bluuebunny/crossref_metadata_2025 huggingface dataset:
CD4 and Major Histocompatibility Complex Class I Downregulation by the Human Immunodeficiency Virus Type 1 Nef Protein in Pediatric AIDS Progression
Authors: Casartelli Nicoletta , Di Matteo Gigliola , Potestà Marina , Rossi Paolo , Doria Margherita
Published: October 2003

The human immunodeficiency virus type 1 (HIV-1)
nef
gene is a crucial determinant in AIDS disease progression. Although several in vitro activities have been attributed to the Nef protein, identifying the one critical for in vivo pathogenicity remains elusive. In this study, we examined a large number of
nef
alleles derived at various time points from 13 perinatally infected children showing different progression rates: six nonprogressors (NPs), three slow progressors (SPs), and four rapid progressors (RPs). The patient-derived
nef
alleles were analyzed for their steady-state expression of a Nef protein, for their relative ability to downregulate cell surface expression of CD4 and major histocompatibility complex class I (MHC-I) and for their capacity to bind the clathrin adaptor AP-1 complex. We found that NP-derived
nef
alleles, compared to
nef
alleles isolated from SPs and RPs, had reduced CD4 and MHC-I downregulation activities. In contrast, SP- and RP-derived
nef
alleles did not differ and efficiently downregulated both CD4 and MHC-I. AP-1 binding was a conserved function of primary
nef
alleles not correlated with clinical progression. Defective Nef proteins from NPs, rather than sharing common specific changes in their sequences, accumulated various amino acid substitutions, mainly located outside the conserved domains previously associated with Nef biological properties. Our data indicate that Nef-mediated downregulation of cell surface CD4 and MHC-I significantly contributes to the expression of the pathogenic potential of HIV-1.
A Molecularly Cloned Schwarz Strain of Measles Virus Vaccine Induces Strong Immune Responses in Macaques and Transgenic Mice
Authors: Combredet Chantal , Labrousse Valérie , Mollet Lucile , Lorin Clarisse , Delebecque Frédéric , Hurtrel Bruno , McClure Harold , Feinberg Mark B. , Brahic Michel , Tangy Frédéric
Published: October 2003

Live attenuated RNA viruses make highly efficient vaccines. Among them, measles virus (MV) vaccine has been given to a very large number of children and has been shown to be highly efficacious and safe. Therefore, this vaccine might be a very promising vector to immunize children against both measles and other infectious agents, such as human immunodeficiency virus. A vector was previously derived from the Edmonston B strain of MV, a vaccine strain abandoned 25 years ago. Sequence analysis revealed that the genome of this vector diverges from Edmonston B by 10 amino acid substitutions not related to any Edmonston subgroup. Here we describe an infectious cDNA for the Schwarz/Moraten strain, a widely used MV vaccine. This cDNA was constructed from a batch of commercial vaccine. The extremities of the cDNA were engineered in order to maximize virus yield during rescue. A previously described helper cell-based rescue system was adapted by cocultivating transfected cells on primary chicken embryo fibroblasts, the cells used to produce the Schwarz/Moraten vaccine. After two passages the sequence of the rescued virus was identical to that of the cDNA and of the published Schwarz/Moraten sequence. Two additional transcription units were introduced in the cDNA for cloning foreign genetic material. The immunogenicity of rescued virus was studied in macaques and in mice transgenic for the CD46 MV receptor. Antibody titers and T-cell responses (ELISpot) in animals inoculated with low doses of rescued virus were identical to those obtained with commercial Schwarz MV vaccine. In contrast, the immunogenicity of the previously described Edmonston B strain-derived MV clone was much lower. This new molecular clone will allow for the production of MV vaccine without having to rely on seed stocks. The additional transcription units allow expressing heterologous antigens, thereby providing polyvalent vaccines based on an approved, safe, and efficient MV vaccine strain that is used worldwide.
Expression of the Zinc-Finger Antiviral Protein Inhibits Alphavirus Replication
Authors: Bick Matthew J. , Carroll John-William N. , Gao Guangxia , Goff Stephen P. , Rice Charles M. , MacDonald Margaret R.
Published: October 2003

The rat zinc-finger antiviral protein (ZAP) was recently identified as a host protein conferring resistance to retroviral infection. We analyzed ZAP's ability to inhibit viruses from other families and found that ZAP potently inhibits the replication of multiple members of the
Alphavirus
genus within the
Togaviridae
, including Sindbis virus, Semliki Forest virus, Ross River virus, and Venezuelan equine encephalitis virus. However, expression of ZAP did not induce a broad-spectrum antiviral state as some viruses, including vesicular stomatitis virus, poliovirus, yellow fever virus, and herpes simplex virus type 1, replicated to normal levels in ZAP-expressing cells. We determined that ZAP expression inhibits Sindbis virus replication after virus penetration and entry, but before the amplification of newly synthesized plus strand genomic RNA. Using a temperature-sensitive Sindbis virus mutant expressing luciferase, we further showed that translation of incoming viral RNA is blocked by ZAP expression. Elucidation of the antiviral mechanism by which ZAP inhibits Sindbis virus translation may lead to the development of agents with broad activity against alphaviruses.
Multigene DNA Priming-Boosting Vaccines Protect Macaques from Acute CD4+-T-Cell Depletion after Simian-Human Immunodeficiency Virus SHIV89.6P Mucosal Challenge
Authors: Doria-Rose N. A. , Ohlen C. , Polacino P. , Pierce C. C. , Hensel M. T. , Kuller L. , Mulvania T. , Anderson D. , Greenberg P. D. , Hu S.-L. , Haigwood N. L.
Published: October 2003

We evaluated four priming-boosting vaccine regimens for the highly pathogenic simian human immunodeficiency virus SHIV89.6P inMacaca nemestrina. Each regimen included gene gun delivery of a DNA vaccine expressing all SHIV89.6 genes plus Env gp160 of SHIV89.6P. Additional components were two recombinant vaccinia viruses, expressing SHIV89.6 Gag-Pol or Env gp160, and inactivated SHIV89.6 virus. We compared (i) DNA priming/DNA boosting, (ii) DNA priming/inactivated virus boosting, (iii) DNA priming/vaccinia virus boosting, and (iv) vaccinia virus priming/DNA boosting versus sham vaccines in groups of 6 macaques. Prechallenge antibody responses to Env and Gag were strongest in the groups that received vaccinia virus priming or boosting. Cellular immunity to SHIV89.6 peptides was measured by enzyme-linked immunospot assay; strong responses to Gag and Env were found in 9 of 12 vaccinia virus vaccinees and 1 of 6 DNA-primed/inactivated-virus-boosted animals. Vaccinated macaques were challenged intrarectally with 50 50% animal infectious doses of SHIV89.6P 3 weeks after the last immunization. All animals became infected. Five of six DNA-vaccinated and 5 of 6 DNA-primed/particle-boosted animals, as well as all 6 controls, experienced severe CD4+-T-cell loss in the first 3 weeks after infection. In contrast, DNA priming/vaccinia virus boosting and vaccinia virus priming/DNA boosting vaccines both protected animals from disease: 11 of 12 macaques had no loss of CD4+T cells or moderate declines. Virus loads in plasma at the set point were significantly lower in vaccinia virus-primed/DNA-boosted animals versus controls (P= 0.03). We conclude that multigene vaccines delivered by a combination of vaccinia virus and gene gun-delivered DNA were effective against SHIV89.6P viral challenge inM. nemestrina.
Caprine Arthritis-Encephalitis Virus Envelope Surface Glycoprotein Regions Interacting with the Transmembrane Glycoprotein: Structural and Functional Parallels with Human Immunodeficiency Virus Type 1 gp120
Authors: Hötzel Isidro , Cheevers William P.
Published: October 2003

A sequence similarity between surface envelope glycoprotein (SU) gp135 of the lentiviruses maedi-visna virus and caprine arthritis-encephalitis virus (CAEV) and human immunodeficiency virus type 1 (HIV-1) gp120 has been described. The regions of sequence similarity are in the second and fifth conserved regions of gp120, and the similarity is highest in sequences coinciding with β-strands 4 to 8 and 25, which are located in the most virion-proximal region of the gp120 inner domain. A subset of this structure, formed by gp120 β-strands 4, 5, and 25, is conserved in most or all lentiviruses. Because of the orientation of gp120 on the virion, this highly conserved virion-proximal region of the gp120 core may interact with the transmembrane glycoprotein (TM) together with the amino and carboxy termini of full-length gp120. Therefore, interactions between SU and TM of lentiviruses may be structurally related. Here we tested whether the amino acid residues in the putative virion-proximal region of CAEV gp135 comprising putative β-strands 4, 5, and 25, as well as its amino and carboxy termini, are important for stable interactions with TM. An amino acid change at gp135 position 119 or 521, located in the turn between putative β-strands 4 and 5 and near β-strand 25, respectively, specifically disrupted the epitope recognized by monoclonal antibody 29A. Thus, similar to the corresponding gp120 regions, these gp135 residues are located in close proximity to each other in the folded protein, supporting the hypothesis of a structural similarity between the gp120 virion-proximal inner domain and gp135. Amino acid changes in the amino- and carboxy-terminal and putative virion-proximal regions of gp135 increased gp135 shedding from the cell surface, indicating that these gp135 regions are involved in interactions with TM. Our results indicate structural and functional parallels between CAEV gp135 and HIV-1 gp120 that may be more broadly applicable to the SU of other lentiviruses.
Replication of an E1B 55-Kilodalton Protein-Deficient Adenovirus (ONYX-015) Is Restored by Gain-of-Function Rather than Loss-of-Function p53 Mutants
Authors: Hann Byron , Balmain Allan
Published: October 2003

ONYX-015 (
dl
1520) is an E1B 55-kilodalton protein-deficient replicating adenovirus that is currently in clinical trials as an antitumor agent. On the basis of the observation that the
E1B 55kD
gene product is able to bind to and inactivate p53, ONYX-015's mechanism of action is proposed to involve selective replication in and killing of p53-deficient cells. While its efficacy as a therapeutic agent appears evident, the virus's mechanism of cellular selectivity, including a possible role of p53 in this regard, is less clear. Indeed, there have been a number of recent reports suggesting that the p53 status of target cells does not reliably predict ONYX-015 replication or cell killing. To address the role of p53 in ONYX-015 selectivity, we have undertaken a rigorous analysis of the behavior of this virus in small airway-derived primary human epithelial cells expressing either dominant-negative or gain-of-function mutant
p53
genes. Examination of small airway epithelial cells expressing a variety of
p53
mutant alleles revealed that while all were able to inhibit endogenous p53 activity, only one allele examined, 248W, demonstrated a markedly increased ability to facilitate ONYX-015 replication. This allele is a member of a group of
p53
mutants (know as class I mutants) characterized by retention of global structural conformation but loss of DNA-binding activity. These observations indicate that the nature of the
p53
mutation affects ONYX-015 replication, help reconcile disparate published findings, and may provide criteria by which to direct clinical application of ONYX-015.
Optimal Induction of T-Cell Responses against Hepatitis C Virus E2 by Antigen Engineering in DNA Immunization
Authors: Youn Jin-Won , Park Su-Hyung , Cho Jae Ho , Sung Young Chul
Published: October 2003

Although DNA immunization is a safe and efficient method for inducing cellular immune responses, it generates relatively weak and slow immune responses. Here, we investigated the effect of hepatitis C virus (HCV) antigen modifications on the induction of T-cell responses in DNA immunization. It is likely that the strength of T-cell responses has an inverse relationship with the length of the insert DNA. Interestingly, a mixture of several plasmids carrying each gene induced a higher level of T-cell responses than a single plasmid expressing a long polyprotein. Moreover, the presence of a transmembrane domain in HCV E2 resulted in stronger T-cell responses against E2 protein than its absence. Taken together, our results indicate that the tailored modifications of DNA-encoded antigens are capable of optimizing the induction of T-cell responses which is required for eliminating the cells chronically infected with highly variable viruses such as HCV and human immunodeficiency virus.
The 3′ End of Norwalk Virus mRNA Contains Determinants That Regulate the Expression and Stability of the Viral Capsid Protein VP1: a Novel Function for the VP2 Protein
Authors: Bertolotti-Ciarlet Andrea , Crawford Sue E. , Hutson Anne M. , Estes Mary K.
Published: October 2003

Norwalk virus (NV) is the prototype strain of a group of noncultivable human caliciviruses responsible for epidemic outbreaks of acute gastroenteritis. The capsid protein VP1 is synthesized from a subgenomic RNA that contains two open reading frames (ORFs), ORF2 and ORF3, and the 3′ untranslated region (UTR). ORF2 and ORF3 code for the capsid protein (VP1) and a small structural basic protein (VP2), respectively. We discovered that the yields of virus-like particles (VLPs) composed of VP1 are significantly reduced when this protein is expressed from ORF2 alone. To determine how the 3′ terminus of the NV subgenomic RNA regulates VP1 expression, we compared VP1 expression levels by using recombinant baculovirus constructs containing different 3′ elements. High VP1 levels were detected by using a recombinant baculovirus that contained ORF2, ORF3, and the 3′UTR (ORF2+3+3′UTR). In contrast, expression of VP1 from constructs that lacked the 3′UTR (ORF2+3), ORF3 (ORF2+3′UTR), or both (ORF2 alone) was highly reduced. Elimination of VP2 synthesis from the subgenomic RNA by mutation resulted in VP1 levels similar to those obtained with the ORF2 construct alone, suggesting a
cis
role for VP2 in upregulation of VP1 expression levels. Comparisons of the kinetics of RNA and capsid protein expression levels by using constructs with or without ORF3 or the 3′UTR revealed that the 3′UTR increased the levels of VP1 RNA, whereas the presence of VP2 resulted in increased levels of VP1. Furthermore, VP2 increased VP1 stability and protected VP1 from disassembly and protease degradation. The increase in VP1 expression levels caused by the presence of VP2 in
cis
was also observed in mammalian cells.
Changes in Mumps Virus Gene Sequence Associated with Variability in Neurovirulent Phenotype
Authors: Rubin Steven A. , Amexis Georgios , Pletnikov Mikhail , Vanderzanden Jacqueline , Mauldin Jeremy , Sauder Christian , Malik Tahir , Chumakov Konstantin , Carbone Kathryn M.
Published: October 2003

Mumps virus is highly neurotropic and, prior to widespread vaccination programs, was the major cause of viral meningitis in the United States. Nonetheless, the genetic basis of mumps virus neurotropism and neurovirulence was until recently not understood, largely due to the lack of an animal model. Here, nonneurovirulent (Jeryl Lynn vaccine) and highly neurovirulent (88-1961 wild type) mumps virus strains were passaged in human neural cells or in chicken fibroblast cells with the goal of neuroadapting or neuroattenuating the viruses, respectively. When tested in our rat neurovirulence assay against the respective parental strains, a Jeryl Lynn virus variant with an enhanced propensity for replication (neurotropism) and damage (neurovirulence) in the brain and an 88-1961 wild-type virus variant with decreased neurotropic and neurovirulent properties were recovered. To determine the molecular basis for the observed differences in neurovirulence and neuroattenuation, the complete genomes of the parental strains and their variants were fully sequenced. A comparison at the nucleotide level associated three amino acid changes with enhanced neurovirulence of the neuroadapted vaccine strain: one each in the nucleoprotein, matrix protein, and polymerase and three amino acid changes with reduced neurovirulence of the neuroattenuated wild-type strain: one each in the fusion protein, hemagglutinin-neuraminidase protein, and polymerase. The potential role of these amino acid changes in neurotropism, neurovirulence, and neuroattenuation is discussed.
Identification of a Human Papillomavirus Type 16-Specific Epitope on the C-Terminal Arm of the Major Capsid Protein L1
Authors: Carter Joseph J. , Wipf Greg C. , Benki Sarah F. , Christensen Neil D. , Galloway Denise A.
Published: October 2003

To characterize epitopes on human papillomavirus (HPV) virus-like particles (VLPs), a panel of mutated HPV-16 VLPs was created. Each mutated VLP had residues substituted from HPV-31 or HPV-52 L1 sequences to the HPV-16 L1 backbone. Mutations were created on the HPV-31 and −52 L1 proteins to determine if HPV-16 type-specific recognition could be transferred. Correct folding of the mutated proteins was verified by resistance to trypsin digestion and by binding to one or more conformation-dependent monoclonal antibodies. Several of the antibodies tested were found to bind to regions already identified as being important for HPV VLP recognition (loops DE, EF, FG, and HI). Sequences at both ends of the long FG loop (amino acids 260 to 290) were required for both H16.V5 and H16.E70 reactivity. A new antibody-binding site was discovered on the C-terminal arm of L1 between positions 427 and 445. Recognition of these residues by the H16.U4 antibody suggests that this region is surface exposed and supports a recently proposed molecular model of HPV VLPs.
Murine Cytomegalovirus m41 Open Reading Frame Encodes a Golgi-Localized Antiapoptotic Protein
Authors: Brune Wolfram , Nevels Michael , Shenk Thomas
Published: October 2003

Viruses have evolved various strategies to prevent premature apoptosis of infected host cells. Some of the viral genes mediating antiapoptotic functions have been identified by their homology to cellular genes, but others are structurally unrelated to genes of known function. In this study, we used a random, unbiased approach to identify such genes in the murine cytomegalovirus genome. From a library of random transposon insertion mutants, a mutant virus that caused premature cell death was isolated. The transposon was inserted within open reading frame m41. An independently constructed m41 deletion mutant showed the same phenotype, whereas deletion mutants lacking the adjacent genes m40 and M42 did not. Apoptosis occurred in different cell types, could be blocked by caspase inhibitors, and did not require p53. Within the murine cytomegalovirus genome, m41, m40, and m39 form a small cluster of genes of unknown function. They are homologous to r41, r40, and r39 of rat cytomegalovirus, but lack sequence homology to UL41, UL40, and UL37 exon 1 (UL37x1) which are located at the corresponding positions of the human cytomegalovirus genome. Unlike UL37x1 of human cytomegalovirus, which encodes a mitochondrion-localized inhibitor of apoptosis that is essential for virus replication, m41 encodes a protein that localizes to the Golgi apparatus. The murine cytomegalovirus m41 product is the first example of a Golgi-localized protein that prevents premature apoptosis and thus extends the life span of infected cells.
Cell Surface Expression of Major Histocompatibility Complex Class I Molecules Is Reduced in Hepatitis C Virus Subgenomic Replicon-Expressing Cells
Authors: Tardif Keith D. , Siddiqui Aleem
Published: October 2003

The hepatitis C virus (HCV) causes chronic hepatitis in most infected individuals by evading host immune defenses. In this investigation, we show that HCV-infected cells may go undetected in the immune system by suppressing major histocompatibility complex (MHC) class I antigen presentation to cytotoxic T lymphocytes. Cells expressing HCV subgenomic replicons have lower MHC class I cell surface expression. This is due to reduced levels of properly folded MHC class I molecules. HCV replicons induce endoplasmic reticulum (ER) stress (K. Tardif, K. Mori, and A. Siddiqui, J. Virol. 76:7453-7459, 2002), which results from a decline in protein glycosylation. Decreasing protein glycosylation can disrupt protein folding, preventing the assembly of MHC class I molecules. This results in the accumulation of unfolded MHC class I. Therefore, the persistence and pathogenesis of HCV may depend upon the ER stress-mediated interference of MHC class I assembly and cell surface expression.
Murine Leukemia Virus Particle Assembly Quantitated by Fluorescence Microscopy: Role of Gag-Gag Interactions and Membrane Association
Authors: Andrawiss Mariam , Takeuchi Yasuhiro , Hewlett Lindsay , Collins Mary
Published: October 2003

In order to track the assembly of murine leukemia virus (MLV), we used fluorescence microscopy to visualize particles containing Gag molecules fused to fluorescent proteins (FPs). Gag-FP chimeras budded from cells to produce fluorescent spots, which passed through the same pore-size filters and sedimented at the same velocity as authentic MLV. N-terminal myristylation of Gag-FPs was necessary for particle formation unless wild-type Gag was coexpressed. By labeling nonmyristylated Gag with yellow FP and wild-type Gag with cyan FP, we could quantitate the coincorporation of two proteins into single particles. This experiment showed that nonmyristylated Gag was incorporated into mixed particles at approximately 50% the efficiency of wild-type Gag. Mutations that inhibit Gag-Gag interactions (K. Alin and S. P. Goff, Virology
216:
418-424, 1996; K. Alin and S. P. Goff, Virology
222:
339-351, 1996) were then introduced into the capsid (CA) region of Gag-FPs. The mutations P150L and R119C/P133L inhibited fluorescent particle formation by these Gag-FPs, but Gag-FPs containing these mutations could be efficiently incorporated into particles when coexpressed with wild-type Gag. When these mutations were introduced into nonmyristylated Gag-FPs, no incorporation into particles in the presence of wild-type Gag was detected. These data suggest that two independent mechanisms, CA interactions and membrane association following myristylation, cooperate in MLV Gag assembly and budding.
A Locus on Mouse Chromosome 6 That Determines Resistance to Herpes Simplex Virus Also Influences Reactivation, While an Unlinked Locus Augments Resistance of Female Mice
Authors: Lundberg Patric , Welander Paula , Openshaw Harry , Nalbandian Christina , Edwards Carl , Moldawer Lyle , Cantin Edouard
Published: October 2003

During studies to determine a role for tumor necrosis factor (TNF) in herpes simplex virus type 1 (HSV-1) infection using TNF receptor null mutant mice, we discovered a genetic locus, closely linked to the TNF p55 receptor (Tnfrsf1a) gene on mouse chromosome 6 (c6), that determines resistance or susceptibility to HSV-1. We named this locus the herpes resistance locus,Hrl, and showed that it also mediates resistance to HSV-2.Hrlhas at least two alleles,Hrlr, expressed by resistant strains like C57BL/6 (B6), andHrls, expressed by susceptible strains like 129S6 (129) and BALB/c. AlthoughHrlis inherited as an autosomal dominant gene, resistance to HSV-1 is strongly sex biased such that female mice are significantly more resistant than male mice. Analysis of backcrosses between resistant B6 and susceptible 129 mice revealed that a second locus, tentatively named the sex modifier locus,Sml, functions to augment resistance of female mice. Besides determining resistance,Hrlis one of several genes involved in the control of HSV-1 replication in the eye and ganglion. Remarkably,Hrlalso affects reactivation of HSV-1, possibly by interaction with some unknown gene(s). We showed thatHrlis distinct fromCmv1, the gene that determines resistance to murine cytomegalovirus, which is encoded in the major NK cell complex just distal of p55 on c6.Hrlhas been mapped to a roughly 5-centimorgan interval on c6, and current efforts are focused on obtaining a high-resolution map forHrl.
Mutational Inactivation of Two Distinct Negative RNA Elements in the Human Papillomavirus Type 16 L2 Coding Region Induces Production of High Levels of L2 in Human Cells
Authors: Öberg Daniel , Collier Brian , Zhao Xiaomin , Schwartz Stefan
Published: October 2003

Here we show that the 5′ end and the middle region of the L2 coding sequence of human papillomavirus type 16 contain strong inhibitory RNA sequences termed inhibitory regions I and II. This is in contrast to L1, which contains one inhibitory region in the 5′ end of the coding region. Inhibitory regions I and II acted in
cis
to reduce L2 mRNA levels and to inhibit the use of the mRNA. In tandem, the two regions reduced L2 mRNA production to undetectable levels. Specific mutational inactivation of the two inhibitory elements in the 5′ end and in the middle region of L2 by the introduction of nucleotide substitutions that changed the nucleotide sequence but not the protein sequence resulted in production of high levels of L2 mRNA and protein. In contrast to L2, a partial L1 mutant in which only the first one third of L1 was mutated produced levels of L1 mRNA and protein similar to those in a full L1 mutant. In addition, the constitutive transport element of simian retrovirus type 1 overcomes the effect of the inhibitory sequences of L1 but not L2.
Distinct Domains in the Adenovirus E3 RIDα Protein Are Required for Degradation of Fas and the Epidermal Growth Factor Receptor
Authors: Zanardi Tom A. , Yei Soonpin , Lichtenstein Drew L. , Tollefson Ann E. , Wold William S. M.
Published: October 2003

Adenovirus (Ad) types 2 and 5 encode at least five proteins within the E3 transcription unit that help the virus evade the immune system. Two such proteins, RIDα (formerly E3-10.4K) and RIDβ (formerly E3-14.5K), form the RID (receptor internalization and degradation) complex (formerly E3-10.4K/14.5K). RID mediates clearance from the cell surface and lysosomal degradation of a number of important members in the tumor necrosis factor receptor (TNFR) superfamily and the receptor tyrosine kinase receptor family. Affected receptors include Fas, TRAIL (TNF-related apoptosis-inducing ligand) receptor 1 (TR1), TR2, and epidermal growth factor receptor (EGFR). Degradation of Fas and TRAIL receptors protects Ad-infected cells from apoptosis. To investigate the mechanism of action of RIDα, 14 mutant RIDα proteins, each containing a three- to five-amino-acid deletion, were constructed and then expressed from the E3 region of a replication-competent recombinant Ad in the same context as wild-type RIDα. Each mutant protein was characterized with regard to five physical properties associated with wild-type RIDα, namely, protein stability, proteolytic cleavage, insertion into the membrane, complex formation with RIDβ, and transport to the cell surface. Additionally, the mutant proteins were tested for their ability to mediate internalization and degradation of EGFR and Fas and to protect cells from Fas-mediated apoptosis. The majority of mutant RIDα proteins (8 out of 14) were physically similar to wild-type RIDα. With regard to functional characteristics, the cytoplasmic domain of RIDα is largely unimportant for receptor internalization and degradation and the extracellular domain of RIDα is important for down-regulation of EGFR but not Fas.
Hypovirus Papain-Like Protease p29 Functions in
trans
To Enhance Viral Double-Stranded RNA Accumulation and Vertical Transmission
Authors: Suzuki Nobuhiro , Maruyama Kazuyuki , Moriyama Miho , Nuss Donald L.
Published: October 2003

The prototypic hypovirus CHV1-EP713 attenuates virulence (hypovirulence) and alters several physiological processes of the chestnut blight fungus
Cryphonectria parasitica
. The papain-like protease, p29, and the highly basic protein, p40, derived, respectively, from the N-terminal and C-terminal portions of the CHV1-EP713-encoded open reading frame (ORF) A polyprotein, p69, both contribute to reduced pigmentation and sporulation. The p29 coding region was shown to suppress pigmentation and asexual sporulation in the absence of virus infection in transformed
C. parasitica
, whereas transformants containing the p40-coding domain exhibited a wild-type, untransformed phenotype. Deletion of either p29 or p40 from the viral genome also results in reduced accumulation of viral RNA. We now show that p29, but not p40, functions in
trans
to enhance genomic RNA accumulation and vertical transmission of p29 deletion mutant viruses. The frequency of virus transmission through conidia was found to decrease with reduced accumulation of viral genomic double-stranded RNA (dsRNA): from almost 100% for wild-type virus to ∼50% for Δp29, and 10 to 20% for Δp69. When expressed from a chromosomally integrated cDNA copy, p29 elevated viral dsRNA accumulation and transmission for Δp29 mutant virus to the level shown by wild-type virus. Increased viral RNA accumulation levels were also observed for a Δp69 mutant lacking almost the entire ORF A sequence. Such enhancements were not detected in transgenic fungal colonies expressing p40. Mutation of p29 residues Cys
70
or Cys
72
, strictly conserved in hypovirus p29 and potyvirus HC-Pro, resulted in the loss of both p29-mediated suppressive activity in virus-free transgenic
C. parasitica
and in
trans
enhancement of RNA accumulation and transmission, suggesting a linkage between these functional activities. These results suggest that p29 is an enhancer of viral dsRNA accumulation and vertical virus transmission through asexual spores.
Severe CD4+T-Cell Depletion in Gut Lymphoid Tissue during Primary Human Immunodeficiency Virus Type 1 Infection and Substantial Delay in Restoration following Highly Active Antiretroviral Therapy
Authors: Guadalupe Moraima , Reay Elizabeth , Sankaran Sumathi , Prindiville Thomas , Flamm Jason , McNeil Andrew , Dandekar Satya
Published: October 2003

Gut-associated lymphoid tissue (GALT) harbors the majority of T lymphocytes in the body and is an important target for human immunodeficiency virus type 1 (HIV-1). We analyzed longitudinal jejunal biopsy samples from HIV-1-infected patients, during both primary and chronic stages of HIV-1 infection, prior to and following the initiation of highly active antiretroviral therapy (HAART) to determine the onset of CD4+T-cell depletion and the effect of HAART on the restoration of CD4+T cells in GALT. Severe depletion of intestinal CD4+T cells occurred during primary HIV-1 infection. Our results showed that the restoration of intestinal CD4+T cells following HAART in chronically HIV-1-infected patients was substantially delayed and incomplete. In contrast, initiation of HAART during early stages of infection resulted in near-complete restoration of intestinal CD4+T cells, despite the delay in comparison to peripheral blood CD4+T-cell recovery. DNA microarray analysis of gene expression profiles and flow-cytometric analysis of lymphocyte homing and cell proliferation markers demonstrated that cell trafficking to GALT and not local proliferation contributed to CD4+T-cell restoration. Evaluation of jejunal biopsy samples from long-term HIV-1-infected nonprogressors showed maintenance of normal CD4+T-cell levels in both GALT and peripheral blood. Our results demonstrate that near-complete restoration of mucosal immune system can be achieved by initiating HAART early in HIV-1 infection. Monitoring of the restoration and/or maintenance of CD4+T cells in GALT provides a more accurate assessment of the efficacy of antiviral host immune responses as well as HAART.
Array Analysis of Viral Gene Transcription during Lytic Infection of Cells in Tissue Culture with Varicella-Zoster Virus
Authors: Cohrs Randall J. , Hurley Michael P. , Gilden Donald H.
Published: October 2003

Varicella-zoster virus (VZV), a neurotropic alphaherpesvirus, causes childhood chickenpox (varicella), becomes latent in dorsal root and autonomic ganglia, and reactivates decades later to cause shingles (zoster) and other neurologic complications. Although the sequence and configuration of VZV DNA have been determined, relatively little is known about viral gene expression in productively infected cells. This is in part because VZV is highly cell associated, and sufficient titers of cell-free virus for use in synchronizing infection do not develop. PCR-based transcriptional arrays were constructed to simultaneously determine the relative abundance of the ≈70 predicted VZV open reading frames (ORFs). Fragments (250 to 600 bp) from the 5′ and 3′ end of each ORF were PCR amplified and inserted into plasmid vectors. The virus DNA inserts were amplified, quantitated, and spotted onto nylon membranes. Probing the arrays with radiolabeled cDNA synthesized from VZV-infected cells revealed an increase in the magnitude of the expressed VZV genes from days 1 to 3 after low-multiplicity virus infection but little change in their relative abundance. The most abundant VZV transcripts mapped to ORFs 9/9A, 64, 33/33A, and 49, of which only ORF 9 corresponded to a previously identified structural gene. Array analysis also mapped transcripts to three large intergenic regions previously thought to be transcriptionally silent, results subsequently confirmed by Northern blot and reverse transcription-PCR analysis. Array analysis provides a formidable tool to analyze transcription of an important ubiquitous human pathogen.
The AIDS-Like Disease of CD4C/Human Immunodeficiency Virus Transgenic Mice Is Associated with Accumulation of Immature CD11bHiDendritic Cells
Authors: Poudrier Johanne , Weng Xiaoduan , Kay Denis G. , Hanna Zaher , Jolicoeur Paul
Published: October 2003

CD4C/human immunodeficiency virus (HIV) transgenic mice develop an AIDS-like disease. We used this model to study the effects of HIV-1 on dendritic cells (DC). We found a progressive decrease in total DC numbers in the lymph nodes, with a significant accumulation of CD11bHiDC. In the thymus, the recovery of transgenic CD8α+DC had a tendency to be lower. Spleen DC were augmented in the marginal zone. Transgenic DC showed a decreased capacity to present antigen in vitro, consistent with their reduced major histocompatibility complex class II expression and impaired maturation profile. The accumulation of immature DC may contribute to disease and may reflect an adaptive advantage for the virus by favoring its replication and preventing the generation of fully functional antiviral responses.
Oligodendrocyte-Specific Expression of Human Immunodeficiency Virus Type 1 Nef in Transgenic Mice Leads to Vacuolar Myelopathy and Alters Oligodendrocyte Phenotype In Vitro
Authors: Radja Fatiha , Kay Denis G. , Albrecht Steffen , Jolicoeur Paul
Published: October 2003

Vacuolar myelopathy (VM) is a frequent central nervous system complication of human immunodeficiency virus type 1 (HIV-1) infection. We report here that transgenic (Tg) mice expressing even low levels of Nef in oligodendrocytes under the regulation of the myelin basic protein (MBP) promoter (MBP/HIV
Nef
) developed VM similar to the human disease in its appearance and topography. The spinal cords of these Tg mice showed lower levels of the myelin proteins MAG and CNPase and of the 21-kDa isoform of MBP prior to the development of vacuoles. In addition, Tg oligodendrocytes in primary in vitro cultures appeared morphologically more mature but, paradoxically, exhibited a less mature phenotype based on O4, O1, CNPase, and MBP staining. In particular, mature CNPase
+
MBP
+
Tg oligodendrocytes were less numerous than non-Tg oligodendrocytes. Therefore, Nef appears to affect the proper differentiation of oligodendrocytes. These data suggest that even low levels of Nef expression in human oligodendrocytes may be responsible for the development of VM in HIV-1-infected individuals.
Evidence against an Essential Role of COPII-Mediated Cargo Transport to the Endoplasmic Reticulum-Golgi Intermediate Compartment in the Formation of the Primary Membrane of Vaccinia Virus
Authors: Husain Matloob , Moss Bernard
Published: October 2003

Vaccinia virus assembles two distinct lipoprotein membranes. The primary membrane contains nonglycosylated proteins, appears as crescents in the cytoplasm, and delimits immature and mature intracellular virions. The secondary or wrapping membrane contains glycoproteins, is derived from virus-modified trans-Golgi or endosomal cisternae, forms a loose coat around some intracellular mature virions, and becomes the envelope of extracellular virions. Although the mode of formation of the wrapping membrane is partially understood, we know less about the primary membrane. Recent reports posit that the primary membrane originates from the endoplasmic reticulum-Golgi intermediate compartment (ERGIC). According to this model, viral primary membrane proteins are cotranslationally inserted into the ER and accumulate in the ERGIC. To test the ERGIC model, we employed Sar1
H79G
, a dominant negative form of the Sar1 protein, which is an essential component of coatomer protein II (COPII)-mediated cargo transport from the ER to the ERGIC and other post-ER compartments. Overexpression of Sar1
H79G
by transfection or by a novel recombinant vaccinia virus with an inducible Sar1
H79G
gene resulted in retention of ERGIC 53 in the ER but did not interfere with localization of viral primary membrane proteins in factory regions or with formation of viral crescent membranes and infectious intracellular mature virions. Wrapping of intracellular mature virions and formation of extracellular virions did not occur, however, because some proteins that are essential for the secondary membrane were retained in the ER as a consequence of Sar1
H79G
overexpression. Our data argue against an essential role of COPII-mediated cargo transport and the ERGIC in the formation of the viral primary membrane. Instead, viral membranes may be derived directly from the ER or by a novel mechanism.
Metaphase Chromosome Tethering Is Necessary for the DNA Synthesis and Maintenance of
oriP
Plasmids but Is Insufficient for Transcription Activation by Epstein-Barr Nuclear Antigen 1
Authors: Sears John , Kolman John , Wahl Geoffrey M. , Aiyar Ashok
Published: October 2003

Epstein-Barr Virus (EBV) infects resting B cells, within which it establishes latency as a stable, circular episome with only two EBV components, the
cis
element
oriP
and the latently expressed protein EBNA1. It is believed that EBNA1's ability to tether
oriP
episomes to metaphase chromosomes is required for its stable replication. We created fusions between the DNA-binding domain (DBD) of EBNA1 and the cellular chromatin-binding proteins HMGA1a and HMG1 to determine the minimal requirements for stable maintenance of an
oriP
-based episome. These two proteins differ in that HMGA1a can associate with metaphase chromosomes but HMG1 cannot. Interestingly, coinciding with metaphase chromosome association, HMGA1a-DBD but not HMG1-DBD supported both the transient replication and stable maintenance of
oriP
plasmids, with efficiencies quantitatively similar to that of EBNA1. However, HMGA1a-DBD activated transcription from EBNA1-dependent episomal reporter to only 20% of the level of EBNA1. Furthermore, EBNA1 but not HMGA1a-DBD activated transcription from a chromosomally integrated EBNA1-dependent transcription reporter. This indicates that EBNA1 possesses functional domains that support transcription activation independent of its ability to tether episomal
oriP
plasmids to cellular chromosomes. We provide evidence that metaphase chromosome tethering is a fundamental requirement for maintenance of an
oriP
plasmid but is insufficient for EBNA1 to activate transcription.
Active Borna Disease Virus Polymerase Complex Requires a Distinct Nucleoprotein-to-Phosphoprotein Ratio but No Viral X Protein
Authors: Schneider Urs , Naegele Melanie , Staeheli Peter , Schwemmle Martin
Published: October 2003

Analysis of the composition and regulation of the Borna disease virus (BDV) polymerase complex has so far been limited by the lack of a functional assay. To establish such an assay on the basis of an artificial minigenome, we constructed expression vectors encoding either nucleoprotein (N), phosphoprotein (P), X protein, or polymerase (L) of BDV under the control of the chicken β-actin promoter. A Flag-tagged version of L colocalized with virus-encoded N and P in characteristic nuclear dots of BDV-infected cells and increased viral N-protein levels in persistently infected Vero cells. Vector-driven expression of L, N, and P in BSR-T7 cells together with a negative-sense BDV minigenome carrying a chloramphenicol acetyltransferase (CAT) reporter gene resulted in efficient synthesis of CAT protein. Induction of CAT protein synthesis strongly depended on a 10- to 30-fold molar excess of the N-encoding plasmid over the P-encoding plasmid. Cotransfection of even small amounts of plasmid encoding the viral X protein reduced CAT synthesis to background levels. Thus, the N-to-P stoichiometry seems to play a central role in the regulation of the BDV polymerase complex. Our data further suggest a negative regulatory function for the X protein of BDV.
Norwalk Virus Nonstructural Protein p48 Forms a Complex with the SNARE Regulator VAP-A and Prevents Cell Surface Expression of Vesicular Stomatitis Virus G Protein
Authors: Ettayebi Khalil , Hardy Michele E.
Published: October 2003

Norwalk virus
(NV), a reference strain of human calicivirus in the
Norovirus
genus of the family
Caliciviridae
, contains a positive-strand RNA genome with three open reading frames. ORF1 encodes a 1,789-amino-acid polyprotein that is processed into nonstructural proteins that include an NTPase, VPg, protease, and RNA-dependent RNA polymerase. The N-terminal protein p48 of ORF1 shows no significant sequence similarity to viral or cellular proteins, and its function in the human calicivirus replication cycle is not known. The lack of sequence similarity to any protein in the public databases suggested that p48 may have a unique function in the NV replication cycle or, alternatively, may perform a characterized function in replication by a unique mechanism. In this report, it is shown that p48 displays a vesicular localization pattern in transfected cells when fused to the fluorescent reporter EYFP. A predicted transmembrane domain at the C terminus of p48 was not necessary for the observed localization pattern, but this domain was sufficient to redirect localization of EYFP to a fluorescent pattern consistent with the Golgi apparatus. A yeast two-hybrid screen identified the SNARE regulator vesicle-associated membrane protein-associated protein A (VAP-A) as a binding partner of p48. Biochemical assays confirmed that p48 and VAP-A interact and form a stable complex in mammalian cells. Furthermore, expression of the vesicular stomatitis virus G glcyoprotein on the cell surface was inhibited when cells coexpressed p48, suggesting that p48 disrupts intracellular protein trafficking.
Astrovirus Induces Diarrhea in the Absence of Inflammation and Cell Death
Authors: Koci Matthew D. , Moser Lindsey A. , Kelley Laura A. , Larsen Diane , Brown Corrie C. , Schultz-Cherry Stacey
Published: October 2003

Astroviruses are a leading cause of infantile viral gastroenteritis worldwide. Very little is known about the mechanisms of astrovirus-induced diarrhea. One reason for this is the lack of a small-animal model. Recently, we isolated a novel strain of astrovirus (TAstV-2) from turkeys with the emerging infectious disease poult enteritis mortality syndrome. In the present studies, we demonstrate that TAstV-2 causes growth depression, decreased thymus size, and enteric infection in infected turkeys. Infectious TAstV-2 can be recovered from multiple tissues, including the blood, suggesting that there is a viremic stage during infection. In spite of the severe diarrhea, histopathologic changes in the intestine were mild and there was a surprising lack of inflammation. This may be due to the increased activation of the potent immunosuppressive cytokine transforming growth factor beta during astrovirus infection. These studies suggest that the turkey will be a useful small-animal model with which to study astrovirus pathogenesis and immunity.
Adenovirus E1B 55-Kilodalton Oncoprotein Binds to Daxxand Eliminates Enhancement of p53-Dependent Transcription byDaxx
Authors: Zhao Lisa Y. , Colosimo April L. , Liu Yue , Wan Yanping , Liao Daiqing
Published: October 2003

The
adenovirus E1B 55-kDa protein impairs the p53 pathway and enhances
transformation, although the underlying mechanisms remain to be
defined. We found that Daxx binds to the E1B 55-kDa protein in a yeast
two-hybrid screen. The two proteins interact through their C termini.
Mutation of three potential phosphorylation sites (S489/490 and T494 to
alanine) within the E1B 55-kDa protein did not affect its interaction
with Daxx, although such mutations were previously shown to inhibit
E1B's ability to repress p53-dependent transcription and to
enhance transformation. In addition to their coimmunoprecipitation in
293 extracts, purified Daxx interacted with the E1B 55-kDa protein in
vitro, indicating their direct interaction. In 293 cells, Daxx
colocalized with the E1B 55-kDa protein within discrete nuclear dots,
where p53 was also found. Such structures were distinct from PML
(promyelocytic leukemia protein) bodies, and it appeared that Daxx was
displaced from PML bodies. Thus, the Daxx concentration was diminished
in dots with a prominent presence of PML and vice versa. Indeed, PML
overexpression led to dramatic redistribution of Daxx from
p53-E1B 55-kDa protein complexes to PML bodies. Additionally,
expression of the E1B 55-kDa protein in Saos2 osteosarcoma cells
reduced the number of PML bodies. Our data suggest that E1B and PML
compete for available Daxx in the cell. Surprisingly, Daxx
significantly augmented p53-mediated transcription and the E1B 55-kDa
protein eliminated this effect. Thus, it is likely that the E1B 55-kDa
protein sequesters Daxx and p53 in specific nuclear locations, where
p53 cannot activate transcription. One consequence of the Daxx-E1B
interaction might be an alteration of normal interactions of Daxx, PML,
and p53, which may contribute to cell
transformation.
Dengue Virus Induces Novel Changes in Gene Expression of Human Umbilical Vein Endothelial Cells
Authors: Warke Rajas V. , Xhaja Kris , Martin Katherine J. , Fournier Marcia F. , Shaw Sunil K. , Brizuela Nathaly , de Bosch Norma , Lapointe David , Ennis Francis A. , Rothman Alan L. , Bosch Irene
Published: October 2003

Endothelial cells are permissive to dengue virus (DV) infection in vitro, although their importance as targets of DV infection in vivo remains a subject of debate. To analyze the virus-host interaction, we studied the effect of DV infection on gene expression in human umbilical vein endothelial cells (HUVECs) by using differential display reverse transcription-PCR (DD-RTPCR), quantitative RT-PCR, and Affymetrix oligonucleotide microarrays. DD identified eight differentially expressed cDNAs, including inhibitor of apoptosis-1, 2′-5′ oligoadenylate synthetase (OAS), a 2′-5′ OAS-like (OASL) gene, galectin-9, myxovirus protein A (MxA), regulator of G-protein signaling, endothelial and smooth muscle cell-derived neuropilin-like protein, and phospholipid scramblase 1. Microarray analysis of 22,000 human genes confirmed these findings and identified an additional 269 genes that were induced and 126 that were repressed more than fourfold after DV infection. Broad functional responses that were activated included the stress, defense, immune, cell adhesion, wounding, inflammatory, and antiviral pathways. These changes in gene expression were seen after infection of HUVECs with either laboratory-adapted virus or with virus isolated directly from plasma of DV-infected patients. Tumor necrosis factor alpha, OASL, and MxA and h-IAP1 genes were induced within the first 8 to 12 h after infection, suggesting a direct effect of DV infection. These global analyses of DV effects on cellular gene expression identify potentially novel mechanisms involved in dengue disease manifestations such as hemostatic disturbance.