bluuebunny/crossref_metadata_2025 huggingface dataset:
Exacerbation of Autoantibody-Mediated Hemolytic Anemia by Viral Infection
Authors: Meite Mory , Léonard Sabine , Idrissi Mohammed El Azami El , Izui Shozo , Masson Pierre L. , Coutelier Jean-Paul
Published: July 2002

Strong enhancement of the pathogenicity of an antierythrocyte monoclonal antibody was observed after infection of mice with lactate dehydrogenase-elevating virus. While injection of the antierythrocyte antibody alone induced only moderate anemia, concomitant infection with this virus, which is harmless in most normal mice, led to a dramatic drop in the hematocrit and to death of infected animals. In vitro and in vivo analyses showed a dramatic increase in the ability of macrophages from infected mice to phagocytose antibody-coated erythrocytes. These results indicate that viruses can trigger the onset of autoimmune disease by enhancing the pathogenicity of autoantibodies. They may explain how unrelated viruses could be implicated in the etiology of autoantibody-mediated autoimmune diseases.
Quantitation of CD8+T-Lymphocyte Responses to Multiple Epitopes from Simian Virus 40 (SV40) Large T Antigen in C57BL/6 Mice Immunized with SV40, SV40 T-Antigen-Transformed Cells, or Vaccinia Virus Recombinants Expressing Full-Length T Antigen or Epitope Minigenes
Authors: Mylin Lawrence M. , Schell Todd D. , Roberts Debra , Epler Melanie , Boesteanu Alina , Collins Edward J. , Frelinger Jeffrey A. , Joyce Sebastian , Tevethia Satvir S.
Published: July 2002

The cytotoxic T-lymphocyte response to wild-type simian virus 40 large tumor antigen (Tag) in C57BL/6 (H2b) mice is directed against threeH2-Db-restricted epitopes, I, II/III, and V, and oneH2-Kb-restricted epitope, IV. Epitopes I, II/III, and IV are immunodominant, while epitope V is immunorecessive. We investigated whether this hierarchical response was established in vivo or was due to differential expansion in vitro by using direct enumeration of CD8+T lymphocytes with Tag epitope/major histocompatibility complex class I tetramers and intracellular gamma interferon staining. The results demonstrate that epitope IV-specific CD8+T cells dominated the Tag-specific response in vivo following immunization with full-length Tag while CD8+T cells specific for epitopes I and II/III were detected at less than one-third of this level. The immunorecessive nature of epitope V was apparent in vivo, since epitope V-specific CD8+T cells were undetectable following immunization with full-length Tag. In contrast, high levels of epitope V-specific CD8+T lymphocytes were recruited in vivo following immunization and boosting with a Tag variant in which epitopes I, II/III, and IV had been inactivated. In addition, analysis of the T-cell receptor β (TCRβ) repertoire of Tag epitope-specific CD8+cells revealed that multiple TCRβ variable regions were utilized for each epitope except Tag epitope II/III, which was limited to TCRβ10 usage. These results indicate that the hierarchy of Tag epitope-specific CD8+T-cell responses is established in vivo.
Mature Dendritic Cells Infected with Herpes Simplex Virus Type 1 Exhibit Inhibited T-Cell Stimulatory Capacity
Authors: Kruse Monika , Rosorius Olaf , Krätzer Friedrich , Stelz Gerhard , Kuhnt Christine , Schuler Gerold , Hauber Joachim , Steinkasserer Alexander
Published: July 2002

Mature dendritic cells (DC) are the most potent antigen-presenting cells within the entire immune system. Interference with the function of these cells therefore constitutes a very powerful mechanism for viruses to escape immune responses. Several members of theHerpesviridaefamily have provided examples of such escape strategies, including interference with antigen presentation and production of homologous cytokines. In this study we investigated the infection of mature DC with herpes simplex virus type 1 (HSV-1) and the way in which infection alters the phenotype and function of mature DC. Interestingly, the T-cell-stimulatory capacity of these DC was strongly impaired. Furthermore, we demonstrated that HSV-1 leads to the specific degradation of CD83, a cell surface molecule which is specifically upregulated during DC maturation. These data indicate that HSV-1 has developed yet another novel mechanism to escape immune responses.
Efficient Homologous RNA Recombination and Requirement for an Open Reading Frame during Replication of Equine Arteritis Virus Defective Interfering RNAs
Authors: Molenkamp Richard , Greve Sophie , Spaan Willy J. M. , Snijder Eric J.
Published: July 2002

Equine arteritis virus
(EAV), the prototype arterivirus, is an enveloped plus-strand RNA virus with a genome of approximately 13 kb. Based on similarities in genome organization and protein expression, the arteriviruses have recently been grouped together with the coronaviruses and toroviruses in the newly established order
Nidovirales
. Previously, we reported the construction of pEDI, a full-length cDNA copy of EAV DI-b, a natural defective interfering (DI) RNA of 5.6 kb (R. Molenkamp et al., J. Virol. 74:3156–3165, 2000). EDI RNA consists of three noncontiguous parts of the EAV genome fused in frame with respect to the replicase gene. As a result, EDI RNA contains a truncated replicase open reading frame (EDI-ORF) and encodes a truncated replicase polyprotein. Since some coronavirus DI RNAs require the presence of an ORF for their efficient propagation, we have analyzed the importance of the EDI-ORF in EDI RNA replication. The EDI-ORF was disrupted at different positions by the introduction of frameshift mutations. These were found either to block DI RNA replication completely or to be removed within one virus passage, probably due to homologous recombination with the helper virus genome. Using recombination assays based on EDI RNA and full-length EAV genomes containing specific mutations, the rates of homologous RNA recombination in the 3′- and 5′-proximal regions of the EAV genome were studied. Remarkably, the recombination frequency in the 5′-proximal region was found to be approximately 100-fold lower than that in the 3′-proximal part of the genome.
Herpes Simplex Virus Type 1 ICP0 Protein Does Not Accumulate in the Nucleus of Primary Neurons in Culture
Authors: Chen Xiao-Ping , Li Jia , Mata Marina , Goss James , Wolfe Darren , Glorioso Joseph C. , Fink David J.
Published: July 2002

Infected-cell protein 0 (ICP0), the product of the herpes simplex virus (HSV) immediate-early (IE) α0 gene, is a promiscuous transactivator of viral early (E) and late (L) gene expression. HSV mutants lacking ICP0 function are severely deficient in viral growth and protein synthesis, particularly at low multiplicities of infection. Early in the infectious process in vitro, ICP0 protein accumulates in distinct domains within the nucleus to form characteristic structures active in the transcription of viral genes. However, following infection of primary trigeminal ganglion cells in vitro with a recombinant HSV mutant that expresses only ICP0, we observed that ICP0 protein accumulated in the characteristic intranuclear distribution only in the nuclei of Schwann cells; neurons in the culture did not accumulate ICP0 despite expression of ICP0 RNA in those cells. The same phenomenon was observed in PC12 cells differentiated to assume a neuronal phenotype. In primary neurons in culture, the amount of ICP0 protein could be increased by pharmacologic inhibition of calcium-activated protease (calpain) activity or by inhibition of protein phosphatase 2B (calcineurin). The failure of ICP0 protein to accumulate in the nucleus of neurons suggests that one mechanism which may impair efficient replication of the virus in neurons, and thus favor the establishment of viral latency in those cells, may be found in the cell-specific processing of that IE gene product.
Incorporation of HLA-DR into the Envelope of Human Immunodeficiency Virus Type 1 In Vivo: Correlation with Stage of Disease and Presence of Opportunistic Infection
Authors: Lawn Stephen D. , Butera Salvatore T.
Published: July 2002

Human immunodeficiency virus type 1 (HIV-1) bearing HLA-DR in its envelope was detected in plasma from all patients with chronic HIV-1 infection (n= 16) and was present at higher levels in patients with active tuberculosis coinfection (n= 6). Intriguingly, however, HLA-DR was not detectable in HIV-1 from patients during primary viremia (n= 6), suggesting the possibility of virus replication in less-activated cells.
RNA Binding Properties of Bunyamwera Virus Nucleocapsid Protein and Selective Binding to an Element in the 5′ Terminus of the Negative-Sense S Segment
Authors: Osborne Jane C. , Elliott Richard M.
Published: July 2002

The genome ofBunyamwera virus(BUN) (familyBunyaviridae, genusBunyavirus) comprises three negative-sense RNA segments which act as transcriptional templates for the viral polymerase only when encapsidated by the nucleocapsid protein (N). Previous studies have suggested that the encapsidation signal may reside within the 5′ terminus of each segment. The BUN N protein was expressed as a 6-histidine-tagged fusion protein inEscherichia coliand purified by metal chelate chromatography. An RNA probe containing the 5′-terminal 32 and 3′-terminal 33 bases of the BUN S (small) genome segment was used to investigate binding by the N protein in vitro using gel mobility shift and filter binding assays. On acrylamide gels a number of discrete RNA-N complexes were resolved, and analysis of filter binding data indicated a degree of cooperativity in N protein binding. RNA-N complexes were resistant to digestion with up to 1 μg of RNase A per ml. Competition assays with a variety of viral and nonviral RNAs identified a region within the 5′ terminus of the BUN S segment for which N had a high preference for binding. This site may constitute the signal for initiation of encapsidation by N.
Importance of Membrane Fusion Mediated by Human Immunodeficiency Virus Envelope Glycoproteins for Lysis of Primary CD4-Positive T Cells
Authors: LaBonte Jason A. , Patel Trushar , Hofmann Wolfgang , Sodroski Joseph
Published: July 2002

In established T-cell lines, the membrane-fusing capacity of the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins mediates cytopathic effects, both syncytium formation and single-cell lysis. Furthermore, changes in the HIV-1 envelope glycoproteins are responsible for the increased CD4+T-cell-depleting ability observed in infected monkeys upon in vivo passage of simian-human immunodeficiency virus (SHIV) chimeras. In this study, a panel of SHIV envelope glycoproteins and their mutant counterparts defective in membrane-fusing capacity were expressed in primary human CD4+T cells. Compared with controls, all of the functional HIV-1 envelope glycoproteins induced cell death in primary CD4+T-cell cultures, whereas the membrane fusion-defective mutants did not. Death occurred almost exclusively in envelope glycoprotein-expressing cells and not in bystander cells. Under standard culture conditions, most dying cells underwent lysis as single cells. When the cells were cultured at high density to promote syncytium formation, the envelope glycoproteins of the passaged, pathogenic SHIVs induced more syncytia than those of the respective parental SHIV. These results demonstrate that the HIV-1 envelope glycoproteins induce the death of primary CD4+T lymphocytes by membrane fusion-dependent processes.
DNA-Dependent Protein Kinase Is Not Required for Efficient Lentivirus Integration
Authors: Baekelandt Veerle , Claeys Anje , Cherepanov Peter , De Clercq Erik , De Strooper Bart , Nuttin Bart , Debyser Zeger
Published: July 2002

How DNA is repaired after retrovirus integration is not well understood. DNA-dependent protein kinase (DNA-PK) is known to play a central role in the repair of double-stranded DNA breaks. Recently, a role for DNA-PK in retroviral DNA integration has been proposed (R. Daniel, R. A. Katz, and A. M. Skalka, Science 284:644–647, 1999). Reduced transduction efficiency and increased cell death by apoptosis were observed upon retrovirus infection of cultured
scid
cells. We have used a human immunodeficiency virus (HIV) type 1 (HIV-1)-derived lentivirus vector system to further investigate the role of DNA-PK during integration. We measured lentivirus transduction of
scid
mouse embryonic fibroblasts (MEF) and xrs-5 or xrs-6 cells. These cells are deficient in the catalytic subunit of DNA-PK and in Ku, the DNA-binding subunit of DNA-PK, respectively. At low vector titers, efficient and stable lentivirus transduction was obtained, excluding an essential role for DNA-PK in lentivirus integration. Likewise, the efficiency of transduction of HIV-derived vectors in
scid
mouse brain was as efficient as that in control mice, without evidence of apoptosis. We observed increased cell death in
scid
MEF and xrs-5 or xrs-6 cells, but only after transduction with high vector titers (multiplicity of infection [MOI], >1 transducing unit [TU]/cell) and subsequent passage of the transduced cells. At an MOI of <1 TU/cell, however, transduction efficiency was even higher in DNA-PK-deficient cells than in control cells. Taken together, the data suggest a protective role of DNA-PK against cellular toxicity induced by high levels of retrovirus integrase or integration. Another candidate cellular enzyme that has been claimed to play an important role during retrovirus integration is poly(ADP-ribose) polymerase (PARP). However, no inhibition of lentivirus vector-mediated transduction or HIV-1 replication by 3-methoxybenzamide, a known PARP inhibitor, was observed. In conclusion, DNA-PK and PARP are not essential for lentivirus integration.
Memory in Viral Quasispecies
Authors: Ruiz-Jarabo Carmen M. , Arias Armando , Baranowski Eric , Escarmís Cristina , Domingo Esteban
Published: July 2002

Biological adaptive systems share some common features: variation among their constituent elements and continuity of core information. Some of them, such as the immune system, are endowed with memory of past events. In this study we provide direct evidence that evolving viral quasispecies possess a molecular memory in the form of minority components that populate their mutant spectra. The experiments have involved foot-and-mouth disease virus populations with known evolutionary histories. The composition and behavior of the viral population in response to a selective constraint were influenced by past evolutionary history in a way that could not be predicted from examination of consensus nucleotide sequences of the viral populations. The molecular memory of the viral quasispecies influenced both the nature and the intensity of the response of the virus to a selective constraint.
Envelope Glycoprotein Determinants of Increased Fusogenicity in a Pathogenic Simian-Human Immunodeficiency Virus (SHIV-KB9) Passaged In Vivo
Authors: Etemad-Moghadam Bijan , Sun Ying , Nicholson Emma K. , Fernandes Mark , Liou Kwa , Gomila Raul , Lee Juliette , Sodroski Joseph
Published: July 2002

Changes in the envelope glycoprotein ectodomains of a nonpathogenic simian-human immunodeficiency virus (SHIV-89.6) that was serially passaged in vivo have been shown to be responsible for the increased pathogenicity of the resulting virus, SHIV-KB9 (G. B. Karlsson, et al., J. Exp. Med. 188:1159–1171, 1998). The 12 amino acid changes in the envelope glycoprotein ectodomains resulted in increased chemokine receptor-binding and syncytium-forming abilities. Here we identify the envelope glycoprotein determinants of these properties. A single amino acid change in the gp120 third variable (V3) loop was both necessary and sufficient for the observed increase in the binding of the SHIV-KB9 gp120 glycoprotein to the CCR5 chemokine receptor. The increased syncytium-forming ability of SHIV-KB9 involved, in addition to the V3 loop change, changes in the second conserved (C2) region of gp120 (residue 225) and in the gp41 ectodomain (residues 564 and 567). The C2 and gp41 ectodomain changes influenced syncytium formation in a cooperative manner. Changes in the V1/V2 gp120 variable loops exerted a negative effect on syncytium formation and chemokine receptor binding, supporting a previously described role of these changes in immune evasion. The definition of the passage-associated changes that determine the efficiency of chemokine receptor binding and membrane fusogenicity will allow evaluation of the contribution of these properties to in vivo CD4-positive lymphocyte depletion.
Envelope Protein-Mediated Down-Regulation of Hepatitis B Virus Receptor in Infected Hepatocytes
Authors: Breiner Klaus M. , Urban Stephan , Glass Bärbel , Schaller Heinz
Published: July 2002

Entry of duck hepatitis B virus (DHBV) is initiated by specific interaction of its large envelope protein (L) with a cellular entry receptor, recently identified as carboxypeptidase D (CPD; historically gp180). In this report, we present evidence demonstrating that this receptor is down-regulated as a result of DHBV infection: (i) receptor levels determined by Western blot were much reduced in DHBV-infected duck livers and undetectable by immunostaining in infected cultured hepatocytes; (ii) results from metabolic labeling experiments indicate enhanced receptor protein turnover; (iii) the kinetics of receptor loss from newly infected cells correlated with the accumulation of newly synthesized viral protein; (iv) expression of DHBV L protein, transduced from a recombinant adenovirus, was sufficient to eliminate gp180/CPD from the Golgi compartment, its normal predominant location; (v) gp180/CPD remained absent from the Golgi compartment in infected hepatocytes, even after overexpression from a recombinant adenovirus, while residual amounts subsequently became detectable in a perinuclear compartment, containing DHBV L protein; (vi) expression of DHBV L protein in a HepG2 cell line, stably expressing gp180/CPD, leads to incomplete receptor maturation and induces its degradation. Taken together, these data are consistent with a model in which the virus receptor interacts early in the biosynthetic pathway with the viral L protein, leading to its retention in a pre-Golgi compartment and to subsequent degradation, thus preventing receptor interference with the export of DHBV via the secretory pathway which it shares with its receptor. Accordingly, and analogously with receptor down-regulation in retroviral systems, DHBV receptor down-modulation may account for the much-reduced efficiency of DHBV superinfection of preinfected hepatocytes.
Integrin αvβ1 Is an Adenovirus Coreceptor
Authors: Li Erguang , Brown Swati L. , Stupack Dwayne G. , Puente Xose S. , Cheresh David A. , Nemerow Glen R.
Published: July 2002

The human embryonic kidney (HEK293) cell line, commonly used for recombinant adenovirus (Ad) propagation, does not express the Ad coreceptor αvβ3 or αvβ5 integrins, yet these cells are efficiently infected by Ad vectors. Here we demonstrate that Ad binds to HEK293 cells via the fiber receptor CAR and is subsequently internalized via interaction with integrin αvβ1. Function-blocking antibodies directed against αv or β1, but not β3, β5, or α5, integrin subunits block Ad infection and viral endocytosis. Therefore, αvβ1 serves as a coreceptor for Ad infection, and the lack of β3 and/or β5 but the relatively high expression of αvβ1 integrins on certain tumor cell types may explain why these cells are readily transduced by Ad vectors.
Axonal Damage Is T Cell Mediated and Occurs Concomitantly with Demyelination in Mice Infected with a Neurotropic Coronavirus
Authors: Dandekar Ajai A. , Wu Gregory F. , Pewe Lecia , Perlman Stanley
Published: July 2002

Mice infected with mouse hepatitis virus (MHV) strain JHM develop primary demyelination. Herein we show that axonal damage occurred in areas of demyelination and also in adjacent areas devoid of myelin damage. Immunodeficient MHV-infected RAG1−/− mice (mice defective in recombinase activating gene 1 expression) do not develop demyelination unless they receive splenocytes from a mouse previously immunized against MHV (G. F. Wu, A. Dandekar, L. Pewe, and S. Perlman, J. Immunol. 165:2278–2286, 2000). In the present study, we show that adoptive transfer of T cells was also required for the majority of the axonal injury observed in these animals. Both demyelination and axonal damage were apparent by 7 days posttransfer. Recent data suggest that axonal injury is a major factor in the long-term disability observed in patients with multiple sclerosis. Our data demonstrate that immune system-mediated damage to axons is also a common feature in mice with MHV-induced demyelination. Remarkably, there appeared to be a minimal, if any, interval of time between the appearance of demyelination and that of axonal injury.
Nef from Human Immunodeficiency Virus Type 1
F12
Inhibits Viral Production and Infectivity
Authors: Fackler Oliver T. , d'Aloja Paola , Baur Andreas S. , Federico Maurizio , Peterlin B. Matija
Published: July 2002

Human immunodeficiency virus type 1
F12
(HIV-1
F12
) interferes with the replication of other strains of HIV. Its accessory protein, Nef, is sufficient for this phenotype, where the production and infectivity of HIV are impaired significantly. The analysis of three rare mutations in this Nef protein revealed that these effects could be separated genetically. Moreover, the defect in virus production correlated with the lack of processing of the p55
Gag
precursor in the presence of Nef from HIV-1
F12
. Importantly, the introduction of one of these mutations (E177G) into Nef from HIV-1
NL4-3
also created a dominant-negative Nef protein. Effects of Nef from HIV-1
F12
on virus production and Gag processing correlated with its altered subcellular distribution. Moreover, the association with two new cellular proteins with molecular masses of 74 and 75 kDa, which do not interact with other Nef proteins, correlated with the decreased virion infectivity. The identification of a dominant-negative protein for the production and infectivity of HIV suggests that Nef plays an active role at this stage of the viral replicative cycle.
OX40 Stimulation by gp34/OX40 Ligand Enhances Productive Human Immunodeficiency Virus Type 1 Infection
Authors: Takahashi Yoshiaki , Tanaka Yuetsu , Yamashita Atsuya , Koyanagi Yoshio , Nakamura Masataka , Yamamoto Naoki
Published: July 2002

OX40 is a member of the tumor necrosis factor (TNF) receptor superfamily and known to be an important costimulatory molecule expressed on activated T cells. To investigate the role of costimulation of OX40 in human immunodeficiency virus type 1 (HIV-1) infection by its natural ligand, gp34, the OX40-transfected ACH-2 cell line, ACH-2/OX40, chronically infected with HIV-1, was cocultured with paraformaldehyde (PFA)-fixed gp34-transfected mouse cell line, SV-T2/gp34. The results showed that HIV-1 production was strongly induced. This was followed by apparent apoptosis, and both processes were specifically inhibited by the gp34-specific neutralizing monoclonal antibody 5A8. Endogenous TNF alpha (TNF-α) and TNF-β production were not involved in the enhanced HIV-1 production. Furthermore, enhanced HIV-1 transcription in gp34-stimulated ACH-2/OX40 cells was dependent on the κB site of the HIV-1 long terminal repeat, and the OX40-gp34 interaction activated NF-κB consisting of p50 and p65 subunits. When primary activated CD4+T cells acutely infected with HIV-1NL4-3(CXCR4-using T-cell-line-tropic) were cocultured with PFA-fixed gp34+human T-cell leukemia virus type 1-bearing MT-2 cells or SV-T2/gp34 cells, HIV-1 production was also markedly enhanced. The enhancement was again significantly inhibited by 5A8. The present study first shows that OX40-gp34 interaction stimulates HIV-1 expression and suggests that OX40 triggering by gp34 may play an important role in enhancing HIV-1 production in both acutely and latently infected CD4+T cells in vivo.
The Vaccinia Virus Superoxide Dismutase-Like Protein (A45R) Is a Virion Component That Is Nonessential for Virus Replication
Authors: Almazán Fernando , Tscharke David C. , Smith Geoffrey L.
Published: July 2002

A characterization of the A45R gene from vaccinia virus (VV) strain Western Reserve is presented. The open reading frame is predicted to encode a 125-amino-acid protein (Mr, of 13,600) with 39% amino acid identity to copper-zinc superoxide dismutase (Cu-Zn SOD). Sequencing of the A45R gene from other orthopoxviruses, here and by others, showed that the protein is highly conserved in all viruses sequenced, including 16 strains of VV, 2 strains of cowpox virus, camelpox virus, and 4 strains of variola virus. In all cases the protein lacks key residues involved in metal ion binding that are important for the catalytic activity. The A45R protein was expressed inEscherichia coli, purified, and tested for SOD activity, but neither enzymatic nor inhibitory SOD activity was detected. Additionally, no virus-encoded SOD activity was detected in infected cells or purified virions. A monoclonal antibody raised against the A45R protein expressed inE. coliidentified the A45R gene product as a 13.5-kDa protein that is expressed late during VV infection. Confocal microscopy of VV-infected cells indicated that the A45R protein accumulated predominantly in cytoplasmic viral factories. Electron microscopy and biochemical analyses showed that the A45R protein is incorporated into the virion core. A deletion mutant lacking the majority of the A45R gene and a revertant virus in which the deleted gene was restored were constructed and characterized. The growth properties of the deletion mutant virus were indistinguishable from those of wild-type and revertant viruses in all cell lines tested, including macrophages. Additionally, the virulence and pathogenicity of the three viruses were also comparable in murine and rabbit models of infection. A45R is unusual in being the first VV core protein described that affects neither virus replication nor virulence.
Hydrophobic Amino Acids in the Human Immunodeficiency Virus Type 1 p2 and Nucleocapsid Proteins Can Contribute to the Rescue of Deleted Viral RNA Packaging Signals
Authors: Rong Liwei , Russell Rodney S. , Hu Jing , Guan Yongjun , Kleiman Lawrence , Liang Chen , Wainberg Mark A.
Published: July 2002

An RNA fragment of 75 nucleotides, which is located between the primer binding site and the 5′ major splice donor site in human immunodeficiency virus type 1, has been shown to participate in specific encapsidation of viral RNA. Compensation studies have identified two second-site mutations, namely, MP2 (a T12I substitution in p2) and MNC (a T24I substitution in the nucleocapsid [NC] protein) that were involved in the rescue of various deletions in the aforementioned RNA region (i.e., BH-D1, BH-D2, and BH-LD3). To study whether the MP2 and MNC point mutations exert their compensatory effects in a
cis
manner, production of Gag proteins was blocked by insertion of stop codons into LD3, LD3-MP2-MNC, and wild-type BH10 such that the constructs generated, i.e., LD3-DG, LD3-MP2-MNC-DG, and BH-DG, only provided RNA transcripts for packaging. The results of cotransfection experiments showed that the LD3-MP2-MNC-DG viral RNA was packaged as inefficiently as LD3-DG; in contrast, BH-DG was efficiently packaged. Therefore, nucleotide substitutions in MP2 and MNC did not act in a
cis
manner to correct the packaging deficits in LD3. Next, we deliberately changed the T12 in p2 or the T24 in the NC to each of 19 other amino acids. We found that amino acids with long hydrophobic side chains, i.e., V, L, I, and M, were favored at either position 12 in p2 or at position 24 in NC to compensate for the above-mentioned deletions. Further studies showed that only a few amino acids could not be used at these two sites by the wild-type virus due to decreased RNA levels in the virion or abnormal Gag protein processing. In this case, W, D, and E could not substitute for T12 in p2, and S, D, and N could not substitute for T24 in NC, without affecting viral infectivity. Therefore, the long hydrophobic side chains of V, L, I, and M are necessary for these amino acids to rescue the BH-D1, BH-D2, and BH-LD3 mutated viruses.
Feline Leukemia Virus DNA Vaccine Efficacy Is Enhanced by Coadministration with Interleukin-12 (IL-12) and IL-18 Expression Vectors
Authors: Hanlon Linda , Argyle David , Bain Derek , Nicolson Lesley , Dunham Stephen , Golder Matthew C. , McDonald Michael , McGillivray Christine , Jarrett Oswald , Neil James C. , Onions David E.
Published: July 2002

The expectation that cell-mediated immunity is important in the control of feline leukemia virus (FeLV) infection led us to test a DNA vaccine administered alone or with cytokines that favored the development of a Th1 immune response. The vaccine consisted of two plasmids, one expressing thegag/polgenes and the other expressing theenvgene of FeLV-A/Glasgow-1. The genetic adjuvants were plasmids encoding the feline cytokines interleukin-12 (IL-12), IL-18, or gamma interferon (IFN-γ). Kittens were immunized by three intramuscular inoculations of the FeLV DNA vaccine alone or in combination with plasmids expressing IFN-γ, IL-12, or both IL-12 and IL-18. Control kittens were inoculated with empty plasmid. Following immunization, anti-FeLV antibodies were not detected in any kitten. Three weeks after the final immunization, the kittens were challenged by the intraperitoneal inoculation of FeLV-A/Glasgow-1 and were then monitored for a further 15 weeks for the presence of virus in plasma and, at the end of the trial, for latent virus in bone marrow. The vaccine consisting of FeLV DNA with the IL-12 and IL-18 genes conferred significant immunity, protecting completely against transient and persistent viremia, and in five of six kittens protecting against latent infection. None of the other vaccines provided significant protection.
Expression and Function of Chemokine Receptors on Human Thymocytes: Implications for Infection by Human Immunodeficiency Virus Type 1
Authors: Taylor James R. , Kimbrell Katherine C. , Scoggins Robert , Delaney Marie , Wu Lijun , Camerini David
Published: July 2002

The presence or absence of the receptor CD4 and the coreceptors CCR5 and CXCR4 restrict the cell tropism of human immunodeficiency virus type 1 (HIV-1). Despite the importance of thymic infection by HIV-1, conflicting reports regarding the expression of HIV-1 coreceptors on human thymocytes have not been resolved. We assayed the expression and function of the major HIV-1 coreceptors, CCR5 and CXCR4, as well as CCR4 and CCR7 as controls, on human thymocytes. We detected CCR5 on 2.5% of thymocytes, CXCR4 on 53% of the cells, and CCR4 on 16% and CCR7 on 11% of human thymocytes. Moreover, infection by R5 HIV-1 did not significantly induce expression of CCR5. We found that two widely used anti-CCR5 monoclonal antibodies cross-reacted with CCR8, which may account for discrepancies among published reports of CCR5 expression on primary cells. This cross-reactivity could be eliminated by deletion of amino acids 2 through 4 of CCR8. Chemotaxis assays showed that SDF-1, which binds CXCR4; MDC, which binds CCR4; and ELC, which binds CCR7, mediated significant chemotaxis of thymocytes. In contrast, MIP-1β, whose receptor is CCR5, did not induce significant chemotaxis. Our results indicate that CXCR4, CCR4, CCR7, and their chemokine ligands may be involved in thymocyte migration during development in the thymus. CCR5 and its ligands, however, are likely not involved in these processes. Furthermore, the pattern of CCR5 and CXCR4 expression that we found may explain the greater susceptibility of human thymocytes to infection by HIV-1 isolates capable of using CXCR4 in cell entry compared to those that use only CCR5.
The Synthetic Peptide P-197 Inhibits Human T-Cell Leukemia Virus Type 1 Envelope-Mediated Syncytium Formation by a Mechanism That Is Independent of Hsc70
Authors: Brighty David W. , Jassal Sushma R.
Published: July 2002

Entry of human T-cell leukemia virus type 1 (HTLV-1) into cells is mediated by the viral envelope glycoproteins gp46 and gp21. The gp46 surface glycoprotein binds to a poorly characterized cell surface receptor, thereby promoting the gp21-dependent fusion of the viral and cellular membranes. Interestingly, a synthetic peptide (P-197) simulating amino acids 197 to 216 of gp46 strongly inhibits envelope-dependent membrane fusion with Molt-4 target cells. It has been suggested that this peptide acts by competitively binding to Hsc70, a putative cellular receptor for HTLV-1. We now demonstrate that P-197 inhibits membrane fusion among diverse HTLV-1-permissive target cells. Importantly, most of these cells lack detectable levels of Hsc70, indicating that P-197 inhibits membrane fusion by a mechanism that is Hsc70 independent. We now suggest that competition for primary receptor binding is unlikely to account for the inhibitory activity of P-197. Understanding the mechanism by which P-197 functions may reveal concepts of general relevance to antiretroviral chemotherapy.
Widespread Gene Delivery and Structure-Specific Patterns of Expression in the Brain after Intraventricular Injections of Neonatal Mice with an Adeno-Associated Virus Vector
Authors: Passini Marco A. , Wolfe John H.
Published: July 2002

Developing a system for widespread somatic gene transfer in the central nervous system (CNS) would be beneficial for understanding the global influence of exogenous genes on animal models. We injected an adeno-associated virus serotype 2 (AAV2) vector into the cerebral lateral ventricles at birth and mapped its distribution and transduction pattern from a promoter capable of expression in multiple targets. The injections resulted in structure-specific patterns of expression that were maintained for at least 1 year in most regions, with efficient targeting of some of the major principal neuron layers. The patterns of transduction were explained by circulation of the viral vector in the subarachnoid space via CSF flow, followed by transduction of underlying structures, rather than by progenitor cell infection and subsequent migration. This study demonstrates that gene transfer throughout the CNS can be achieved without germ line transmission and establishes an experimental strategy for introducing genes to somatic cells in a highly predictable manner.
Sorting of Marburg Virus Surface Protein and Virus Release Take Place at Opposite Surfaces of Infected Polarized Epithelial Cells
Authors: Sänger Christian , Mühlberger Elke , Ryabchikova Elena , Kolesnikova Larissa , Klenk Hans-Dieter , Becker Stephan
Published: July 2002

Marburg virus, a filovirus, causes severe hemorrhagic fever with hitherto poorly understood molecular pathogenesis. We have investigated here the vectorial transport of the surface protein GP of Marburg virus in polarized epithelial cells. To this end, we established an MDCKII cell line that was able to express GP permanently (MDCK-GP). The functional integrity of GP expressed in these cells was analyzed using vesicular stomatitis virus pseudotypes. Further experiments revealed that GP is transported in MDCK-GP cells mainly to the apical membrane and is released exclusively into the culture medium facing the apical membrane. When MDCKII cells were infected with Marburg virus, the majority of GP was also transported to the apical membrane, suggesting that the protein contains an autonomous apical transport signal. Release of infectious progeny virions, however, took place exclusively at the basolateral membrane of the cells. Thus, vectorial budding of Marburg virus is presumably determined by factors other than the surface protein.
Protein-DNA Binding and CpG Methylation at Nucleotide Resolution of Latency-Associated Promoters Qp, Cp, and LMP1p of Epstein-Barr Virus
Authors: Salamon Daniel , Takacs Maria , Ujvari Dorina , Uhlig Jörg , Wolf Hans , Minarovits Janos , Niller Hans Helmut
Published: July 2002

Epstein-Barr viral (EBV) latency-associated promoters Qp, Cp, and LMP1p are crucial for the regulated expression of the EBNA and LMP transcripts in dependence of the latency type. By transient transfection and in vitro binding analyses, many promoter elements and transcription factors have previously been shown to be involved in the activities of these promoters. However, the latency promoters have only partially been examined at the nucleotide level in vivo. Therefore, we undertook a comprehensive analysis of in vivo protein binding and CpG methylation patterns at these promoters in five representative cell lines and correlated the results with the known in vitro binding data and activities of these promoters from previous transfection experiments. Promoter activity inversely correlated with the methylation state of promoters, although Qp was a remarkable exception. Novel protein binding data were obtained for all promoters. For Cp, binding correlated well with promoter activity; for LMP1p and Qp, binding patterns looked similar regardless of promoter activity.
Modified FGF4 Signal Peptide Inhibits Entry of Herpes Simplex Virus Type 1
Authors: Bultmann Hermann , Busse James S. , Brandt Curtis R.
Published: July 2002

Entry of herpes simplex virus type 1 (HSV-1) into host cells occurs through fusion of the viral envelope with the plasma membrane and involves complex and poorly understood interactions between several viral and cellular proteins. One strategy for dissecting the function of this fusion machine is through the use of specific inhibitors. We identified a peptide with antiviral activity that blocks HSV-1 infection at the entry stage and during cell-to-cell spreading. This peptide (called EB for “entry blocker”) consists of the FGF4 signal sequence with an RRKK tetramer at the amino terminus to improve solubility. The activity of EB depends exclusively but not canonically on the signal sequence. Inhibition of virus entry (hrR3) and plaque formation (KOS) strongly depend on virus concentrations and serum addition, with 50% inhibitory concentrations typically ranging from 1 to 10 μM. Blocking preadsorbed virus requires higher EB concentrations. Cytotoxic effects (trypan blue exclusion) are first noted at 50 μM EB in serum-free medium and at ≥200 μM in the presence of serum. EB does not affect gC-dependent mechanisms of virus attachment and does not block virus attachment at 4°C. Instead, EB directly interacts with virions and inactivates them irreversibly without, however, disrupting their physical integrity as judged by electron microscopy. At subvirucidal concentrations, EB changes the adhesive properties of virions, causing aggregation at high virus concentrations. This peptide may be a useful tool for studying viral entry mechanisms.
Multiple Groups of Novel Retroviral Genomes in Pigs and Related Species
Authors: Patience Clive , Switzer William M. , Takeuchi Yasuhiro , Griffiths David J. , Goward Melanie E. , Heneine Walid , Stoye Jonathan P. , Weiss Robin A.
Published: July 2002

In view of the concern over potential infection hazards in the use of porcine tissues and organs for xenotransplantation to humans, we investigated the diversity of porcine endogenous retrovirus (PERV) genomes in the DNA of domestic pigs and related species. In addition to the three known envelope subgroups of infectious gamma retroviruses (PERV-A, -B, and -C), classed together here as PERV group γ1, four novel groups of gamma retrovirus (γ2 to γ5) and four novel groups of beta retrovirus (β1 to β4) genomes were detected in pig DNA using generic and specific PCR primers. PCR quantification indicated that the retroviral genome copy number in the Landrace × Duroc F
1
hybrid pig ranged from 2 (β2 and γ5) to approximately 50 (γ1). The γ1, γ2, and β4 genomes were transcribed into RNA in adult kidney tissue. Apart from γ1, the retroviral genomes are not known to be infectious, and sequencing of a small number of amplified genome fragments revealed stop codons in putative open reading frames in several cases. Analysis of DNA from wild boar and other species of Old World pigs (
Suidae
) and New World peccaries (
Tayassuidae
) showed that one retrovirus group, β2, was common to all species tested, while the others were present among all Old World species but absent from New World species. The PERV-C subgroup of γ1 genomes segregated among domestic pigs and were absent from two African species (red river hog and warthog). Thus domestic swine and their phylogenetic relatives harbor multiple groups of hitherto undescribed PERV genomes.
Efficient Translation Initiation Is Required for Replication of Bovine Viral Diarrhea Virus Subgenomic Replicons
Authors: Myers Tina M. , Kolupaeva Victoria G. , Mendez Ernesto , Baginski Scott G. , Frolov Ilya , Hellen Christopher U. T. , Rice Charles M.
Published: July 2002

An internal ribosome entry site (IRES) mediates translation initiation of bovine viral diarrhea virus (BVDV) RNA. Studies have suggested that a portion of the N
pro
open reading frame (ORF) is required, although its exact function has not been defined. Here we show that a subgenomic (sg) BVDV RNA in which the NS3 ORF is preceded only by the 5′ nontranslated region did not replicate to detectable levels following transfection. However, RNA synthesis and cytopathic effects were observed following serial passage in the presence of a noncytopathic helper virus. Five sg clones derived from the passaged virus contained an identical, silent substitution near the beginning of the NS3 coding sequence (G400U), as well as additional mutations. Four of the reconstructed mutant RNAs replicated in transfected cells, and in vitro translation showed increased levels of NS3 for the mutant RNAs compared to that of wild-type (wt) MetNS3. To more precisely dissect the role of these mutations, we constructed two sg derivatives: ad3.10, which contains only the G400U mutation, and ad3.7, with silent substitutions designed to minimize RNA secondary structure downstream of the initiator AUG. Both RNAs replicated and were translated in vitro to similar levels. Moreover, ad3.7 and ad3.10, but not wt MetNS3, formed toeprints downstream of the initiator AUG codon in an assay for detecting the binding of 40S ribosomal subunits and 43S ribosomal complexes to the IRES. These results suggest that a lack of stable RNA secondary structure(s), rather than a specific RNA sequence, immediately downstream of the initiator AUG is important for optimal translation initiation of pestivirus RNAs.
Analysis of an
Autographa californica
Multicapsid Nucleopolyhedrovirus
lef-6
-Null Virus: LEF-6 Is Not Essential for Viral Replication but Appears To Accelerate Late Gene Transcription
Authors: Lin Guangyun , Blissard Gary W.
Published: July 2002

The
Autographa californica
multicapsid nucleopolyhedrovirus (Ac
M
NPV)
lef-6
gene was previously shown to be necessary for optimal transcription from an Ac
M
NPV late promoter in transient late expression assays. In the present study, we examined the expression and cellular localization of
lef-6
during the Ac
M
NPV infection cycle and generated a
lef-6
-null virus for studies of the role of
lef-6
in the infection cycle. Transcription of
lef-6
was detected from 4 to 48 h postinfection, and the LEF-6 protein was identified in dense regions of infected cell nuclei, a finding consistent with its potential role as a late transcription factor. To examine
lef-6
in the context of the Ac
M
NPV infection cycle, we deleted the
lef-6
gene from an Ac
M
NPV genome propagated as an infectious BACmid in
Escherichia coli
. Unexpectedly, the resulting Ac
M
NPV
lef-6
-null BACmid (vAc
lef6KO
) was able to propagate in cell culture, although virus yields were substantially reduced. Thus, the
lef-6
gene is not essential for viral replication in Sf9 cells. Two “repair” Ac
M
NPV BACmids (vAc
lef6KO-REP-P
and vAc
lef6KO-REP-ie1P
) were generated by transposition of the
lef-6
gene into the polyhedrin locus of the vAc
lef6KO
BACmid. Virus yields from the two repair viruses were similar to those from wild-type Ac
M
NPV or a control (BACmid-derived) virus. The
lef-6
-null BACmid (vAc
lef6KO
) was further examined to determine whether the deletion of
lef-6
affected DNA replication or late gene transcription in the context of an infection. The
lef-6
deletion did not appear to affect viral DNA replication. Using Northern blot analysis, we found that although early transcription was apparently unaffected, both late and very late transcription were delayed in cells infected with the
lef-6
-null BACmid. This phenotype was rescued in viruses containing the
lef-6
gene reinserted into the
polyhedrin
locus. Thus, the
lef-6
gene was not essential for either viral DNA replication or late gene transcription, but the absence of
lef-6
resulted in a substantial delay in the onset of late transcription. Therefore,
lef-6
appears to accelerate the infection cycle of Ac
M
NPV.
Targeting and Hematopoietic Suppression of Human CD34+Cells by Measles Virus
Authors: Manchester Marianne , Smith Kent A. , Eto Danelle S. , Perkin Hugh B. , Torbett Bruce E.
Published: July 2002

The major cause of mortality in measles is generalized suppression of cell-mediated immunity that persists following virus clearance and results in secondary infections. The mechanisms contributing to this long-term immunosuppression are not clear. Herein we present evidence that measles virus (MV) disrupts hematopoiesis by infecting human CD34+cells and human bone marrow stroma. MV infection does not affect the hematopoietic capability of hematopoietic stem cells (HSCs) directly; rather, the infection impairs the ability of stroma to support development of HSCs. These results suggest that MV-mediated defects in hematopoiesis contribute to the long-term immunosuppression seen in measles.
Determination of a Statistically Valid Neutralization Titer in Plasma That Confers Protection against Simian-Human Immunodeficiency Virus Challenge following Passive Transfer of High-Titered Neutralizing Antibodies
Authors: Nishimura Yoshiaki , Igarashi Tatsuhiko , Haigwood Nancy , Sadjadpour Reza , Plishka Ron J. , Buckler-White Alicia , Shibata Riri , Martin Malcolm A.
Published: July 2002

We previously reported that high-titered neutralizing antibodies directed against the human immunodeficiency virus type 1 (HIV-1) envelope can block the establishment of a simian immunodeficiency virus (SIV)/HIV chimeric virus (SHIV) infection in two monkeys following passive transfer (R. Shibata et al., Nat. Med. 5:204-210, 1999). In the present study, increasing amounts of neutralizing immunoglobulin G (IgG) were administered to 15 pig-tailed macaques in order to obtain a statistically valid protective neutralization endpoint titer in plasma. Using an in vitro assay which measures complete neutralization of the challenge SHIV, we correlated the titers of neutralizing antibodies in plasma at the time of virus inoculation (which ranged from 1:3 to 1:123) with the establishment of infection in virus-challenged animals. Ten of 15 monkeys in the present experiment were virus free as a result of neutralizing IgG administration as monitored by DNA PCR (peripheral blood mononuclear cells and lymph node cells), RNA PCR (plasma), virus isolation, and the transfer of lymph node cell suspensions (10
8
cells) plus 8 ml of whole blood from protected animals to naïve macaques. The titer of neutralizing antibodies in the plasma calculated to protect 99% of virus-challenged monkeys was 1:38.