates that NBCCV is a peripheral membrane protein. Analysis of NBCCV truncation mutants revealed that the 141-amino-acid C-terminal portion of this protein was capable of targeting green fluorescent protein to the perinuclear region. The difference in the intracellular localization between the NBCCV and NLACV proteins suggests that the mechanisms involved in the morphogenesis of New World hantaviruses are distinct from that documented for other members of the
Bunyaviridae
family.
Increased Expression of MIP-1α and MIP-1β mRNAs in the Brain Correlates Spatially and Temporally with the Spongiform Neurodegeneration Induced by a Murine Oncornavirus
Authors: Askovic Srdjan , Favara Cynthia , McAtee Frank J. , Portis John L.
Published: July 2002

The chimeric murine oncornavirus FrCasEcauses a rapidly progressive paralytic disease associated with spongiform neurodegeneration throughout the neuroaxis. Neurovirulence is determined by the sequence of the viral envelope gene and by the capacity of the virus to infect microglia. The neurocytopathic effect of this virus appears to be indirect, since the cells which degenerate are not infected. In the present study we have examined the possible role of inflammatory responses in this disease and have used as a control the virus F43. F43 is an highly neuroinvasive but avirulent virus which differs from FrCasEonly in 3′polandenvsequences. Like FrCasE, F43 infects large numbers of microglial cells, but it does not induce spongiform neurodegeneration. RNAase protection assays were used to detect differential expression of genes encoding a variety of cytokines, chemokines, and inflammatory cell-specific markers. Tumor necrosis factor alpha (TNF-α) and TNF-β mRNAs were upregulated in advanced stages of disease but not early, even in regions with prominent spongiosis. Surprisingly there was no evidence for upregulation of the cytokines interleukin-1α (IL-1α), IL-1β, and IL-6 or of the microglial marker F4/80 at any stage of this disease. In contrast, increased levels of the β-chemokines MIP-1α and -β were seen early in the disease and were concentrated in regions of the brain rich in spongiosis, and the magnitude of responses was similar to that observed in the brains of mice injected with the glutamatergic neurotoxin ibotenic acid. MIP-1α and MIP-1β mRNAs were also upregulated in F43-inoculated mice, but the responses were three- to fivefold lower and occurred later in the course of infection than was observed in FrCasE-inoculated mice. These results suggest that the robust increase in expression of MIP-1α and MIP-1β in the brain represents a correlate of neurovirulence in this disease, whereas the TNF responses are likely secondary events.
Random, Asynchronous, and Asymmetric Transcriptional Activity of Enhancer-Flanking Major Immediate-Early Genes
ie1/3
and
ie2
during Murine Cytomegalovirus Latency in the Lungs
Authors: Grzimek Natascha K. A. , Dreis Doris , Schmalz Susanne , Reddehase Matthias J.
Published: July 2002

The lungs are a major organ site of cytomegalovirus (CMV) pathogenesis, latency, and recurrence. Previous work on murine CMV latency has documented a high load and an even distribution of viral genomes in the lungs after the resolution of productive infection. Initiation of the productive cycle requires expression of the
ie1/3
transcription unit, which is driven by the immediate-early (IE) promoter P
1/3
and generates IE1 and IE3 transcripts by differential splicing. Latency is molecularly defined by the absence of IE3 transcripts specifying the essential transactivator protein IE3. In contrast, IE1 transcripts were found to be generated focally and randomly, reflecting sporadic P
1/3
activity. Selective generation of IE1 transcripts implies molecular control of latency operating after
ie1/3
transcription initiation. P
1/3
is regulated by an upstream enhancer. It is widely assumed that the viral transcriptional program is started by activation of the enhancer through the binding of transcription factors. Accordingly, stochastic transcription during latency might reflect episodes of enhancer activation by the “noise” activity of intrinsic transcription factors. In addition to
ie1/3
, the enhancer controls gene
ie2
, which has its own promoter, P
2
, and is transcribed in opposite direction. We show here that
ie2
is also randomly transcribed during latency. Notably, however,
ie1
and
ie2
were found to be expressed independently. We infer from this finding that expression of the major IE genes is regulated asymmetrically and asynchronously via the combined control unit P
1/3
-E-P
2
. Our data are consistent with a stochastic nature of enhancer action as it is proposed by the “binary” or probability model.
Mitochondrial Aconitase Binds to the 3′ Untranslated Region of the Mouse Hepatitis Virus Genome
Authors: Nanda Santosh K. , Leibowitz Julian L.
Published: July 2002

Mouse hepatitis virus (MHV), a member of theCoronaviridae, contains a polyadenylated positive-sense single-stranded genomic RNA which is 31 kb long. MHV replication and transcription take place via the synthesis of negative-strand RNA intermediates from a positive-strand genomic template. Acis-acting element previously identified in the 3′ untranslated region binds totrans-acting host factors from mouse fibroblasts and forms at least three RNA-protein complexes. The largest RNA-protein complex formed by thecis-acting element and the lysate from uninfected mouse fibroblasts has a molecular weight of about 200 kDa. The complex observed in gel shift assays has been resolved by second-dimension sodium dodecyl sulfate-polyacrylamide gel electrophoresis into four proteins of approximately 90, 70, 58, and 40 kDa after RNase treatment. Specific RNA affinity chromatography also has revealed the presence of a 90-kDa protein associated with RNA containing thecis-acting element bound to magnetic beads. The 90-kDa protein has been purified from uninfected mouse fibroblast crude lysates. Protein microsequencing identified the 90-kDa protein as mitochondrial aconitase. Antibody raised against purified mitochondrial aconitase recognizes the RNA-protein complex and the 90-kDa protein, which can be released from the complex by RNase digestion. Furthermore, UV cross-linking studies indicate that highly purified mitochondrial aconitase binds specifically to the MHV 3′ protein-binding element. Increasing the intracellular level of mitochondrial aconitase by iron supplementation resulted in increased RNA-binding activity in cell extracts and increased virus production as well as viral protein synthesis at early hours of infection. These results are particularly interesting in terms of identification of an RNA target for mitochondrial aconitase, which has a cytoplasmic homolog, cytoplasmic aconitase, also known as iron regulatory protein 1, a well-recognized RNA-binding protein. The binding properties of mitochondrial aconitase and the functional relevance of RNA binding appear to parallel those of cytoplasmic aconitase.
Herpes Simplex Virus Type 1 Promoter Activity during Latency Establishment, Maintenance, and Reactivation in Primary Dorsal Root Neurons In Vitro
Authors: Arthur Jane L. , Scarpini Cinzia G. , Connor Vivienne , Lachmann Robin H. , Tolkovsky Aviva M. , Efstathiou Stacey
Published: July 2002

A neonatal rat dorsal root ganglion-derived neuronal culture system has been utilized to study herpes simplex virus (HSV) latency establishment, maintenance, and reactivation. We present our initial characterization of viral gene expression in neurons following infection with replication-defective HSV recombinants carrying β-galactosidase and/or green fluorescent protein reporter genes under the control of lytic cycle- or latency-associated promoters. In this system lytic virus reporter promoter activity was detected in up to 58% of neurons 24 h after infection. Lytic cycle reporter promoters were shut down over time, and long-term survival of neurons harboring latent virus genomes was demonstrated. Latency-associated promoter-driven reporter gene expression was detected in neurons from early times postinfection and was stably maintained in up to 83% of neurons for at least 3 weeks. In latently infected cultures, silent lytic cycle promoters could be activated in up to 53% of neurons by nerve growth factor withdrawal or through inhibition of histone deacetylases by trichostatin A. We conclude that the use of recombinant viruses containing reporter genes, under the regulation of lytic and latency promoter control in neuronal cultures in which latency can be established and reactivation can be induced, is a potentially powerful system in which to study the molecular events that occur during HSV infection of neurons.
Envelope Glycoprotein Determinants of Neutralization Resistance in a Simian-Human Immunodeficiency Virus (SHIV-HXBc2P 3.2) Derived by Passage in Monkeys
Authors: Si Zhihai , Cayabyab Mark , Sodroski Joseph
Published: July 2002

The simian-human immunodeficiency virus SHIV-HXBc2 contains the envelope glycoproteins of a laboratory-adapted, neutralization-sensitive human immunodeficiency virus type 1 variant, HXBc2. Serial in vivo passage of the nonpathogenic SHIV-HXBc2 generated SHIV KU-1, which causes rapid CD4+T-cell depletion and an AIDS-like illness in monkeys. A molecularly cloned pathogenic SHIV, SHIV-HXBc2P 3.2, was derived from the SHIV KU-1 isolate and differs from the parental SHIV-HXBc2 by only 12 envelope glycoprotein amino acid residues. Relative to SHIV-HXBc2, SHIV-HXBc2P 3.2 was resistant to neutralization by all of the antibodies tested with the exception of the 2G12 antibody. The sequence changes responsible for neutralization resistance were located in variable regions of the gp120 exterior envelope glycoprotein and in the gp41 transmembrane envelope glycoprotein. The 2G12 antibody, which neutralized SHIV-HXBc2 and SHIV-HXBc2P 3.2 equally, bound the HXBc2 and HXBc2P 3.2 envelope glycoproteins on the cell surface comparably. The ability of the other tested antibodies to achieve saturation was less for the HXBc2P 3.2 envelope glycoproteins than for the HXBc2 envelope glycoproteins, even though the affinity of the antibodies for the two envelope glycoproteins was similar. Thus, a highly neutralization-sensitive SHIV, by modifying both gp120 and gp41 glycoproteins, apparently achieves a neutralization-resistant state by decreasing the saturability of its envelope glycoproteins by antibodies.
Discordance between Frequency of Human Immunodeficiency Virus Type 1 (HIV-1)-Specific Gamma Interferon-Producing CD4+T Cells and HIV-1-Specific Lymphoproliferation in HIV-1-Infected Subjects with Active Viral Replication
Authors: Palmer B. E. , Boritz E. , Blyveis N. , Wilson C. C.
Published: July 2002

One hallmark of uncontrolled, chronic human immunodeficiency virus type 1 (HIV-1) infection is the absence of strong HIV-1-specific, CD4+T-cell-proliferative responses, yet the mechanism underlying this T helper (Th)-cell defect remains controversial. To better understand the impact of HIV-1 replication on Th-cell function, we compared the frequency of CD4+Th-cell responses based on production of gamma interferon to lymphoproliferative responses directed against HIV-1 proteins in HIV-1-infected subjects with active in vivo viral replication versus those on suppressed highly active antiretroviral therapy (HAART). No statistically significant differences in the frequencies of cytokine-secreting, HIV-1-specific CD4+T cells between the donor groups were found, despite differences in viral load and treatment status. However, HIV-1-specific lymphoproliferative responses were significantly greater in the subjects with HAART suppression than in subjects with active viral replication. Similar levels of HIV-1 RNA were measured in T-cell cultures stimulated with HIV-1 antigens regardless of donor in vivo viral loads, but only HIV-1-specific CD4+T cells from subjects with HAART suppression proliferated in vitro, suggesting that HIV-1 replication in vitro does not preclude HIV-1-specific lymphoproliferation. This study demonstrates a discordance between the frequency and proliferative capacity of HIV-1-specific CD4+T cells in subjects with ongoing in vivo viral replication and suggests that in vivo HIV-1 replication contributes to the observed defect in HIV-1-specific CD4+T-cell proliferation.