bluuebunny/crossref_metadata_2025 huggingface dataset:
Real-Time PCR Quantification of Human Cytomegalovirus DNA in Amniotic Fluid Samples from Mothers with Primary Infection
Authors: Gouarin S. , Gault E. , Vabret A. , Cointe D. , Rozenberg F. , Grangeot-Keros L. , Barjot P. , Garbarg-Chenon A. , Lebon P. , Freymuth F.
Published: July 2002

A real-time PCR assay was developed to quantify human cytomegalovirus (HCMV) DNA in amniotic fluid (AF) samples collected from 30 pregnant women with primary HCMV infection as detected either from HCMV-immunoglobulin G (IgG) seroconversion or by the presence of HCMV-specific IgG and IgM associated with a low IgG avidity. Clinical information available for each case included ultrasonographic examination and fetal or newborn outcome. HCMV infection of fetuses or newborns was confirmed for the 30 studied cases. AF samples were subdivided into three groups. In group A (
n
= 13), fetuses presented major ultrasound abnormalities, and pregnancy was terminated. In group B (
n
= 13), fetuses had normal ultrasound findings, the pregnancy went to term, and the newborns were asymptomatic at birth. In group C (
n
= 4), fetuses had no or minor ultrasonographic signs, and pregnancy was terminated. The HCMV DNA load values in AF samples were significantly higher in group A (median, 2.8 × 10
5
genome equivalents [GE]/ml) than in group B (median, 8 × 10
3
GE/ml) (
P
= 0.014). Our findings suggest that HCMV load level in AF samples correlates with fetal clinical outcome but might also be dependent on other factors, such as the gestational age at the time of AF sampling and the time elapsed since maternal infection.
Prevalence and Genetic Characterization of Toxin A Variant Strains of
Clostridium difficile
among Adults and Children with Diarrhea in France
Authors: Barbut Frédéric , Lalande Valérie , Burghoffer Béatrice , Thien Huong Vu , Grimprel Emmanuel , Petit Jean-Claude
Published: July 2002

Toxin A variant strains (toxin A-negative, toxin B-positive strains) of
Clostridium difficile
have been reported to be responsible for diarrhea or pseudomembranous colitis in humans. These strains lack parts of the repeating sequences of the toxin A gene (
tcdA
) and are toxin A negative by commercial enzyme immunoassays (EIA). Here, we report the prevalence of the toxin A variant strains in 334 patients with
C. difficile
-associated diarrhea in France. The repeating segment of the
tcdA
gene (1,200 bp) was amplified by PCR using the primers NK9 and NK11 (H. Kato et al., J. Clin. Microbiol.
36:
2178-2182, 1998). In the case of amplified fragments of unexpected size, the entire
tcdA
gene was studied by PCRs A1, A2, and A3 (Rupnik et al., J. Clin. Microbiol. 36:2240-2247, 1998), and strains were characterized by serotyping, pulsed-field gel electrophoresis and PCR ribotyping. By PCR with primers NK9 and NK11,
C. difficile
variant strains were detected in 2.7% of patients. Several variant types were found. A deletion of approximately 1,700 bp was observed in six strains from five patients. These strains belonged to serotype F and were characterized by the same pulsotype and the same PCR ribotype. They were toxin A negative by EIA and exhibited an atypical cytopathic effect on MRC-5 cells. Two other
tcdA
variant types that exhibited a positive result for toxin A by EIA were identified: one from serotype H with a longer amplified fragment (insertion of 200 bp) and one with a deletion of 600 bp. Diagnosis of
C. difficile
-associated diseases would have been missed in five patients (1.5%) by laboratories that screen the stools only for the presence of toxin A. This result underlines the need for testing stool by the cytotoxicity assay in patients with a high suspicion
of C. difficile
-associated diarrhea but a negative immunoassay for toxin A.
A Frequent, Naturally Occurring Mutation (P130T) of Human Hepatitis B Virus Core Antigen Is Compensatory for Immature Secretion Phenotype of Another Frequent Variant (I97L)
Authors: Yuan Thomas Ta-Tung , Shih Chiaho
Published: July 2002

A frequent mutation at codon 97 of human hepatitis B virus core antigen has been shown to cause an “immature secretion” phenotype, featuring nonselective and excessive secretions of virions containing immature viral genome. Our current study demonstrates that this abnormality can be efficiently offset by another frequent core mutation, P130T.
High Levels of Viral Replication during Primary Simian Immunodeficiency Virus SIVagm Infection Are Rapidly and Strongly Controlled in African Green Monkeys
Authors: Diop Ousmane Madiagne , Gueye Aïssatou , Dias-Tavares Marisa , Kornfeld Christopher , Faye Abdourahmane , Ave Patrick , Huerre Michel , Corbet Sylvie , Barre-Sinoussi Françoise , Müller-Trutwin Michaela C.
Published: July 2002

In contrast to pathogenic human immunodeficiency virus and simian immunodeficiency virus (SIV) infections, chronic SIVagm infections in African green monkeys (AGMs) are characterized by persistently low peripheral and tissue viral loads that correlate with the lack of disease observed in these animals. We report here data on the dynamics of acute SIVagm infection in AGMs that exhibit remarkable similarities with viral replication patterns observed in peripheral blood during the first 2 weeks of pathogenic SIVmac infections. Plasma viremia was evident at day 3 postinfection (p.i.) in AGMs, and rapid viral replication led by days 7 to 10 to peak viremias characterized by high levels of antigenemia (1.2 to 5 ng of p27/ml of plasma), peripheral DNA viral load (10
4
to 10
5
DNA copies/10
6
peripheral blood mononuclear cells [PBMC]), and plasma RNA viral load (2 × 10
6
to 2 × 10
8
RNA copies/ml). The lymph node (LN) RNA and DNA viral load patterns were similar to those in blood, with peaks observed between day 7 and day 14. These values in LNs (ranging from 3 × 10
5
to 3 × 10
6
RNA copies/10
6
LN cell [LNC] and 10
3
to 10
4
DNA copies/10
6
LNC) were at no time point higher than those observed in the blood. Both in LNs and in blood, rapid and significant decreases were observed in all infected animals after this peak of viral replication. Within 3 to 4 weeks p.i., antigenemia was no longer detectable and peripheral viral loads decreased to values similar to those characteristic of the chronic phase of infection (10
2
to 10
3
DNA copies/10
6
PBMC and 2 × 10
3
to 2 × 10
5
RNA copies/ml of plasma). In LNs, viral loads declined to 5 × 10
1
to 10
3
DNA copies and 10
4
to 3 × 10
5
RNA copies per 10
6
LNC at day 28 p.i. and continued to decrease until day 84 p.i. (<10 to 3 × 10
4
RNA copies/10
6
LNC). Despite extensive viremia during primary infection, neither follicular hyperplasia nor CD8
+
cell infiltration into LN germinal centers was detected. Altogether, these results indicate that the nonpathogenic outcome of SIVagm infection in its natural host is associated with a rapidly induced control of viral replication in response to SIVagm infection, rather than with a poorly replicating virus or a constitutive host genetic resistance to virus replication.
Expression of Secreted Cytokine and Chemokine Inhibitors by Ectromelia Virus
Authors: Smith Vincent P. , Alcami Antonio
Published: July 2002

The production of secreted proteins that bind cytokines and block their activity has been well characterized as an immune evasion strategy of the orthopoxviruses vaccinia virus (VV) and cowpox virus (CPV). However, very limited information is available on the expression of similar cytokine inhibitors by ectromelia virus (EV), a virulent natural mouse pathogen that causes mousepox. We have characterized the expression and binding properties of three major secreted immunomodulatory activities in 12 EV strains and isolates. Eleven of the 12 EVs expressed a soluble, secreted 35-kDa viral chemokine binding protein with properties similar to those of homologous proteins from VV and CPV. All of the EVs expressed soluble, secreted receptors that bound to mouse, human, and rat tumor necrosis factor alpha. We also detected the expression of a soluble, secreted interleukin-1β (IL-1β) receptor (vIL-1βR) by all of the EVs. EV differed from VV and CPV in that binding of human125I-IL-1β to the EV vIL-1βR could not be detected. Nevertheless, the EV vIL-1βR prevented the interaction of human and mouse IL-1β with cellular receptors. There are significant differences in amino acid sequence between the EV vIL-1βR and its VV and CPV homologs which may account for the results of the binding studies. The conservation of these activities in EV suggests evolutionary pressure to maintain them in a natural poxvirus infection. Mousepox represents a useful model for the study of poxvirus pathogenesis and immune evasion. These findings will facilitate future study of the role of EV immunomodulatory factors in the pathogenesis of mousepox.
Human Herpesvirus 8 Open Reading Frame 21 Is a Thymidine and Thymidylate Kinase of Narrow Substrate Specificity That Efficiently Phosphorylates Zidovudine but Not Ganciclovir
Authors: Gustafson Erik A. , Schinazi Raymond F. , Fingeroth Joyce D.
Published: July 2002

Human herpesvirus 8 (HHV8) open reading frame (ORF) 21 is predicted to encode a protein similar to the thymidine kinase (TK) enzyme of other herpesviruses. Expressed in mammalian cells, ORF 21 was found to have low TK activity, based on poor growth in media containing hypoxanthine-aminopterin-thymidine (HAT) and low incorporation of [
3
H]thymidine into high-molecular-weight DNA. Kinetic analysis using HHV8 TK as a purified glutathione
S
-transferase (GST) fusion protein showed that the enzyme has a comparatively high

K
m

for thymidine (dThd) of ∼33.2 μM. Nearly 50% of the phosphorylated product of the reaction with dThd was thymidylate. This monophosphate kinase activity was more pronounced with 3′-azido-3′-deoxythymidine (AZT), in which 78% of the reaction product was AZT diphosphate. Thymidine analogs competitively inhibited dThd phosphorylation by HHV8 TK, while 2′-deoxyguanosine, 2′-deoxyadenosine, 2′-deoxycytidine, and corresponding analogs did not. Further competition experiments revealed that the nucleoside analog ganciclovir (GCV), at up to 1,000-fold molar excess, could not significantly inhibit dThd phosphorylation by the enzyme. In support of these data, 143B TK
−
cells expressing HHV8 TK phosphorylated GCV very poorly and were not susceptible to GCV toxicity compared to parental cells. Phosphorylation of [
3
H]GCV by a purified GST-HHV8 TK fusion protein was not detected by high-pressure liquid chromatography analysis. Structural features of HHV8 TK substrate recognition were investigated. Therapeutic implications of these findings are discussed.
Pseudorabies Virus Expressing Bovine Herpesvirus 1 Glycoprotein B Exhibits Altered Neurotropism and Increased Neurovirulence
Authors: Gerdts Volker , Beyer Jörg , Lomniczi Béla , Mettenleiter Thomas C.
Published: July 2002

Herpesvirus glycoproteins play dominant roles in the initiation of infection of target cells in culture and thus may also influence viral tropism in vivo. Whereas the relative contribution of several nonessential glycoproteins to neurovirulence and neurotropism of
Pseudorabies virus
(PrV), an alphaherpesvirus which causes Aujeszky's disease in pigs, has recently been uncovered in studies using viral deletion mutants, the importance of essential glycoproteins is more difficult to assess. We isolated an infectious PrV mutant, PrV-9112C2, which lacks the gene encoding the essential PrV glycoprotein B (gB) but stably carries in its genome and expresses the homologous gene of bovine herpesvirus 1 (BHV-1) (A. Kopp and T. C. Mettenleiter, J. Virol. 66:2754–2762, 1992). Apart from exhibiting a slight delay in penetration kinetics, PrV-9112C2 was similar in its growth characteristics in cell culture to wild-type PrV. To analyze the effect of the exchange of these homologous glycoproteins in PrV's natural host, swine, 4-week-old piglets were intranasally infected with 10
6
PFU of either wild-type PrV strain Kaplan (PrV-Ka), PrV-9112C2, or PrV-9112C2R, in which the PrV gB gene was reinserted instead of the BHV-1 gB gene. Animals infected with PrV-Ka and PrV-9112C2R showed a similar course of disease, i.e., high fever, marked respiratory symptoms but minimal neurological disorders, and excretion of high amounts of virus. All animals survived the infection. In contrast, animals infected with PrV-9112C2 showed no respiratory symptoms and developed only mild fever. However, on day 5 after infection, all piglets developed severe central nervous system (CNS) symptoms leading to death within 48 to 72 h. Detailed histological analyses showed that PrV-9112C2R infected all regions of the nasal mucosa and subsequently spread to the CNS preferentially by the trigeminal route. In contrast, PrV-9112C2 primarily infected the olfactory epithelium and spread via the olfactory route. In the CNS, more viral antigen and significantly more pronounced histological changes resulting in more severe encephalitis were found after PrV-9112C2 infection. Thus, our results demonstrate that replacement of PrV gB by the homologous BHV-1 glycoprotein resulted in a dramatic increase in neurovirulence combined with an alteration in the route of neuroinvasion, indicating that the essential gB is involved in determining neurotropism and neurovirulence of PrV.
Human Immunodeficiency Virus Type 1 Spinoculation Enhances Infection through Virus Binding
Authors: O'Doherty Una , Swiggard William J. , Malim Michael H.
Published: July 2002

The study of early events in the human immunodeficiency virus type 1 (HIV-1) life cycle can be limited by the relatively low numbers of cells that can be infected synchronously in vitro. Although the efficiency of HIV-1 infection can be substantially improved by centrifugal inoculation (spinoculation or shell vial methods), the underlying mechanism of enhancement has not been defined. To understand spinoculation in greater detail, we have used real-time PCR to quantitate viral particles in suspension, virions that associate with cells, and the ability of those virions to give rise to reverse transcripts. We report that centrifugation of HIV-1
IIIB
virions at 1,200 ×
g
for 2 h at 25°C increases the number of particles that bind to CEM-SS T-cell targets by ∼40-fold relative to inoculation by simple virus-cell mixing. Following subsequent incubation at 37°C for 5 h to allow membrane fusion and uncoating to occur, the number of reverse transcripts per target cell was similarly enhanced. Indeed, by culturing spinoculated samples for 24 h, ∼100% of the target cells were reproducibly shown to be productively infected, as judged by the expression of p24

gag

. Because the modest
g
forces employed in this procedure were found to be capable of sedimenting viral particles and because CD4-specific antibodies were effective at blocking virus binding, we propose that spinoculation works by depositing virions on the surfaces of target cells and that diffusion is the major rate-limiting step for viral adsorption under routine in vitro pulsing conditions. Thus, techniques that accelerate the binding of viruses to target cells not only promise to facilitate the experimental investigation of postentry steps of HIV-1 infection but should also help to enhance the efficacy of virus-based genetic therapies.
A Role for SKIP in EBNA2 Activation of CBF1-Repressed Promoters
Authors: Zhou Sifang , Fujimuro Masahiro , Hsieh James J.-D. , Chen Lin , Hayward S. Diane
Published: July 2002

EBNA2 is essential for Epstein-Barr virus (EBV) immortalization of B lymphocytes. EBNA2 functions as a transcriptional activator and targets responsive promoters through interaction with the cellular DNA binding protein CBF1. We have examined the mechanism whereby EBNA2 overcomes CBF1-mediated transcriptional repression. A yeast two-hybrid screen performed using CBF1 as the bait identified a protein, SKIP, which had not previously been recognized as a CBF1-associated protein. Protein-protein interaction assays demonstrated contacts between SKIP and the SMRT, CIR, Sin3A, and HDAC2 proteins of the CBF1 corepressor complex. Interestingly, EBNA2 also interacted with SKIP in glutathione
S
-transferase affinity and mammalian two-hybrid assays and colocalized with SKIP in immunofluorescence assays. Interaction with SKIP was not affected by mutation of EBNA2 conserved region 6, the CBF1 interaction region, but was abolished by mutation of conserved region 5. Mutation of conserved region 5 also severely impaired EBNA2 activation of a reporter containing CBF1 binding sites. Thus, interaction with both CBF1 and SKIP is necessary for efficient promoter activation by EBNA2. A model is presented in which EBNA2 competes with the SMRT-corepressor complex for contacts on SKIP and CBF1.
Herpes Simplex Virus ICP0 Mutants Are Hypersensitive to Interferon
Authors: Mossman Karen L. , Saffran Holly A. , Smiley James R.
Published: July 2002

Interferon (IFN) is an important immune system molecule capable of inducing an antiviral state within cells. Herpes simplex virus type 1 (HSV-1) replication is somewhat reduced in tissue culture in the presence of IFN, presumably due to decreased viral transcription. Here, we show mutations that inactivate immediate-early (IE) gene product ICP0 render HSV-1 exquisitely sensitive to IFN inhibition, resulting in greatly decreased levels of viral mRNA transcripts and the resulting polypeptides and a severe reduction in plaque formation ability. Mutations in other HSV-1 genes, including the genes coding for virion transactivator VP16 and the virion host shutoff protein vhs, IE gene ICP22, and the protein kinase UL13 gene, do not increase the IFN sensitivity of HSV-1. Interestingly, ICP0 mutants demonstrate the same level of sensitivity to IFN as wild-type virus on U2OS cells, an osteosarcoma cell line that is known to complement mutations in ICP0 and VP16. Thus, in some cell types, functional ICP0 is required for HSV-1 to efficiently bypass the inhibitory effects of IFN in order to ensure its replication. The significance of this link between ICP0 and IFN resistance is discussed.
Identification of Contact Residues and Definition of the CAR-Binding Site of Adenovirus Type 5 Fiber Protein
Authors: Kirby Ian , Davison Elizabeth , Beavil Andrew J. , Soh Cecilia P. C. , Wickham Thomas J. , Roelvink Peter W. , Kovesdi Imre , Sutton Brian J. , Santis George
Published: July 2002

The binding of adenovirus (Ad) fiber knob to its cellular receptor, the coxsackievirus and Ad receptor (CAR), promotes virus attachment to cells and is a major determinant of Ad tropism. Analysis of the kinetics of binding of Ad type 5 (Ad5) fiber knob to the soluble extracellular domains of CAR together (sCAR) and each immunoglobulin (Ig) domain (IgV and IgC2) independently by surface plasmon resonance demonstrated that the IgV domain is necessary and sufficient for binding, and no additional membrane components are required to confer high-affinity binding to Ad5 fiber knob. Four Ad5 fiber knob mutations, Ser408Glu and Pro409Lys in the AB loop, Tyr477Ala in the DG loop, and Leu485Lys in β strand F, effectively abolished high-affinity binding to CAR, while Ala406Lys and Arg412Asp in the AB loop and Arg481Glu in β strand E significantly reduced the level of binding. Circular dichroism spectroscopy showed that these mutations do not disorder the secondary structure of the protein, implicating Ser408, Pro409, Tyr477, and Leu485 as contact residues, with Ala406, Arg412, and Arg481 being peripherally or indirectly involved in CAR binding. The critical residues have exposed side chains that form a patch on the surface, which thus defines the high-affinity interface for CAR. Additional site-directed mutagenesis of Ad5 fiber knob suggests that the binding site does not extend to the adjacent subunit or toward the edge of the R sheet. These findings have implications for our understanding of the biology of Ad infection, the development of novel Ad vectors for targeted gene therapy, and the construction of peptide inhibitors of Ad infection.
A Beta-Stranded Motif Drives Capsid Protein Oligomers of the Parvovirus Minute Virus of Mice into the Nucleus for Viral Assembly
Authors: Lombardo Eleuterio , Ramírez Juan C. , Agbandje-McKenna Mavis , Almendral José M.
Published: July 2002

The determinants of nuclear import in the VP-1 and VP-2 capsid proteins of the parvovirus minute virus of mice strain i (MVMi) synthesized in human fibroblasts were sought by genetic analysis in an infectious plasmid. Immunofluorescence of transfected cells revealed that the two proteins were involved in cooperative cytoplasmic interactions for nuclear cotransport. However, while VP-1 translocated regardless of extension of deletions and did not form capsid epitopes by itself, VP-2 seemed to require cytoplasmic folding and the overall conformation for nuclear transport. The sequence
528
KGKLTMRAKLR
538
was found necessary for nuclear uptake of VP-2, even though it was not sufficient to confer a nuclear localization capacity on a heterologous protein. In the icosahaedral MVMi capsid, this sequence forms the carboxy end of the amphipathic beta-strand I (βI), and all its basic residues are contiguously positioned at the face that in the unassembled subunit would be exposed to solvent. Mutations in singly expressed VP-2 that either decrease the net basic charge of the exposed face (K530N-R534T), perturb the hydrophobicity of the opposite face (L531E), or distort the βI conformation (G529P) produced cytoplasmic subviral oligomers. Particle formation by βI mutants indicated that the basic residues clustered at one face of βI drive VP oligomers into the nucleus preceding and uncoupled to assembly and that the nuclear environment is required for MVMi capsid formation in the infected cell. The degree of VP-1/VP-2 transport cooperativity suggests that VP trimers are the morphogenetic intermediates translocating through the nuclear pore. The results support a model in which nuclear transport signaling preserves the VP-1/VP-2 stoichiometry necessary for efficient intranuclear assembly and in which the beta-stranded VP-2 nuclear localization motif contributes to the quality control of viral morphogenesis.
Adenovirus Vector-Induced Expression of the C-X-C Chemokine IP-10 Is Mediated through Capsid-Dependent Activation of NF-κB
Authors: Borgland Stephanie L. , Bowen Gloria P. , Wong Norman C. W. , Libermann Towia A. , Muruve Daniel A.
Published: July 2002

The use of adenovirus vectors for gene therapy has been limited by well-defined cellular and humoral immune responses. We have previously shown that adenovirus vectors rapidly induce the expression of the C-X-C chemokine, interferon-inducible protein 10 (IP-10), in vivo. Various first-generation, type 5 adenovirus vectors, including adCMVβgal and UV-psoralen-inactivated adenovirus, equally induced the expression of IP-10 mRNA as early as 3 h following infection in mouse renal epithelial cells (REC). Luciferase reporter experiments using deletional mutants of the murine IP-10 5′-flanking region revealed that transcriptional activation of the IP-10 promoter by adCMVβgal was dependent on the −161- to −96-bp region upstream of the transcription start site. In electrophoretic mobility shift assays, adCMVβgal, adCMV-GFP, FG140, and transcription-defective adenovirus induced protein binding to oligonucleotides containing a consensus sequence for NF-κB at position −113 of the IP-10 promoter. Supershift assays confirmed an increase in binding activity of NF-κB p65 but not p50 or cRel in REC cells infected with various replication-deficient adenoviruses. Coinfection of REC cells with adCMVβgal and an adenoviral vector expressing IκBα resulted in suppression of adCMVβgal-induced expression of IP-10 at 6 and 16 h, further strengthening the conclusion that adenovirus-induced activation of IP-10 is dependent on NF-κB. The induction of IP-10 appeared to be direct because infection with adenovirus vectors failed to induce the expression of the potent IP-10 stimulators, interferon gamma and tumor necrosis factor alpha. Together, these findings demonstrate that adenovirus vectors directly induce the expression of IP-10 through capsid dependent activation of NF-κB.
Markedly Increased Susceptibility to Natural Sheep Scrapie of Transgenic Mice Expressing Ovine PrP
Authors: Vilotte Jean-Luc , Soulier Solange , Essalmani Rachid , Stinnakre Marie-George , Vaiman Daniel , Lepourry Laurence , Da Silva Jose Costa , Besnard Nathalie , Dawson Mike , Buschmann Anne , Groschup Martin , Petit Stephanie , Madelaine Marie-Francoise , Rakatobe Sabine , Le Dur Annick , Vilette Didier , Laude Hubert
Published: July 2002

The susceptibility of sheep to scrapie is known to involve, as a major determinant, the nature of the prion protein (PrP) allele, with the VRQ allele conferring the highest susceptibility to the disease. Transgenic mice expressing in their brains three different ovine PrP
VRQ
-encoding transgenes under an endogenous PrP-deficient genetic background were established. Nine transgenic (tgOv) lines were selected and challenged with two scrapie field isolates derived from VRQ-homozygous affected sheep. All inoculated mice developed neurological signs associated with a transmissible spongiform encephalopathy (TSE) disease and accumulated a protease-resistant form of PrP (PrPres) in their brains. The incubation duration appeared to be inversely related to the PrP steady-state level in the brain, irrespective of the transgene construct. The survival time for animals from the line expressing the highest level of PrP was reduced by at least 1 year compared to those of two groups of conventional mice. With one isolate, the duration of incubation was as short as 2 months, which is comparable to that observed for the rodent TSE models with the briefest survival times. No survival time reduction was observed upon subpassaging of either isolate, suggesting no need for adaptation of the agent to its new host. Overexpression of the transgene was found not to be required for transmission to be accelerated compared to that observed with wild-type mice. Conversely, transgenic mice overexpressing murine PrP were found to be less susceptible than tgOv lines expressing ovine PrP at physiological levels. These data argue that ovine PrP
VRQ
provided a better substrate for sheep prion replication than did mouse PrP. Altogether, these tgOv mice could be an improved model for experimental studies on natural sheep scrapie.
Longer and Shorter Forms of Sendai Virus C Proteins Play Different Roles in Modulating the Cellular Antiviral Response
Authors: Garcin Dominique , Curran Joseph , Itoh Masae , Kolakofsky Daniel
Published: July 2002

The Sendai virus (SeV) C gene codes for a nested set of four C proteins that carry out several functions, including the modulation of viral RNA synthesis and countering of the cellular antiviral response. Using mutant C genes (and in particular a C gene with a deletion of six amino acids present only in the larger pair of C proteins) and recombinant SeV carrying these mutant C genes, we find that the nested set of C proteins carry out a nested set of functions. All of the C proteins interdict interferon (IFN) signaling to IFN-stimulated genes (ISGs) and prevent pY701-Stat1 formation. However, only the larger C proteins can induce STAT1 instability, prevent IFN from inducing an antiviral state, or prevent programmed cell death. Remarkably, interdiction of IFN signaling to ISGs and the absence of pY701-Stat1 formation did not prevent IFN-α from inducing an anti-Vesicular stomatitis virus (VSV) state. It is possible that IFN-α signaling to induce an anti-VSV state can occur independently of the well-established Jak/Stat/ISGF3 pathway and that it is this parallel pathway that is targeted by the longer C proteins.
Salicylates Inhibit Flavivirus Replication Independently of Blocking Nuclear Factor Kappa B Activation
Authors: Liao Ching-Len , Lin Yi-Ling , Wu Bi-Ching , Tsao Chang-Huei , Wang Mei-Chuan , Liu Chiu-I , Huang Yue-Ling , Chen Jui-Hui , Wang Jia-Pey , Chen Li-Kuang
Published: July 2002

Flaviviruses comprise a positive-sense RNA genome that replicates exclusively in the cytoplasm of infected cells. Whether flaviviruses require an activated nuclear factor(s) to complete their life cycle and trigger apoptosis in infected cells remains elusive. Flavivirus infections quickly activate nuclear factor kappa B (NF-κB), and salicylates have been shown to inhibit NF-κB activation. In this study, we investigated whether salicylates suppress flavivirus replication and virus-induced apoptosis in cultured cells. In a dose-dependent inhibition, we found salicylates within a range of 1 to 5 mM not only restricted flavivirus replication but also abrogated flavivirus-triggered apoptosis. However, flavivirus replication was not affected by a specific NF-κB peptide inhibitor, SN50, and a proteosome inhibitor, lactacystin. Flaviviruses also replicated and triggered apoptosis in cells stably expressing IκBα-ΔN, a dominant-negative mutant that antagonizes NF-κB activation, as readily as in wild-type BHK-21 cells, suggesting that NF-κB activation is not essential for either flavivirus replication or flavivirus-induced apoptosis. Salicylates still diminished flavivirus replication and blocked apoptosis in the same IκBα-ΔN cells. This inhibition of flaviviruses by salicylates could be partially reversed by a specific p38 mitogen-activated protein (MAP) kinase inhibitor, SB203580. Together, these results show that the mechanism by which salicylates suppress flavivirus infection may involve p38 MAP kinase activity but is independent of blocking the NF-κB pathway.
Activation of Interferon Response Factor-3 in Human Cells Infected with Herpes Simplex Virus Type 1 or Human Cytomegalovirus
Authors: Preston Chris M. , Harman Andrew N. , Nicholl Mary Jane
Published: July 2002

Activation of cellular interferon-stimulated genes (ISGs) after infection with herpes simplex virus type 1 (HSV-1) or human cytomegalovirus (HCMV) was investigated. The level of ISG54-specific RNA in human fetal lung (HFL) or human foreskin (BJ) fibroblasts increased substantially after infection with either virus in the presence of cycloheximide. HSV-1 particles lacking glycoprotein D or glycoprotein H failed to induce ISG54-specific RNA synthesis, demonstrating that entry of virus particles rather than binding of virions to the cell surface was required for the effect. A DNA-binding complex that recognized an interferon-responsive sequence motif was induced upon infection with HSV-1 or HCMV in the presence of cycloheximide, and the complex was shown to contain the cell proteins interferon response factor 3 (IRF-3) and CREB-binding protein. IRF-3 was modified after infection with HSV-1 or HCMV to a form of lower electrophoretic mobility, consistent with phosphorylation. De novo transcription of viral or cellular genes was not required for the activation of IRF-3, since the effect was not sensitive to inhibition by actinomycin D. Infection of HFL fibroblasts with HSV-1 under conditions in which viral replication proceeded normally resulted in severely reduced levels of the IRF-3-containing complex, defining the activation of IRF-3 as a target for viral interference with ISG induction. In BJ fibroblasts, however, significant activation of IRF-3 was detected even when the viral gene expression program progressed to later stages, demonstrating that the degree of inhibition of the response was dependent on host cell type. As a consequence of IRF-3 activation, endogenous interferon was released from BJ cells and was capable of triggering the appropriate signal transduction pathway in both infected and uninfected cells. Activation of ISG54-specific RNA synthesis was not detected after infection of human U-373MG glioblastoma cells, showing that the induction of the response by infection is cell type dependent.
Human Immunodeficiency Virus Type 1 Protease Cleavage Site Mutations Associated with Protease Inhibitor Cross-Resistance Selected by Indinavir, Ritonavir, and/or Saquinavir
Authors: Côté Hélène C. F. , Brumme Zabrina L. , Harrigan P. Richard
Published: July 2002

We examined the prevalence of cleavage site mutations, both within and outside the
gag
region, in 28 protease inhibitor (PI) cross-resistant patients treated with indinavir, ritonavir, and/or saquinavir compared to control patients treated with reverse transcriptase inhibitors. Three human immunodeficiency virus protease cleavage sites within
gag
(p2/NC, NC/p1, and NC/TFP) showed considerable in vivo evolution before and after therapy with indinavir, ritonavir, and/or saquinavir. Another
gag
cleavage site (p1/p6

gag

) showed a trend compared to matched controls. The other eight recognized cleavage sites showed relatively little difference between PI-resistant cases and controls. An A→V substitution at the P2 position of the NC/p1 and NC/TFP cleavage sites was the most common (29%) change selected by the PIs used in this study.
Neutralizing Antibodies in Persistent Borna Disease Virus Infection: Prophylactic Effect of gp94-Specific Monoclonal Antibodies in Preventing Encephalitis
Authors: Furrer Esther , Bilzer Thomas , Stitz Lothar , Planz Oliver
Published: July 2002

Borna disease virus (BDV) infection triggers an immune-mediated encephalomyelitis and results in a persistent infection. The immune response in the acute phase of the disease is characterized by a cellular response in which CD8+T cells are responsible for the destruction of virus-infected brain cells. CD4+T cells function as helper cells and support the production of antiviral antibodies. Antibodies generated in the acute phase of the disease against the nucleoprotein and the phosphoprotein are nonneutralizing. In the chronic phase of the disease, neutralizing antibodies directed against the matrix protein and glycoprotein are synthesized. In the present work, the biological role of the neutralizing-antibody response to BDV was further investigated. By analyzing the blood of rats infected intracerebrally with BDV, a highly neurotropic virus, nucleic acid could be detected between 30 and 50 days after infection. Neutralizing antibodies were found between 60 and 100 days after infection. Furthermore, we produced hybridomas secreting BDV-specific neutralizing monoclonal antibodies. These antibodies, directed against the major glycoprotein (gp94) of BDV, were able to prevent Borna disease if given prophylactically. These data suggest that the late appearance of BDV-specific neutralizing antibodies is due to the presence of BDV in the blood of chronically infected rats. Furthermore, these antibodies have the potential to neutralize the infectious virus when given early, which is an important finding with respect to the development of a vaccine.
Isolation and Characterization of Noncytopathic Pestivirus Mutants Reveals a Role for Nonstructural Protein NS4B in Viral Cytopathogenicity
Authors: Qu Lin , McMullan Laura K. , Rice Charles M.
Published: July 2002

Isolates of bovine viral diarrhea virus (BVDV), the prototype pestivirus, are divided into cytopathic (cp) and noncytopathic (ncp) biotypes according to their effect on cultured cells. The cp viruses also differ from ncp viruses by the production of viral nonstructural protein NS3. However, the mechanism by which cp viruses induce cytopathic effect in cell culture remains unknown. Here we used a genetic approach to isolate ncp variants that arose from a cp virus at low frequency. A bicistronic BVDV (cp strain NADL) was created that expressed puromycin acetyltransferase as a dominant selectable marker. This bicistronic virus exhibited slightly slower growth kinetics and smaller plaques than NADL but remained cp. A number of independent ncp variants were isolated by puromycin selection. Remarkably, these ncp variants produced NS3 and viral RNA at levels comparable to those of the cp parent. Sequence analyses uncovered no change in NS3, but for all ncp variants a Y2441C substitution at residue 15 of NS4B was found. Introduction of the Y2441C substitution into the NADL or bicistronic cp viruses reconstituted the ncp phenotype. Y2441 is highly conserved among pestiviruses and is located in a region of NS4B predicted to be on the cytosolic side of the endoplasmic reticulum membrane. Other engineered substitutions for Y2441 also affected viral cytopathogenicity and viability, with Y2441V being cp, Y2441A being ncp, and Y2441D rendering the virus unable to replicate. The ncp substitutions for Y2441 resulted in slightly increased levels of NS2-3 relative to NS3. We also showed that NS3, NS4B, and NS5A could be chemically cross-linked in NADL-infected cells, indicating that they are associated as components of a multiprotein complex. Although the mechanism remains to be elucidated, these results demonstrate that mutations in NS4B can attenuate BVDV cytopathogenicity despite NS3 production.
High-Dose Borna Disease Virus Infection Induces a Nucleoprotein-Specific Cytotoxic T-Lymphocyte Response and Prevention of Immunopathology
Authors: Furrer Esther , Bilzer Thomas , Stitz Lothar , Planz Oliver
Published: July 2002

Experimental Borna disease virus (BDV) infection of rats and natural infection of horses and sheep leads to severe central nervous system disease based on immunopathological pathways. The virus replicates slowly, and the cellular immune response results in immunopathology. CD8+T cells exert effector cell functions, and their activity results in the destruction of virus-infected cells. Previously, Oldach and colleagues (D. Oldach, M. C. Zink, J. M. Pyper, S. Herzog, R. Rott, O. Narayan, and J. E. Clements, Virology 206:426–434, 1995) have reported protection against Borna disease after inoculation of high-dose cell-adapted BDV. Here we show that the outcome of the infection, i.e., immunopathology versus protection, is simply dependent on the amount of virus used for infection. High-dose BDV (106FFU) triggers an early virus-specific reaction of the immune system, as demonstrated by strong cellular and humoral responses. In particular, the early presence and function of nucleoprotein-specific CD8+T cells could be demonstrated in the brain. We present evidence that in a noncytolytic and usually persistent virus infection, high-dose input virus mediates early control of the pathogen due to an efficient induction of an antiviral immune mechanism. From these data, we conclude that immune reactivity, in particular the cytotoxic T-cell response, determines whether the virus is controlled with prevention of the ensuing immunopathological disease or whether a persistent infection is established.
Herpes Simplex Virus Virion Host Shutoff Protein Requires a Mammalian Factor for Efficient In Vitro Endoribonuclease Activity
Authors: Lu Patricia , Jones Frank E. , Saffran Holly A. , Smiley James R.
Published: July 2002

The virion host shutoff protein (vhs) of herpes simplex virus (HSV) triggers global shutoff of host protein synthesis and accelerated mRNA turnover during virus infection and induces endoribonucleolytic cleavage of exogenous RNA substrates when it is produced in a rabbit reticulocyte (RRL) in vitro translation system. Although vhs induces RNA turnover in the absence of other HSV gene products, it is not yet known whether cellular factors are required for its activity. As one approach to addressing this question, we expressed vhs in the budding yeast
Saccharomyces cerevisiae
. Expression of vhs inhibited colony formation, and the severity of this effect varied with the carbon source. The biological relevance of this effect was assessed by examining the activity of five mutant forms of vhs bearing previously characterized in-frame linker insertions. The results indicated a complete concordance between the growth inhibition phenotype in yeast and mammalian host cell shutoff. Despite these results, expression of vhs did not trigger global mRNA turnover in vivo, and cell extracts of yeast expressing vhs displayed little if any vhs-dependent endoribonuclease activity. However, activity was readily detected when such extracts were mixed with RRL. These data suggest that the vhs-dependent endoribonuclease requires one or more mammalian macromolecular factors for efficient activity.
V Domain of Human SLAM (CDw150) Is Essential for Its Function as a Measles Virus Receptor
Authors: Ono Nobuyuki , Tatsuo Hironobu , Tanaka Kotaro , Minagawa Hiroko , Yanagi Yusuke
Published: July 2002

Human signaling lymphocytic activation molecule (SLAM; also known as CDw150) has been shown to be a cellular receptor for measles virus (MV). Chinese hamster ovary cells transfected with a mouse SLAM cDNA were not susceptible to MV and the vesicular stomatitis virus pseudotype bearing MV envelope proteins alone, indicating that mouse SLAM cannot act as an MV receptor. To determine the functional domain of the receptor, we tested the abilities of several chimeric SLAM proteins to function as MV receptors. The ectodomain of SLAM comprises the two immunoglobulin superfamily domains (V and C2). Various chimeric transmembrane proteins possessing the V domain of human SLAM were able to act as MV receptors, whereas a chimera consisting of human SLAM containing the mouse V domain instead of the human V domain no longer acted as a receptor. To examine the interaction between SLAM and MV envelope proteins, recombinant soluble forms of SLAM were produced. The soluble molecules possessing the V domain of human SLAM were shown to bind to cells expressing the MV hemagglutinin (H) protein but not to cells expressing the MV fusion protein or irrelevant envelope proteins. These results indicate that the V domain of human SLAM is necessary and sufficient to interact with the MV H protein and allow MV entry.
Detection of Infectious Baboon Cytomegalovirus after Baboon-to-Human Liver Xenotransplantation
Authors: Michaels Marian G. , Jenkins Frank J. , St. George Kirsten , Nalesnik Michael A. , Starzl Thomas E. , Rinaldo Charles R.
Published: July 2002

Xenotransplantation is considered to be a solution for the human donor shortage. However, there is a potential risk of transmitting animal infections from the transplanted organ. The known transmissibility and clinical significance of human cytomegalovirus (HCMV) infection after allotransplantation led us to evaluate whether baboon cytomegalovirus (BCMV) transmission could occur after a baboon-to-human liver xenotransplant. We examined serial blood samples from a baboon liver recipient and isolated replication-competent CMV-like agents on days 29, 36, and 42 after xenotransplantation. BCMV and HCMV DNAs were detected in the day 29 isolate, while only HCMV DNA was detected in the other isolates. This is the first report of detecting a replication-competent virus from a source animal after xenotransplantation and is a concern with regard to potential zoonotic transmission to others.
“Hit-and-Run” Transformation by Adenovirus Oncogenes
Authors: Nevels Michael , Täuber Birgitt , Spruss Thilo , Wolf Hans , Dobner Thomas
Published: July 2002

According to classical concepts of viral oncogenesis, the persistence of virus-specific oncogenes is required to maintain the transformed cellular phenotype. In contrast, the “hit-and-run” hypothesis claims that viruses can mediate cellular transformation through an initial “hit,” while maintenance of the transformed state is compatible with the loss (“run”) of viral molecules. It is well established that the adenovirus E1A and E1B gene products can cooperatively transform primary human and rodent cells to a tumorigenic phenotype and that these cells permanently express the viral oncogenes. Additionally, recent studies have shown that the adenovirus E4 region encodes two novel oncoproteins, the products of E4orf6 and E4orf3, which cooperate with the viral E1A proteins to transform primary rat cells in an E1B-like fashion. Unexpectedly, however, cells transformed by E1A and either E4orf6 or E4orf3 fail to express the viral E4 gene products, and only a subset contain E1A proteins. In fact, the majority of these cells lack E4- and E1A-specific DNA sequences, indicating that transformation occurred through a hit-and-run mechanism. We provide evidence that the unusual transforming activities of the adenoviral oncoproteins may be due to their mutagenic potential. Our results strongly support the possibility that even tumors that lack any detectable virus-specific molecules can be of viral origin, which could have a significant impact on the use of adenoviral vectors for gene therapy.
Region of Herpes Simplex Virus Type 1 Latency-Associated Transcript Sufficient for Wild-Type Spontaneous Reactivation Promotes Cell Survival in Tissue Culture
Authors: Inman Melissa , Perng Guey-Chuen , Henderson Gail , Ghiasi Homayon , Nesburn Anthony B. , Wechsler Steven L. , Jones Clinton
Published: July 2002

The latency-associated transcript (LAT) is the only abundant herpes simplex virus type 1 (HSV-1) transcript expressed during latency. In the rabbit eye model, LAT null mutants do not reactivate efficiently from latency. We recently demonstrated that the LAT null mutant
d
LAT2903 induces increased levels of apoptosis in trigeminal ganglia of infected rabbits compared to LAT
+
strains (G.-C. Perng, C. Jones, J. Ciacci-Zarella, M. Stone, G. Henderson, A. Yokht, S. M. Slanina, F. M. Hoffman, H. Ghiasi, A. B. Nesburn, and C. S. Wechsler, Science 287:1500–1503, 2000).The same study also demonstrated that a plasmid expressing LAT nucleotides 301 to 2659 enhanced cell survival of transfected cells after induction of apoptosis. Consequently, we hypothesized that LAT enhances spontaneous reactivation in part, because it promotes survival of infected neurons. Here we report on the ability of plasmids expressing different portions of the 5′ end of LAT to promote cell survival after induction of apoptosis. A plasmid expressing the first 1.5 kb of LAT (LAT nucleotides 1 to 1499) promoted cell survival in neuro-2A (mouse neuronal) and CV-1 (monkey fibroblast) cells. A plasmid expressing just the first 811 nucleotides of LAT promoted cell survival less efficiently. Plasmids expressing the first 661 nucleotides or less of LAT did not promote cell survival. We previously showed that a mutant expressing just the first 1.5 kb of LAT has wild-type spontaneous reactivation in rabbits, and a mutant expressing just the first 811 nucleotides of LAT has a reactivation frequency higher than that of
d
LAT2903 but lower than that of wild-type virus. In addition, mutants reported here for the first time, expressing just the first 661 or 76 nucleotides of LAT, had spontaneous reactivation indistinguishable from that of the LAT null mutant
d
LAT2903. In summary, these studies provide evidence that there is a functional relationship between the ability of LAT to promote cell survival and its ability to enhance spontaneous reactivation.