bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of Protein Kinases Dysregulated in CD4+T Cells in Pathogenic versus Apathogenic Simian Immunodeficiency Virus Infection
Authors: Bostik Pavel , Wu Peggy , Dodd Geraldine L. , Villinger Francois , Mayne Ann E. , Bostik Vanda , Grimm Bennett D. , Robinson Dan , Kung Hsing-Jien , Ansari Aftab A.
Published: July 2002

Human immunodeficiency virus infection in humans and simian immunodeficiency virus (SIV) infection in rhesus macaques (RM) leads to a generalized loss of immune responses involving perturbations in T-cell receptor (TCR) signaling. In contrast, naturally SIV-infected sooty mangabeys (SM) remain asymptomatic and retain immune responses despite relatively high viral loads. However, SIV infection in both RM and SM led to similar decreases in TCR-induced Lck phosphorylation. In this study, a protein tyrosine kinase (PTK) differential display method was utilized to characterize the effects of in vivo SIV infection on key signaling molecules of the CD4+T-cell signaling pathways. The CD4+T cells from SIV-infected RM, but not SIV-infected SM, showed chronic downregulation of baseline expression of MLK3, PRK, and GSK3, and symptomatically SIV-infected RM showed similar downregulation of MKK3. In vitro TCR stimulation with or without CD28 costimulation of CD4+T cells did not lead to the enhancement of gene transcription of these PTKs. While the CD4+T cells from SIV-infected RM showed a significant increase of the baseline and anti-TCR-mediated ROR2 transcription, SIV infection in SM led to substantially decreased anti-TCR-stimulated ROR2 transcription. TCR stimulation of CD4+T cells from SIV-infected RM (but not SIV-infected SM) led to the repression of CaMKKβ and the induction of gene transcription of MLK2. Studies of the function of these molecules in T-cell signaling may lead to the identification of potential targets for specific intervention, leading to the restoration of T-cell responses.
Individual and Bivalent Vaccines Based on Alphavirus Replicons Protect Guinea Pigs against Infection with Lassa and Ebola Viruses
Authors: Pushko Peter , Geisbert Joan , Parker Michael , Jahrling Peter , Smith Jonathan
Published: July 2002

Lassa and Ebola viruses cause acute, often fatal, hemorrhagic fever diseases, for which no effective vaccines are currently available. Although lethal human disease outbreaks have been confined so far to sub-Saharan Africa, they also pose significant epidemiological concern worldwide as demonstrated by several instances of accidental importation of the viruses into North America and Europe. In the present study, we developed experimental individual vaccines for Lassa virus and bivalent vaccines for Lassa and Ebola viruses that are based on an RNA replicon vector derived from an attenuated strain of Venezuelan equine encephalitis virus. The Lassa and Ebola virus genes were expressed from recombinant replicon RNAs that also encoded the replicase function and were capable of efficient intracellular self-amplification. For vaccinations, the recombinant replicons were incorporated into virus-like replicon particles. Guinea pigs vaccinated with particles expressing Lassa virus nucleoprotein or glycoprotein genes were protected from lethal challenge with Lassa virus. Vaccination with particles expressing Ebola virus glycoprotein gene also protected the animals from lethal challenge with Ebola virus. In order to evaluate a single vaccine protecting against both Lassa and Ebola viruses, we developed dual-expression particles that expressed glycoprotein genes of both Ebola and Lassa viruses. Vaccination of guinea pigs with either dual-expression particles or with a mixture of particles expressing Ebola and Lassa virus glycoprotein genes protected the animals against challenges with Ebola and Lassa viruses. The results showed that immune responses can be induced against multiple vaccine antigens coexpressed from an alphavirus replicon and suggested the possibility of engineering multivalent vaccines based upon alphavirus vectors for arenaviruses, filoviruses, and possibly other emerging pathogens.
Molecular Genetic Analysis of Revertants from a Poliovirus Mutant That Is Specifically Adapted to the Mouse Spinal Cord
Authors: Jia Qingmei , Hogle James M. , Hashikawa Tsutomu , Nomoto Akio
Published: July 2002

SA virus, a mutant of the Mahoney strain of type 1 poliovirus (PV1/Mahoney), replicates specifically in the spinal cords of mice and causes paralysis, although the PV1/Mahoney strain does not show any mouse neurovirulence (Q. Jia, S. Ohka, K. Iwasaki, K. Tohyama, and A. Nomoto, J. Virol. 73:6041–6047, 1999). The key mutation site for the mouse neurovirulence of SA was mapped to nucleotide (nt) 928 of the genome (A to G), resulting in the amino acid substitution of Met for Ile at residue 62 within the capsid protein VP4 (VP4062). A small-plaque phenotype of SA appears to be indicative of its mouse-neurovirulent phenotype. To identify additional amino acid residues involved in the host range determination of PV, a total of 14 large-plaque (LP) variants were isolated from a single point mutant, Mah/I4062M, that showed the SA phenotype. All the LP variants no longer showed any mouse neurovirulence when delivered via an intraspinal inoculation route. Of these, 11 isolates had a back mutation at nt 928 (G to A) that restored the nucleotide of the PV1/Mahoney type. The reversions of the remaining three isolates (LP8, LP9, and LP14) were mediated by a second site mutation. Molecular genetic analysis involving recombinants between Mah/I4062M and the LP variants revealed that the mere substitution of an amino acid residue at position 107 in VP1 (Val to Leu) (LP9), position 33 in VP2 (Val to Ile) (LP14), or position 231 in VP3 (Ile to Thr) (LP8) was sufficient to restore the PV1/Mahoney phenotype. These amino acid residues are located either on the surface or inside of the virus particle. Our results indicate that the mouse neurovirulence of PV is determined by the virion surface structure, which is formed by all four capsid proteins.
Vaccination with Attenuated Simian Immunodeficiency Virus by DNA Inoculation
Authors: Kent Stephen J. , Dale C. Jane , Preiss Scott , Mills John , Campagna Daniella , Purcell Damian F. J.
Published: July 2002

Delivering attenuated lentivirus vaccines as proviral DNA would be simple and inexpensive. Inoculation of macaques with wild-type simian immunodeficiency virus strain mac239 (SIV
mac239
) DNA or SIV
mac239
DNA containing a single deletion in the 3′
nef
-long terminal repeat overlap region (
nef
/LTR) led to sustained SIV infections and AIDS. Injection of SIV
mac239
DNA containing identical deletions in both the 5′ LTR and 3′
nef
/LTR resulted in attenuated SIV infections and substantial protection against subsequent mucosal SIV
mac251
challenge.
Hepatitis C Virus Core and Envelope Proteins Do Not Suppress the Host's Ability To Clear a Hepatic Viral Infection
Authors: Sun Jiaren , Bodola Francis , Fan Xuegong , Irshad Habib , Soong Lynn , Lemon Stanley M. , Chan Teh-Sheng
Published: July 2002

Several hepatitis C virus (HCV) proteins have been shown in vitro to interact with host cellular components that are involved in immune regulation. However, there is a paucity of data supporting the relevance of these observations to the in vivo situation. To test the hypothesis that such an interaction suppresses immune responses, we studied a line of transgenic C57BL/6 mice that express the HCV core and envelope proteins in the liver. The potential effects of these proteins on the hepatic immune response were evaluated by challenging these mice with a hepatotropic adenovirus. Both transgenic and nontransgenic mice developed similar courses of infection and cleared the virus from the liver by 28 days postinfection. Both groups of mice mounted similar immunoglobulin G (IgG), IgG2a, interleukin-2, and tumor necrosis factor alpha responses against the virus. Additionally, BALB/c mice were able to clear infection with recombinant adenovirus that does or does not express the HCV core and envelope 1 proteins in the same manner. These data suggest that HCV core and envelope proteins do not inhibit the hepatic antiviral mechanisms in these murine experimental systems and thus favor a model in which HCV circumvents host responses through a mechanism that does not involve general suppression of intrahepatic immune responses.
Contribution of the Human Parainfluenza Virus Type 3 HN-Receptor Interaction to Pathogenesis In Vivo
Authors: Prince Gregory A. , Ottolini Martin G. , Moscona Anne
Published: July 2002

The envelope of human parainfluenza virus type 3 (HPF3) contains two viral glycoproteins, the hemagglutinin-neuraminidase (HN) protein and the fusion (F) protein. In a previous study, highly fusogenic variant HPF3 viruses were isolated, including two, C-0 and C-22, that exhibit increased avidity for sialic acid receptors due to single amino acid changes in the HN protein and one, C-28, that has decreased neuraminidase activity relative to that of the wild type (wt) and is delayed in the release of virus particles into the supernatant fluid. These variants form very large plaques and destroy a cell monolayer more rapidly than does wt HPF3 in cell culture. These variant viruses allowed us to formulate hypotheses about the roles of HN in pathogenesis. We investigated the behavior of wt HPF3 and the three variant viruses in the cotton rat model. In the cotton rat, there was no delayed clearance of any of the variant viruses compared to that of the wt. The variant plaque morphology was preserved in vivo, and there was no reversion to the wt phenotype in the infected animals. In spite of a slight advantage of wt virus in viral titer, there were no differences in the severities of peribronchiolitis between wt viruses and the variants. However, there were marked differences in severities in alveolitis and interstitial pneumonitis when each of the three variants was compared to the wt, with the variants causing enhanced disease. Thus, despite similar or lower viral titers and similar clearance rates, the variants caused more extensive disease in the lung. The results show that mutations in HN conferring altered fusion properties in cell culture also confer striking differences in the ability of HPF3 to cause extensive disease in the cotton rat lung and that this effect is dissociated from any effect on viral replication.
Identification of Two Prion Protein Regions That Modify Scrapie Incubation Time
Authors: Supattapone Surachai , Muramoto Tamaki , Legname Giuseppe , Mehlhorn Ingrid , Cohen Fred E. , DeArmond Stephen J. , Prusiner Stanley B. , Scott Michael R.
Published: July 2002

A series of prion transmission experiments was performed in transgenic (Tg) mice expressing either wild-type, chimeric, or truncated prion protein (PrP) molecules. Following inoculation with Rocky Mountain Laboratory (RML) murine prions, scrapie incubation times for Tg(MoPrP)4053, Tg(MHM2)294/Prnp
0/0
, and Tg(MoPrP,Δ23–88)9949/Prnp
0/0
mice were ∼50, 120, and 160 days, respectively. Similar scrapie incubation times were obtained after inoculation of these lines of Tg mice with either MHM2(MHM2(RML)) or MoPrP(Δ23–88)(RML) prions, excluding the possibility that sequence-dependent transmission barriers could account for the observed differences. Tg(MHM2)294/Prnp
0/0
mice displayed prolonged scrapie incubation times with four different strains of murine prions. These data provide evidence that the N terminus of MoPrP and the chimeric region of MHM2 PrP (residues 108 through 111) both influence the inherent efficiency of prion propagation.
Ebola Virus Glycoprotein: Proteolytic Processing, Acylation, Cell Tropism, and Detection of Neutralizing Antibodies
Authors: Ito Hiroshi , Watanabe Shinji , Takada Ayato , Kawaoka Yoshihiro
Published: July 2002

Using the vesicular stomatitis virus (VSV) pseudotype system, we studied the functional properties of the Ebola virus glycoprotein (GP). Amino acid substitutions at the GP cleavage site, which reduce glycoprotein cleavability and viral infectivity in some viruses, did not appreciably change the infectivity of VSV pseudotyped with GP. Likewise, removal of two acylated cysteine residues in the transmembrane region of GP showed no discernible effects on infectivity. Although most filoviruses are believed to target endothelial cells and hepatocytes preferentially, the GP-carrying VSV showed greater affinity for epithelial cells than for either of these cell types, indicating that Ebola virus GP does not necessarily have strong tropism toward endothelial cells and hepatocytes. Finally, when it was used to screen for neutralizing antibodies against Ebola virus GP, the VSV pseudotype system allowed us to detect strain-specific neutralizing activity that was inhibited by secretory GP (SGP). This finding provides evidence of shared neutralizing epitopes on GP and SGP molecules and indicates the potential of SGP to serve as a decoy for neutralizing antibodies.
Temperature-Sensitive Transformation by an Abelson Virus Mutant Encoding an Altered SH2 Domain
Authors: Mainville Celine A. , Parmar Kalindi , Unnikrishnan Indira , Gong Li , Raffel Glen D. , Rosenberg Naomi
Published: July 2002

Abelson murine leukemia virus (Ab-MLV) encodes the v-Abl protein tyrosine kinase and induces transformation of immortalized fibroblast lines and pre-B cells. Temperature-sensitive mutations affecting the kinase domain of the protein have demonstrated that the kinase activity is absolutely required for transformation. Despite this requirement, mutations affecting other regions of v-Abl modulate transformation activity. The SH2 domain and the highly conserved FLVRES motif within it form a phosphotyrosine-binding pocket that is required for interactions between the kinase and cellular substrates. To understand the impact of SH2 alterations on Ab-MLV-mediated transformation, we studied the Ab-MLV mutant P120/R273K. This mutant encodes a v-Abl protein in which the βB5 arginine at the base of the phosphotyrosine-binding pocket has been replaced by a lysine. Unexpectedly, infection of NIH 3T3 or pre-B cells with P120/R273K revealed a temperature-dependent transformation phenotype. At 34°C, P120/R273K transformed about 10-fold fewer cells than wild-type virus of equivalent titer; at 39.5°C, 300-fold fewer NIH 3T3 cells were transformed and pre-B cells were refractory to transformation. Temperature-dependent transformation was accompanied by decreased phosphorylation of Shc, a protein that interacts with the v-Abl SH2 and links the protein to Ras, and decreased induction of c-Myc expression. These data suggest that alteration of the FLVRES pocket affects the ability of v-Abl to interact with at least some of its substrates in a temperature-dependent fashion and identify a novel type of temperature-sensitive Abelson virus.
Evidence of TwoLyssavirusPhylogroups with Distinct Pathogenicity and Immunogenicity
Authors: Badrane Hassan , Bahloul Chokri , Perrin Pierre , Tordo Noël
Published: July 2002

The genetic diversity of representative members of theLyssavirusgenus (rabies and rabies-related viruses) was evaluated using the gene encoding the transmembrane glycoprotein involved in the virus-host interaction, immunogenicity, and pathogenicity. Phylogenetic analysis distinguished seven genotypes, which could be divided into two major phylogroups having the highest bootstrap values. Phylogroup I comprises the worldwide genotype 1 (classicRabies virus), theEuropean bat lyssavirus(EBL) genotypes 5 (EBL1) and 6 (EBL2), the African genotype 4 (Duvenhage virus), and theAustralian bat lyssavirusgenotype 7. Phylogroup II comprises the divergent African genotypes 2 (Lagos bat virus) and 3 (Mokola virus). We studied immunogenic and pathogenic properties to investigate the biological significance of this phylogenetic grouping. Viruses from phylogroup I (Rabies virusand EBL1) were found to be pathogenic for mice when injected by the intracerebral or the intramuscular route, whereas viruses from phylogroup II (Mokola and Lagos bat viruses) were only pathogenic by the intracerebral route. We showed that the glycoprotein R333 residue essential for virulence was naturally replaced by a D333 in the phylogroup II viruses, likely resulting in their attenuated pathogenicity. Moreover, cross-neutralization distinguished the same phylogroups. Within each phylogroup, the amino acid sequence of the glycoprotein ectodomain was at least 74% identical, and antiglycoprotein virus-neutralizing antibodies displayed cross-neutralization. Between phylogroups, the identity was less than 64.5% and the cross-neutralization was absent, explaining why the classical rabies vaccines (phylogroup I) cannot protect against lyssaviruses from phylogroup II. Our tree-axial analysis divided lyssaviruses into two phylogroups that more closely reflect their biological characteristics than previous serotypes and genotypes.
Inhibition of Human Immunodeficiency Virus Type 1 (HIV-1) Replication by HIV-1-Based Lentivirus Vectors Expressing Transdominant Rev
Authors: Mautino Mario R. , Keiser Nicholas , Morgan Richard A.
Published: July 2002

Retrovirus vectors expressing transdominant-negative mutants of Rev (TdRev) inhibit human immunodeficiency virus type 1 (HIV-1) replication by preventing the nuclear export of unspliced viral transcripts, thus inhibiting the synthesis of Gag-Pol, Env, and genomic RNA. The use of HIV-1–based vectors to express TdRev would have the advantage of allowing access to nondividing hematopoietic cells. It would also provide additional levels of protection by sequestering the viral regulatory proteins Tat and Rev, competing for encapsidation into wild-type virions, and inhibiting reverse transcription. Here we describe HIV-1-based vectors that express TdRev. These vectors contain mutations in the splicing signals or replacement of the Rev-responsive element by the simian retrovirus type 1 constitutive transport element, making them less sensitive to the inhibitory effects of TdRev. In addition, overexpression of Rev and the use of an HIV-1 helper plasmid that drives high levels of Gag-Pol synthesis were used to transiently overcome the inhibition by TdRev of the synthesis of Gag-Pol during vector production. SupT1 cells transduced with these vectors were more resistant to HIV-1 replication than cells transduced with Moloney murine leukemia virus-based vectors expressing TdRev. Furthermore, we show that these vectors can be mobilized by the wild-type virus, reducing the infectivity of virions escaping inhibition and conferring protection against HIV-1 replication to previously untransduced cells.
In Vivo Analysis of Retroviral Enhancer Mutations in Hematopoietic Cells: SP1/EGR1 and ETS/GATA Motifs Contribute to Long Terminal Repeat Specificity
Authors: Wahlers Anke , Zipfel Peter F. , Schwieger Maike , Ostertag Wolfram , Baum Christopher
Published: July 2002

The objective of this work was to identify, in the context of chromosomally integrated DNA, the contribution of defined transcription factor binding motifs to the function of a complex retrovirus enhancer in hematopoietic cells in vivo. Repopulating murine hematopoietic cells were transduced with equal gene dosages of replication-incompetent retrovirus vectors encoding enhanced green fluorescent protein. Enhancer sequences were derived from mouse spleen focus-forming virus. Destruction of GC-rich sites representing overlapping targets for SP1 or EGR1 uniformly attenuated gene expression (∼25 to 70% of wild-type levels) in all hematopoietic lineages, as shown by multicolor flow cytometry of peripheral blood and bone marrow cells at various time points posttransplantation. In contrast, a point mutation within a dual ETS/GATA motif that abolished transactivation by ETS factors but not by GATA-1 slightly increased activity in erythroid cells and significantly attenuated enhancer function in T lymphocytes. This study shows that controlled gene transfer in transplantable hematopoietic cells allows a functional analysis of distinct
cis
elements within a complex retrovirus enhancer, as required for the characterization and engineering of various cellular and viral regulatory sequences in basic research and gene therapy.
Complete Nucleotide Sequence of the Rhesus Lymphocryptovirus: Genetic Validation for an Epstein-Barr Virus Animal Model
Authors: Rivailler Pierre , Jiang Hua , Cho Young-gyu , Quink Carol , Wang Fred
Published: July 2002

We sequenced the rhesus lymphocryptovirus (LCV) genome in order to determine its genetic similarity to Epstein-Barr virus (EBV). The rhesus LCV encodes a repertoire identical to that of EBV, with 80 open reading frames, including cellular interleukin-10,
bcl-2
, and colony-stimulating factor 1 receptor homologues and an equivalent set of viral glycoproteins. The highly conserved rhesus LCV gene repertoire provides a unique animal model for the study of EBV pathogenesis.
Relationship between Vaccinia Virus Intracellular Cores, Early mRNAs, and DNA Replication Sites
Authors: Mallardo Massimo , Leithe Edward , Schleich Sibylle , Roos Norbert , Doglio Laura , Krijnse Locker Jacomine
Published: July 2002

Virus assembly, a late event in the life cycle of vaccinia virus (VV), is preceded by a number of steps that all occur in the cytoplasm of the infected host cell: virion entry, delivery of the viral core into the cytoplasm, and transcription from these cores of early mRNAs, followed by the process of DNA replication. In the present study the quantitative and structural relationships between these distinct steps of VV morphogenesis were investigated. We show that viral RNA and DNA synthesis increases linearly with increasing amounts of incoming cores. Moreover, at multiplicities of infection that result in 10 to 40 cores per cell, an approximately 1:1 ratio between cores and sites of DNA replication exists, suggesting that each core is infectious. We have shown previously that VV early mRNAs collect in distinct granular structures that recruit components of the host cell translation machinery. Strikingly, these structures appeared to form some distance away from intracellular cores (M. Mallardo, S. Schleich, and J. Krijnse Locker, Mol. Biol. Cell 12:3875-3891, 2001). In the present study the intracellular locations of the sites of early mRNA accumulation and those of the subsequent process of DNA replication were compared. We show that these are distinct structures that have different intracellular locations. Finally, we study the fate of the parental DNA after core uncoating. By electron microscopy, cores were found close to membranes of the endoplasmic reticulum (ER) and the parental DNA, once it had left the core, appeared to associate preferentially with the cytosolic side of those membranes. Since we have previously shown that the process of DNA replication occurs in an ER-enclosed cytosolic “subcompartment” (N. Tolonen, L. Doglio, S. Schleich, and J. Krijnse Locker, Mol. Biol. Cell 12:2031-2046, 2001), the present data suggest that the parental DNA is released into the cytosol and associates with the same membranes where DNA replication is subsequently initiated. The combined data are discussed with respect to the cytosolic organization of VV morphogenesis.
A Fifteen-Amino-Acid TVB Peptide Serves as a Minimal Soluble Receptor for Subgroup B Avian Leukosis and Sarcoma Viruses
Authors: Knauss Daniel J. , Young John A. T.
Published: July 2002

The TVB receptor for subgroup B, D, and E avian sarcoma and leukosis viruses (ASLVs) is a tumor necrosis factor receptor-related protein that is most closely related to the TRAIL receptors. Here we show that the major subgroup B viral interaction determinants of TVB are contained within a linear 15-amino-acid peptide derived from the N-terminal region of the receptor. Moreover, this peptide was sufficient not only for binding to ASLV-B but also for activating viral entry into mammalian cells that lacked the cognate viral receptor. Peptide-dependent viral entry was blocked in the presence of bafilomycin A1, indicating that virions can be trafficked to an acidic endosomal fusion compartment without the need for physical attachment of the viral receptor to a cellular membrane.
Isolation and Analysis of Retroviral Integration Targets by Solo Long Terminal Repeat Inverse PCR
Authors: Jin Yi Feng , Ishibashi Toshio , Nomoto Akio , Masuda Michiaki
Published: July 2002

Upon retroviral infection, the genomic RNA is reverse transcribed to make proviral DNA, which is then integrated into the host chromosome. Although the viral elements required for successful integration have been extensively characterized, little is known about the host DNA structure constituting preferred targets for proviral integration. In order to elucidate the mechanism for the target selection, comparison of host DNA sequences at proviral integration sites may be useful. To achieve simultaneous analysis of the upstream and downstream host DNA sequences flanking each proviral integration site, a Moloney murine leukemia virus-based retroviral vector was designed so that its integrated provirus could be removed by Cre-
loxP
homologous recombination, leaving a solo long terminal repeat (LTR). Taking advantage of the solo LTR, inverse PCR was carried out to amplify both the upstream and downstream cellular flanking DNA. The method called solo LTR inverse PCR, or SLIP, proved useful for simultaneously cloning the upstream and downstream flanking sequences of individual proviral integration sites from the polyclonal population of cells harboring provirus at different chromosomal sites. By the SLIP method, nucleotide sequences corresponding to 38 independent proviral integration targets were determined and, interestingly, atypical virus-host DNA junction structures were found in more than 20% of the cases. Characterization of retroviral integration sites using the SLIP method may provide useful insights into the mechanism for proviral integration and its target selection.
Complete, Long-Lasting Protection against Lethal Infectious Bursal Disease Virus Challenge by a Single Vaccination with an Avian Herpesvirus Vector Expressing VP2 Antigens
Authors: Tsukamoto K. , Saito S. , Saeki S. , Sato T. , Tanimura N. , Isobe T. , Mase M. , Imada T. , Yuasa N. , Yamaguchi S.
Published: July 2002

Marek's disease herpesvirus is a vaccine vector of great promise for chickens; however, complete protection against foreign infectious diseases has not been achieved. In this study, two herpesvirus of turkey recombinants (rHVTs) expressing large amounts of infectious bursal disease virus (IBDV) VP2 antigen under the control of a human cytomegalovirus (CMV) promoter or CMV/β-actin chimera promoter (Pec promoter) (rHVT-cmvVP2 and rHVT-pecVP2) were constructed. rHVT-pecVP2, which expressed the VP2 antigen approximately four times more than did rHVT-cmvVP2 in vitro, induced complete protection against a lethal IBDV challenge in chickens, whereas rHVT-cmvVP2 induced 58% protection. All of the chickens vaccinated with rHVT-pecVP2 had a protective level of antibodies to the VP2 antigen at the time of challenge, whereas only 42 and 67% of chickens vaccinated with rHVT-cmvVP2 or the conventional live IBDV vaccine, respectively, had the antibodies. The antibody level of chickens vaccinated with rHVT-pecVP2 increased for 16 weeks, and the peak antibody level persisted throughout the experiment. The serum antibody titer at 30 weeks of age was about 20 or 65 times higher than that of chickens vaccinated with rHVT-cmvVP2 or the conventional live vaccine, respectively. rHVT-pecVP2, isolated consistently for 30 weeks from the vaccinated chickens, expressed the VP2 antigen after cultivation, and neither nucleotide mutations nor deletion in the VP2 gene was found. These results demonstrate that the amount of VP2 antigen expressed in the HVT vector was correlated with the vaccine efficacy against lethal IBDV challenge, and complete protective immunity that is likely to persist for the life of the chickens was induced.
1-β-
d
-Arabinofuranosylcytosine Inhibits Borna Disease Virus Replication and Spread
Authors: Bajramovic Jeffrey J. , Syan Sylvie , Brahic Michel , de la Torre Juan Carlos , Gonzalez-Dunia Daniel
Published: July 2002

Borna disease virus (BDV) is a nonsegmented, negative-strand RNA virus that causes neurological diseases in a variety of warm-blooded animal species. There is general consensus that BDV can also infect humans, being a possible zoonosis. Although the clinical consequences of human BDV infection are still controversial, experimental BDV infection is a well-described model for human neuropsychiatric diseases. To date, there is no effective treatment against BDV. In this paper, we demonstrate that the nucleoside analog 1-β-
d
-arabinofuranosylcytosine (Ara-C), a known inhibitor of DNA polymerases, inhibits BDV replication. Ara-C treatment inhibited BDV RNA and protein synthesis and prevented BDV cell-to-cell spread in vitro. Replication of other negative-strand RNA viruses such as influenza virus or measles virus was not inhibited by Ara-C, underscoring the particularity of the replication machinery of BDV. Strikingly, Ara-C treatment induced nuclear retention of viral ribonucleoparticles. These findings could not be attributed to known effects of Ara-C on the host cell, suggesting that Ara-C directly inhibits the BDV polymerase. Finally, we show that Ara-C inhibits BDV replication in vivo in the brain of infected rats, preventing persistent infection of the central nervous system as well as the development of clinical disease. These findings open the way to the development of effective antiviral therapy against BDV.
Envelope-Dependent, Cyclophilin-Independent Effects of Glycosaminoglycans on Human Immunodeficiency Virus Type 1 Attachment and Infection
Authors: Zhang Yi-jun , Hatziioannou Theodora , Zang Trinity , Braaten Douglas , Luban Jeremy , Goff Stephen P. , Bieniasz Paul D.
Published: July 2002

Cell surface glycosaminoglycans (GAGs), in particular heparan sulfate (HS), have been proposed to mediate the attachment of human immunodeficiency virus type 1 (HIV-1) to target cells prior to virus entry, and both the viral gp120 envelope protein and virion-associated cyclophilin A (CypA) have been shown to directly interact with HS and its analogues. To determine the role of GAGs in HIV attachment and infection, we generated HIV-susceptible derivatives of CHO cell lines that either express high levels of GAGs (CHO-K1) or lack GAGs (pgsA745). Using a panel of HIV-1 envelopes, we found that cell surface GAG-mediated effects on virion attachment and infection vary in an envelope strain-dependent but coreceptor-independent manner. In fact, cell surface GAG-mediated enhancement of infection is confined to isolates that contain a highly positively charged V3-loop sequence, while infection by most strains is apparently inhibited by the presence of GAGs. Moreover, the enhancing and inhibitory effects of polycations and polyanions on HIV-1 infection are largely dependent on the presence of cell surface GAGs. These observations are consistent with a model in which GAGs influence in vitro HIV-1 infection primarily by modifying the charge characteristics of the target cell surface. Finally, the effects of GAGs on HIV-1 infection are observed to an equivalent extent whether CypA is present in or absent from virions. Overall, these data exclude a major role for GAGs in mediating the attachment of many HIV-1 strains to target cells via interactions with virion-associated gp120 or CypA.
Half-Life of the Duck Hepatitis B Virus Covalently Closed Circular DNA Pool In Vivo following Inhibition of Viral Replication
Authors: Addison William R. , Walters Kathie-Anne , Wong Winnie W. S. , Wilson John S. , Madej Danuta , Jewell Lawrence D. , Tyrrell D. Lorne J.
Published: July 2002

Covalently closed circular DNA (cccDNA) is a crucial intermediate in the replication of hepadnaviruses. We inhibited the replication of duck hepatitis B virus in congenitally infected ducks with a combination of lamivudine and a dideoxyguanosine prodrug. Inhibition of viral replication should prevent renewal of the cccDNA pool, and its decay was measured in liver biopsy samples collected over a 5-month period. In three ducks, the cccDNA pools declined exponentially, with half-lives ranging from 35 to 57 days. In two others, the pools declined exponentially for about 70 days but then stabilized at about 6 copies/diploid genome. The selection of drug-resistant virus mutants is an unlikely explanation for this unexpected stabilization of cccDNA levels. Liver sections stained for the cell division marker PCNA showed that animals in which cccDNA loss was continuous had significantly greater numbers of PCNA-positive nuclei than did those animals in which cccDNA levels had plateaued.
Role of the Varicella-Zoster Virus gB Cytoplasmic Domain in gB Transport and Viral Egress
Authors: Heineman Thomas C. , Hall Susan L.
Published: July 2002

To study the function of the varicella-zoster virus (VZV) gB cytoplasmic domain during viral infection, we produced a VZV recombinant virus that expresses a truncated form of gB lacking the C-terminal 36 amino acids of its cytoplasmic domain (VZV gB-36). VZV gB-36 replicates in noncomplementing cells and grows at a rate similar to that of native VZV. However, cells infected with VZVgB-36 form extensive syncytia compared to the relatively small syncytia formed during native VZV infection. In addition, electron microscopy shows that very little virus is present on the surfaces of cells infected with VZV gB-36, while cells infected with native VZV exhibit abundant virions on the cell surface. The C-terminal 36 amino acids of the gB cytoplasmic domain have been shown in transfection-based experiments to contain both an endoplasmic reticulum-to-Golgi transport signal (the C-terminal 17 amino acids) and a consensus YXXφ (where Y is tyrosine, X is any amino acid, and φ is any bulky hydrophobic amino acid) signal sequence (YSRV) that mediates the internalization of gB from the plasma membrane. As predicted based on these data, gB-36 expressed during the infection of cultured cells is transported inefficiently to the Golgi. Despite lacking the YSRV signal sequence, gB-36 is internalized from the plasma membrane; however, in contrast to native gB, it fails to localize to the Golgi. Therefore, the C-terminal 36 amino acids of the VZV gB cytoplasmic domain are required for normal viral egress and for both the pre- and post-Golgi transport of gB.
Regions of the Herpes Simplex Virus Scaffolding Protein That Are Important for Intermolecular Self-Interaction
Authors: Preston Valerie G. , McDougall Iris M.
Published: July 2002

The herpes simplex virus type 1 (HSV-1) scaffolding protein encoded by gene UL26.5 promotes the formation of the icosahedral capsid shell through its association with the major capsid protein VP5 and through intermolecular interactions with itself. Inside the capsid shell, the UL26.5 product together with the maturational protease, a minor protein, form a spherical structure which is broken down and released from the capsid during packaging of the viral genome. Selected residues from four internal regions of the HSV-1 scaffolding protein that have significant conservation of amino acids within the scaffolding proteins of alphaherpesviruses were mutated, and the properties of the proteins were examined. Only the HSV-1 scaffolding protein with mutations in the conserved N-terminal domain showed reduced interaction with the varicella-zoster virus homologue in a cell-based immunofluorescence assay, providing the first evidence that this domain in the HSV-1 protein is likely to be involved in intermolecular self-interaction. Scaffolding protein with mutations in this domain or in either of two other domains failed to assemble into scaffold-like particles but retained the ability to self-interact, although the aggregates were significant smaller than most of the aggregates formed by the wild-type protein. These results suggest that there are multiple domains involved in the intermolecular self-association of the HSV-1 scaffolding protein that can act independently of one another. This conclusion was supported by the observation that none of the mutant proteins with lesions in an individual domain, including a protein with mutations in a central region previously implicated in self-interaction (A. Pelletier, F. Dô, J. J. Brisebois, L. Lagacé, and M. G. Cordingley, J. Virol. 71:5197–5208, 1997), interfered with capsid assembly in a baculovirus expression system. A protein mutated in the central region and another conserved domain, both of which had been predicted to form coiled coils, was impaired for capsid formation but still retained the capacity to interact with VP5.
Nef-Mediated Resistance of Human Immunodeficiency Virus Type 1 to Antiviral Cytotoxic T Lymphocytes
Authors: Yang Otto O. , Nguyen Phuong Thi , Kalams Spyros A. , Dorfman Tanya , Göttlinger Heinrich G. , Stewart Sheila , Chen Irvin S. Y. , Threlkeld Steven , Walker Bruce D.
Published: July 2002

Although Nef has been proposed to effect the escape of human immunodeficiency virus type 1 (HIV-1) from cytotoxic T lymphocytes (CTL) through downmodulation of major histocompatibility complex class I molecules, little direct data have been presented previously to support this hypothesis. By comparingnef-competent andnef-deleted HIV-1 strains in an in vitro coculture system, we demonstrate that the presence of this viral accessory gene leads to impairment of the ability of HIV-1-specific CTL clones to suppress viral replication. Furthermore, inhibition by genetically modified CTL that do not require major histocompatibility complex class I-presented antigen (expressing the CD4 T-cell receptor [TCR] ζ-chain hybrid receptor) is similar for bothnef-competent and -deleted strains, indicating that Nef does not impair the effector functions of CTL but acts at the level of TCR triggering. In contrast, we note that another accessory gene,vpr, does not induce resistance of HIV-1 to suppression by CTL clones. We conclude that Nef (and not Vpr) contributes to functional HIV-1 immune evasion and that this effect is mediated by diminished antigen presentation to CTL.
Regulated Gene Expression in the Chicken Embryo by Using Replication-Competent Retroviral Vectors
Authors: Sato Noboru , Matsuda Kenji , Sakuma Chie , Foster Douglas N. , Oppenheim Ronald W. , Yaginuma Hiroyuki
Published: July 2002

Rous sarcoma virus (RSV)-derived retroviral vector could efficiently deliver the green fluorescent protein (GFP), which is driven by the internal cytomegalovirus enhancer/promoter, into restricted cell populations in the chicken embryo. RSV-derived vectors coupled with the
tet
regulatory elements also revealed doxycycline-dependent inducible GFP expression in the chicken embryo in ovo.
Construction of a Self-Excisable Bacterial Artificial Chromosome Containing the Human Cytomegalovirus Genome and Mutagenesis of the Diploid TRL/IRL13 Gene
Authors: Yu Dong , Smith Gregory A. , Enquist Lynn W. , Shenk Thomas
Published: July 2002

The full-length genome of human cytomegalovirus strain AD169 was cloned as an infectious bacterial artificial chromosome (BAC) plasmid, pAD/Cre. The BAC vector, flanked by LoxP sites, was inserted immediately after the Us28 open reading frame without deletion of any viral sequences. The BAC vector contained the Cre recombinase-encoding gene disrupted by an intron under control of the simian virus 40 early promoter. When pAD/Cre was transfected into primary human foreskin fibroblast cells, Cre was expressed and mediated site-specific recombination between the two LoxP sites, excising the BAC DNA backbone. This gave rise to progeny virus that was wild type with the exception of an inserted 34-bp LoxP site. We performed site-directed mutagenesis on pAD/Cre to generate a series of viruses in which the TRL/IRL13 diploid genes were disrupted and subsequently repaired. The mutants reach the same titer as the wild-type virus, indicating that the TRL/IRL13 open reading frames are not required for virus growth in cell culture. The sequence of the TRL13 open reading frame in the low-passage Toledo strain of human cytomegalovirus is quite different from the corresponding region in the AD169 strain. One of multiple changes is a frameshift mutation. As a consequence, strain Toledo encodes a putative TRL13 protein whose C-terminal domain is larger (extending through the TRL14 coding region) and encodes in a reading frame different from that of strain AD169. We speculate that the strain AD169 coding region has drifted during passage in the laboratory. We propose that TRL13 has been truncated in strain AD169 and that the partially overlapping TRL14 open reading frame is not functional. This view is consistent with the presence of both TRL13 and -14 on all mRNAs that we have mapped from this region, an organization that would include the much longer strain Toledo TRL13 open reading frame on the mRNAs.
Chronic Subclinical Prion Disease Induced by Low-Dose Inoculum
Authors: Thackray Alana M. , Klein Michael A. , Aguzzi Adriano , Bujdoso Raymond
Published: July 2002

We have compared the transmission characteristics of the two mouse-adapted scrapie isolates, ME7 and Rocky Mountain Laboratory (RML), in tga20 mice. These mice express elevated levels of PrP protein compared to wild-type mice and display a relatively short disease incubation period following intracerebral prion inoculation. Terminal prion disease in tga20 mice induced by ME7 or RML was characterized by a distinct pattern of clinical signs and different incubation times. High-dose RML inoculated intracerebrally into tga20 mice induced the most rapid onset of clinical signs, with mice succumbing to terminal disease after only 58 ± 3 days. In contrast, high-dose ME7 gave a mean time to terminal disease of 74 ± 0 days. Histological examination of brain sections from prion-inoculated tga20 mice at terminal disease showed that ME7 gave rise to a more general and extensive pattern of vacuolation than RML. Low-dose inoculum failed to induce terminal disease but did cause preclinical symptoms, including the appearance of reversible clinical signs. Some mice oscillated between showing no clinical signs and early clinical signs for many months but never progressed to terminal disease. Brain tissue from these mice with chronic subclinical prion disease, sacrificed at >200 days postinoculation, contained high levels of infectivity and showed the presence of PrP
Sc
. Parallel analysis of brain tissue from mice with terminal disease showed similar levels of infectivity and detectable PrP
Sc
. These results show that high levels of infectivity and the presence of the abnormal isomer of PrP can be detected in mice with subclinical disease following low-dose prion inoculation.
Abrogation of Ref1 Retrovirus Restriction in Human Cells
Authors: Towers Greg , Collins Mary , Takeuchi Yasuhiro
Published: July 2002

We have previously described postentry restriction of murine leukemia virus in mammals. Here we characterize the block in human cells. Restricted infection kinetics are multiple hit at high virus dose, and restriction is abrogated by preexposure to restricted virus. We hypothesize that restricted capsid can titrate out the restriction factor.
Specific Inactivation of Inhibitory Sequences in the 5′ End of the Human Papillomavirus Type 16 L1 Open Reading Frame Results in Production of High Levels of L1 Protein in Human Epithelial Cells
Authors: Collier Brian , Öberg Daniel , Zhao Xiaomin , Schwartz Stefan
Published: July 2002

The expression of human papillomavirus type 16 late genes encoding virus capsid proteins L1 and L2 is restricted to terminally differentiated epithelial cells in the superficial layers of the squamous epithelium. We wish to understand the molecular mechanisms that determine the levels of expression of the human papillomavirus type 16 late genes. We have previously shown that the L1 coding region contains inhibitory sequences. Here we extend previous findings to show that the 5′ end of the L1 gene contains strong inhibitory sequences but that the 3′ end does not. We show that the first 514 nucleotides of the L1 coding region contain multiple inhibitory elements that act independently of one another and that the major inhibitory element is located within the first 129 nucleotides of the L1 gene. Introduction of point mutations in the inhibitory elements in the 5′ end of the L1 gene which altered the RNA sequence without affecting the protein sequence specifically inactivated the inhibitory elements and resulted in production of high levels of human papillomavirus type 16 L1 mRNA and protein in human epithelial cells. Furthermore, we show that inhibitory sequences are present in the L1 coding regions of multiple human papillomavirus types, demonstrating that these elements are conserved among the human papillomaviruses, and suggest that they have an important function in the viral life cycle.
Structural Analysis of the Hepatitis C Virus RNA Polymerase in Complex with Ribonucleotides
Authors: Bressanelli Stéphane , Tomei Licia , Rey Félix A. , De Francesco Raffaele
Published: July 2002

We report here the results of a systematic high-resolution X-ray crystallographic analysis of complexes of the hepatitis C virus (HCV) RNA polymerase with ribonucleoside triphosphates (rNTPs) and divalent metal ions. An unexpected observation revealed by this study is the existence of a specific rGTP binding site in a shallow pocket at the molecular surface of the enzyme, 30 Å away from the catalytic site. This previously unidentified rGTP pocket, which lies at the interface between fingers and thumb, may be an allosteric regulatory site and could play a role in allowing alternative interactions between the two domains during a possible conformational change of the enzyme required for efficient initiation. The electron density map at 1.7-Å resolution clearly shows the mode of binding of the guanosine moiety to the enzyme. In the catalytic site, density corresponding to the triphosphates of nucleotides bound to the catalytic metals was apparent in each complex with nucleotides. Moreover, a network of triphosphate densities was detected; these densities superpose to the corresponding moieties of the nucleotides observed in the initiation complex reported for the polymerase of bacteriophage φ6, strengthening the proposal that the two enzymes initiate replication de novo by similar mechanisms. No equivalent of the protein stacking platform observed for the priming nucleotide in the φ6 enzyme is present in HCV polymerase, however, again suggesting that a change in conformation of the thumb domain takes place upon template binding to allow for efficient de novo initiation of RNA synthesis.
Human Immunodeficiency Virus cDNA Metabolism: Notable Stability of Two-Long Terminal Repeat Circles
Authors: Butler Scott L. , Johnson Erik P. , Bushman Frederic D.
Published: July 2002

Early steps of retroviral replication involve reverse transcription of the viral RNA to yield a linear double-stranded cDNA copy and then integration of the viral cDNA into a chromosome of the host cell. A portion of the viral cDNA can also follow nonproductive pathways in which it becomes circularized. In one pathway, the ends of the linear cDNA become joined together by the cellular nonhomologous DNA end-joining system to form two-long terminal repeat (2-LTR) circles. It has been argued that 2-LTR circles are quickly degraded in human immunodeficiency virus (HIV)-infected cells, allowing the presence of 2-LTR circles to be used as a marker for ongoing de novo infection in patients. Following this idea, detection of 2-LTR circles in patients undergoing successful highly active antiretroviral therapy has led to the proposal that viral replication persists despite treatment. We have used fluorescence-monitored PCR (Taqman) to quantitate the metabolism of HIV cDNA early after infection. Contrary to previous work, we find that 2-LTR circles are actually quite stable in experiments where confounding variables are controlled. Thus, studies relying on the lability of 2-LTR circles are open to reinterpretation. We also used the quantitative PCR methods to analyze the effects of MG132, a proteasome inhibitor, which revealed that viral complexes containing mostly completed cDNAs are the primary substrates for proteasome-mediated degradation.