bluuebunny/crossref_metadata_2025 huggingface dataset:
Mutational Analysis of the GB Virus B Internal Ribosome Entry Site
Authors: Rijnbrand René , Abell Geoffrey , Lemon Stanley M.
Published: July 2002

GB virus B
(GBV-B) is a recently discovered hepatotropic flavivirus that is distantly related to hepatitis C virus (HCV). We show here that translation of its polyprotein is initiated by internal entry of ribosomes on GBV-B RNA. We analyzed the translational activity of dicistronic RNA transcripts containing wild-type or mutated 5′ nontranslated GBV-B RNA (5′NTR) segments, placed between the coding sequences of two reporter proteins, in vitro in rabbit reticulocyte lysate and in vivo in transfected BT7-H cells. We related these results to a previously proposed model of the secondary structure of the GBV-B 5′NTR (M. Honda, et al. RNA 2:955–968, 1996). We identified an internal ribosome entry site (IRES) bounded at its 5′ end by structural domain II, a location analogous to the 5′ limit of the IRES in both the HCV and pestivirus 5′NTRs. Mutational analysis confirmed the structure proposed for domain II of GBV-B RNA, and demonstrated that optimal IRES-mediated translation is dependent on each of the putative RNA hairpins in this domain, including two stem-loops not present in the HCV or pestivirus structures. IRES activity was also absolutely dependent on (i) phylogenetically conserved, adenosine-containing bulge loops in domain III and (ii) the primary nucleotide sequence of stem-loop IIIe. IRES-directed translation was inhibited by a series of point mutations predicted to stabilize stem-loop IV, which contains the initiator AUG codon in its loop segment. A reporter gene was translated most efficiently when fused directly to the initiator AUG codon, with no intervening downstream GBV-B sequence. This finding indicates that the 3′ limit of the GBV-B IRES is at the initiator AUG and that it does not require downstream polyprotein-coding sequence as suggested for the HCV IRES. These results show that the GBV-B IRES, while sharing a common general structure, differs both structurally and functionally from other flavivirus IRES elements.
Recombinant Rinderpest Vaccines Expressing Membrane-Anchored Proteins as Genetic Markers: Evidence of Exclusion of Marker Protein from the Virus Envelope
Authors: Walsh Edmund P. , Baron Michael D. , Rennie Louise F. , Monaghan Paul , Anderson John , Barrett Thomas
Published: July 2002

Rinderpest virus (RPV) causes a severe disease of cattle resulting in serious economic losses in parts of the developing world. Effective control and elimination of this disease require a genetically marked rinderpest vaccine that allows serological differentiation between animals that have been vaccinated against rinderpest and those which have recovered from natural infection. We have constructed two modified cDNA clones of the vaccine strain RNA genome of the virus, with the coding sequence of either a receptor site mutant form of the influenza virus hemagglutinin (HA) gene or a membrane-anchored form of the green fluorescent protein (GFP) gene (ANC-GFP), inserted as a potential genetic marker. Infectious recombinant virus was rescued in cell culture from both constructs. The RPVINS-HA and RPVANC-GFP viruses were designed to express either the HA or ANC-GFP protein on the surface of virus-infected cells with the aim of stimulating a strong humoral antibody response to the marker protein. In vitro studies showed that the marker proteins were expressed on the surface of virus-infected cells, although to different extents, but neither was incorporated into the envelope of the virus particles. RPVINS-HA- or RPVANC-GFP-vaccinated cattle produced normal levels of humoral anti-RPV antibodies and significant levels of anti-HA or anti-GFP antibodies, respectively. Both viruses were effective in stimulating protective immunity against RPV and antibody responses to the marker protein in all animals when tested in a cattle vaccination trial.
Reduced Susceptibility of Human Immunodeficiency Virus Type 1 (HIV-1) from Patients with Primary HIV Infection to Nonnucleoside Reverse Transcriptase Inhibitors Is Associated with Variation at Novel Amino Acid Sites
Authors: Brown Andrew J. Leigh , Precious Heather M. , Whitcomb Jeannette M. , Wong Joseph K. , Quigg Marlynne , Huang Wei , Daar Eric S. , D'Aquila Richard T. , Keiser Philip H. , Connick Elizabeth , Hellmann Nicholas S. , Petropoulos Christos J. , Richman Douglas D. , Little Susan J.
Published: July 2002

Recently, significant numbers of individuals with primary human immunodeficiency virus (HIV) infection have been found to harbor viral strains with reduced susceptibility to antiretroviral drugs. In one study, HIV from 16% of such antiretroviral-naive individuals was shown to have a susceptibility to nonnucleoside reverse transcriptase (RT) inhibitors (NNRTIs) between 2.5- and 10-fold lower than that of a wild-type control. Mutations in the RT domain that had previously been associated with antiretroviral resistance were not shared by these strains. We have analyzed by logistic regression 46 variable amino acid sites in RT for their effect on susceptibility and have identified two novel sites influencing susceptibility to NNRTIs: amino acids 135 and 283 in RT. Eight different combinations of amino acids at these sites were observed among these patients. These combinations showed a 14-fold range in mean susceptibility to both nevirapine and delavirdine. In vitro mutagenesis of the control strain combined with a phenotypic assay confirmed the significance of amino acid variation at these sites for susceptibility to NNRTIs.
Infection of Human Dendritic Cells by a Sindbis Virus Replicon Vector Is Determined by a Single Amino Acid Substitution in the E2 Glycoprotein
Authors: Gardner Jason P. , Frolov Ilya , Perri Silvia , Ji Yaying , MacKichan Mary Lee , zur Megede Jan , Chen Minchao , Belli Barbara A. , Driver David A. , Sherrill Scott , Greer Catherine E. , Otten Gillis R. , Barnett Susan W. , Liu Margaret A. , Dubensky Thomas W. , Polo John M.
Published: July 2002

The ability to target antigen-presenting cells with vectors encoding desired antigens holds the promise of potent prophylactic and therapeutic vaccines for infectious diseases and cancer. Toward this goal, we derived variants of the prototype alphavirus, Sindbis virus (SIN), with differential abilities to infect human dendritic cells. Cloning and sequencing of the SIN variant genomes revealed that the genetic determinant for human dendritic cell (DC) tropism mapped to a single amino acid substitution at residue 160 of the envelope glycoprotein E2. Packaging of SIN replicon vectors with the E2 glycoprotein from a DC-tropic variant conferred a similar ability to efficiently infect immature human DC, whereupon those DC were observed to undergo rapid activation and maturation. The SIN replicon particles infected skin-resident mouse DC in vivo, which subsequently migrated to the draining lymph nodes and upregulated cell surface expression of major histocompatibility complex and costimulatory molecules. Furthermore, SIN replicon particles encoding human immunodeficiency virus type 1 p55Gagelicited robust Gag-specific T-cell responses in vitro and in vivo, demonstrating that infected DC maintained their ability to process and present replicon-encoded antigen. Interestingly, human and mouse DC were differentially infected by selected SIN variants, suggesting differences in receptor expression between human and murine DC. Taken together, these data illustrate the tremendous potential of using a directed approach in generating alphavirus vaccine vectors that target and activate antigen-presenting cells, resulting in robust antigen-specific immune responses.
The K-bZIP Protein from Kaposi's Sarcoma-Associated Herpesvirus Interacts with p53 and Represses Its Transcriptional Activity
Authors: Park Junsoo , Seo Taegun , Hwang Seungmin , Lee Daeyoup , Gwack Yousang , Choe Joonho
Published: July 2002

Kaposi's sarcoma-associated herpesvirus (KSHV) is a gammaherpesvirus that has been implicated in the pathogenesis of Kaposi's sarcoma. KSHV encodes K-bZIP (open reading frame K8), a protein that belongs to the basic region-leucine zipper (bZIP) family of transcription factors. Here we show that K-bZIP associates with the cellular transcription factor p53 directly in vitro and in vivo. This interaction requires the bZIP domain of K-bZIP and the carboxy-terminal region (amino acids 300 to 393) of p53. We also show that K-bZIP represses the transcriptional activity of p53 which is required for apoptosis of the host cell. These results imply that K-bZIP blocks p53-mediated host cell death through its interaction with p53.
Simian Homologues of Human Gamma-2 and Betaherpesviruses in Mandrill and Drill Monkeys
Authors: Lacoste Vincent , Mauclere Philippe , Dubreuil Guy , Lewis John , Georges-Courbot Marie-Claude , Rigoulet Jacques , Petit Thierry , Gessain Antoine
Published: July 2002

Recent serological and molecular surveys of different primate species allowed the characterization of several Kaposi's sarcoma-associated herpesvirus (KSHV) homologues in macaques, African green monkeys, chimpanzees, and gorillas. Identification of these new primate rhadinoviruses revealed the existence of two distinct genogroups, called RV1 and RV2. Using a degenerate consensus primer PCR method for the herpesvirus DNA polymerase gene, the presence of KSHV homologues has been investigated in two semi-free-ranging colonies of eight drill (
Mandrillus leucophaeus
), five mandrill (
Mandrillus sphinx
), and two hybrid (
Mandrillus leucophaeus-Mandrillus sphinx
) monkeys, living in Cameroon and Gabon, Central Africa. This search revealed the existence of not only two distinct KSHV homologues, each one belonging to one of the two rhadinovirus genogroups, but also of two new betaherpesvirus sequences, one being close to cytomegaloviruses and the other being related to human herpesviruses 6 and 7 (HHV-6 and -7). The latter viruses are the first simian HHV-6 and -7 homologues identified to date. These data show that mandrill and drill monkeys are the hosts of at least four novel distinct herpesviruses. Moreover, mandrills, like macaques and African green monkeys, harbor also two distinct gamma-2 herpesviruses, thus strongly suggesting that a second gamma-2 herpesvirus, belonging to the RV2 genogroup, may exist in humans.
Recovery and Altered Neutralization Specificities of Chimeric Viruses Containing Capsid Protein Domain Exchanges from Antigenically Distinct Strains of Feline Calicivirus
Authors: Neill John D. , Sosnovtsev Stanislav V. , Green Kim Y.
Published: July 2002

Feline calicivirus (FCV) strains can show significant antigenic variation when tested for cross-reactivity with antisera produced against other FCV strains. Previous work has demonstrated the presence of hypervariable amino acid sequences in the capsid protein of FCV (designated regions C and E) that were postulated to constitute the major antigenic determinants of the virus. To examine the involvement of hypervariable sequences in determining the antigenic phenotype, the nucleotide sequences encoding the E regions from three antigenically distinct parental FCV strains (CFI, KCD, and NADC) were exchanged for the equivalent sequences in an FCV Urbana strain infectious cDNA clone. Two of the three constructs were recovered as viable, chimeric viruses. In six additional constructs, of which three were recovered as viable virus, the E region from the parental viruses was divided into left (N-terminal) and right (C-terminal) halves and engineered into the infectious clone. A final viable construct contained the C, D, and E regions of the NADC parental strain. Recovered chimeric viruses showed considerable antigenic variation from the parental viruses when tested against parental hyperimmune serum. No domain exchange was able to confer complete recognition by parental antiserum with the exception of the KCD E region exchange, which was neutralized at a near-homologous titer with KCD antiserum. These data demonstrate that it is possible to recover engineered chimeric FCV strains that possess altered antigenic characteristics. Furthermore, the E hypervariable region of the capsid protein appears to play a major role in the formation of the antigenic structure of the virion where conformational epitopes may be more important than linear in viral neutralization.
Identification of a Novel Cleavage Activity of the First Papain-Like Proteinase Domain Encoded by Open Reading Frame 1a of the Coronavirus
Avian Infectious Bronchitis Virus
and Characterization of the Cleavage Products
Authors: Lim K. P. , Ng Lisa F. P. , Liu D. X.
Published: July 2002

The coronavirus
Avian infectious bronchitis virus
(IBV) employs polyprotein processing as a strategy to express its gene products. Previously we identified the first cleavage event as proteolysis at the Gly
673
-Gly
674
dipeptide bond mediated by the first papain-like proteinase domain (PLPD-1) to release an 87-kDa mature protein. In this report, we demonstrate a novel cleavage activity of PLPD-1. Expression, deletion, and mutagenesis studies showed that the product encoded between nucleotides 2548 and 8865 was further cleaved by PLPD-1 at the Gly
2265
-Gly
2266
dipeptide bond to release an N-terminal 195-kDa and a C-terminal 41-kDa cleavage product. Characterization of the cleavage activity revealed that the proteinase is active on this scissile bond when expressed in vitro in rabbit reticulocyte lysates and can act on the same substrate in
trans
when expressed in intact cells. Both the N- and C-terminal cleavage products were detected in virus-infected cells and were found to be physically associated. Glycosidase digestion and site-directed mutagenesis studies of the 41-kDa protein demonstrated that it is modified by N-linked glycosylation at the Asn
2313
residue encoded by nucleotides 7465 to 7467. By using a region-specific antiserum raised against the IBV sequence encoded by nucleotides 8865 to 9786, we also demonstrated that a 33-kDa protein, representing the 3C-like proteinase (3CLP), was specifically immunoprecipitated from the virus-infected cells. Site-directed mutagenesis and expression studies showed that a previously predicted cleavage site (Q
2583
-G
2584
) located within the 41-kDa protein-encoding region was not utilized by 3CLP, supporting the conclusion that the 41-kDa protein is a mature viral product.
Nuclear Matrix Attachment Regions of Human Papillomavirus Type 16 Repress or Activate the E6 Promoter, Depending on the Physical State of the Viral DNA
Authors: Stünkel Walter , Huang Zhonghui , Tan Shyh-Han , O'Connor Mark J. , Bernard Hans-Ulrich
Published: July 2002

Two nuclear matrix attachment regions (MARs) bracket a 550-bp segment of the long control region (LCR) containing the epithelial cell-specific enhancer and the E6 promoter of human papillomavirus type 16 (HPV-16). One of these MARs is located in the 5′ third of the LCR (5′-LCR-MAR); the other lies within the E6 gene (E6-MAR). To study their function, we linked these MARs in various natural or artificial permutations to a chimeric gene consisting of the HPV-16 enhancer-promoter segment and a reporter gene. In transient transfections of HeLa cells, the presence of either of these two MARs strongly represses reporter gene expression. In contrast to this, but similar to the published behavior of cellular MARs, reporter gene expression is stimulated strongly by the E6-MAR and moderately by the 5′-LCR-MAR in stable transfectants of HeLa or C33A cells. To search for binding sites of soluble nuclear proteins which may be responsible for repression during transient transfections, we performed electrophoretic mobility shift assays (EMSAs) of overlapping oligonucleotides that represented all sequences of these two MARs. Both MARs contain multiple sites for two strongly binding proteins and weak binding sites for additional factors. The strongest complex, with at least five binding sites in each MAR, is generated by the CCAAT displacement factor (CDP)/Cut, as judged by biochemical purification, by EMSAs with competing oligonucleotides and with anti-CDP/Cut oligonucleotides, and by mutations. CDP/Cut, a repressor that is down-regulated during differentiation, apparently represses HPV-16 transcription in undifferentiated epithelials cells and in HeLa cells, which are rich in CDP/Cut. In analogy to poorly understood mechanisms acting on cellular MARs, activation after physical linkage to chromosomal DNA may result from competition between the nuclear matrix and CDP/Cut. Our observations show that
cis
-responsive elements that regulate the HPV-16 E6 promoter are tightly clustered over at least 1.3 kb and occur throughout the E6 gene. HPV-16 MARs are context dependent transcriptional enhancers, and activated expression of HPV-16 oncogenes dependent on chromosomal integration may positively select tumorigenic cells during the multistep etiology of cervical cancer.
Human Beta Interferon Scaffold Attachment Region Inhibits De Novo Methylation and Confers Long-Term, Copy Number-Dependent Expression to a Retroviral Vector
Authors: Dang Qi , Auten Jennifer , Plavec Ivan
Published: July 2002

Moloney murine leukemia virus-based retroviral vector expression is gradually lost during prolonged in vitro culture of CEMSS T cells. However, when the human beta interferon scaffold attachment region (IFN-SAR) was inserted into the vector immediately upstream of the 3′ long terminal repeat (LTR), expression was maintained for the length of the study (4 months). Clonal analysis of the retrovirus vector-infected CEMSS cells showed that SAR-containing retroviral vector expression levels were positively correlated with the proviral copy numbers (
P
< 0.0001), while there was no correlation between the proviral copy numbers and expression levels in control vector-infected clones. Thirty-three percent of the CEMSS cell clones infected with the control vector showed evidence of partial or complete methylation in the 5′ LTR region. In sharp contrast, we detected no methylation in the clones infected with the SAR-containing vector. To demonstrate a direct inhibitory effect of methylation on retroviral vector expression, we have transfected 293 cells with in vitro-methylated proviral DNA. In transiently transfected cells, expression of methylated LTR was reduced but not completely inhibited, irrespective of the presence of the IFN-SAR sequence. In stably transfected cells, however, methylation completely abolished expression of the control vector but not of the SAR-containing vector. Furthermore, the expression of the SAR-containing vector was stable over time, indicating the ability of the SAR sequence to alleviate methylation-mediated transcriptional repression of a vector. This study extends our understanding of the mechanisms of retroviral vector inactivation by methylation and provides insight into a functional role for the SAR elements.
Complex Effects of Deletions in the 5′ Untranslated Region of Primate Foamy Virus on Viral Gene Expression and RNA Packaging
Authors: Heinkelein Martin , Thurow Jana , Dressler Marco , Imrich Horst , Neumann-Haefelin Dieter , McClure Myra O. , Rethwilm Axel
Published: July 2002

Due to various advantageous features there is current interest in retroviral vectors derived from primate foamy viruses (PFVs). Two PFV
cis
-acting sequences have been mapped in the 5′ region of the RNA (pre-)genome and in the 3′
pol
genomic region. In order to genetically separate PFV packaging constructs from vector constructs, we investigated the effect of deletions in the 5′ untranslated region (UTR) of PFV packaging constructs and vectors on gene expression and RNA incorporation into viral particles. Our results indicate that the 5′ UTR serves different previously unknown functions. First, the R region of the long terminal repeat was found to be required for PFV
gag
gene expression. This regulation of gene expression appeared to be mainly posttranscriptional. Second, constructs with sequence deletions between the R region and the
gag
gene start codon packaged as much viral mRNA into particles as the undeleted construct, and RNA from such a 5′-UTR-deleted packaging construct was copackaged into vector-virus particles, together with vector RNA which was preferentialy packaged. Finally, in the U5 region a sequence was identified that was required to allow cleavage of the Gag precursor protein by the
pol
gene-encoded protease, suggesting a role of RNA in PFV particle formation. Taken together, the results indicate that complex interactions of the viral RNA, capsid, and polymerase proteins take place during PFV particle formation and that a clear separation of PFV vector and packaging construct sequences may be difficult to achieve.
TheBFRF1Gene of Epstein-Barr Virus Encodes a Novel Protein
Authors: Farina Antonella , Santarelli Roberta , Gonnella Roberta , Bei Roberto , Muraro Raffaella , Cardinali Giorgia , Uccini Stefania , Ragona Giuseppe , Frati Luigi , Faggioni Alberto , Angeloni Antonio
Published: July 2002

Computer analysis of the Epstein-Barr virus (EBV) genome indicates there are ∼100 open reading frames (ORFs). Thus far about 30 EBV genes divided into the categories latent and lytic have been identified. TheBamHI F region of EBV is abundantly transcribed during lytic replication. This region is highly conserved among herpesviruses, thus suggesting that some common function could be retained in the ORFs encompassed within this viral fragment. To identify putative novel proteins and possible new markers for viral replication, we focused our attention on the first rightward ORF in theBamHI F region (BFRF1). Histidine and glutathioneS-transferase-tagged BFRF1 fusion proteins were synthesized to produce a mouse monoclonal antibody (MAb). Analysis of human sera revealed a high seroprevalence of antibodies to BFRF1 in patients affected by nasopharyngeal carcinoma or Burkitt's lymphoma, whereas no humoral response to BFRF1 could be detected among healthy donors. An anti-BFRF1 MAb recognizes a doublet migrating at 37 to 38 kDa in cells extracts from EBV-infected cell lines following lytic cycle activation and in an EBV-negative cell line (DG75) transfected with a plasmid expressing theBFRF1gene. Northern blot analysis allowed the detection of a major transcript of 3.7 kb highly expressed in EBV-positive lytic cycle-induced cell lines. Treatment with inhibitors of viral DNA polymerase, such as phosphonoacetic acid and acyclovir, reduced but did not abolish the transcription ofBFRF1, thus indicating thatBFRF1can be classified as an early gene. Cell fractionation experiments, as well as immunolocalization by immunofluorescence microscopy, immunohistochemistry, and immunoelectron microscopy, showed that BFRF1 is localized on the plasma membrane and nuclear compartments of the cells and is a structural component of the viral particle. Identification of BFRF1 provides a new marker with which to monitor EBV infection and might help us better understand the biology of the virus.
Distinctions between Bovine Herpesvirus 1 and Herpes Simplex Virus Type 1 VP22 Tegument Protein Subcellular Associations
Authors: Harms Jerome S. , Ren Xiaodi , Oliveira Sergio C. , Splitter Gary A.
Published: July 2002

The alphaherpesvirus tegument protein VP22 has been characterized with multiple traits including microtubule reorganization, nuclear localization, and nonclassical intercellular trafficking. However, all these data were derived from studies using herpes simplex virus type 1 (HSV-1) and may not apply to VP22 homologs of other alphaherpesviruses. We compared subcellular attributes of HSV-1 VP22 (HVP22) with bovine herpesvirus 1 (BHV-1) VP22 (BVP22) using green fluorescent protein (GFP)-fused VP22 expression vectors. Fluorescence microscopy of cell lines transfected with these constructs revealed differences as well as similarities between the two VP22 homologs. Compared to that of HVP22, the BVP22 microtubule interaction was much less pronounced. The VP22 nuclear interaction varied, with a marbled or halo appearance for BVP22 and a speckled or nucleolus-bound appearance for HVP22. Both VP22 homologs associated with chromatin at various stages of mitosis and could traffic from expressing cells to the nuclei of nonexpressing cells. However, distinct qualitative differences in microtubule, nuclear, and chromatin association as well as trafficking were observed. The differences in VP22 homolog characteristics revealed in this study will help define VP22 function within HSV-1 and BHV-1 infection.
CXCR4 Is Down-Regulated in Cells Infected with the CD4-Independent X4 Human Immunodeficiency Virus Type 1 Isolate m7NDK
Authors: Valente Susana T. , Chanel Chantal , Dumonceaux Julie , Olivier René , Marullo Stephano , Briand Pascale , Hazan Uriel
Published: July 2002

Macrophages and T cells infected in vitro with CD4-dependent human immunodeficiency virus type 1 (HIV-1) isolates have reduced levels of CD4 protein, a phenomenon involved in retroviral interference. We have previously characterized the first CD4-independent HIV-1 X4 isolate m7NDK, which directly interacts with CXCR4 through its mutated gp120. We thus investigate CXCR4 expression in cells infected with this m7NDK CXCR4-dependent HIV-1 mutant. We present evidence of the down-regulation of CXCR4 membrane expression in CD4-positive or -negative cells chronically infected with the HIV-1 m7NDK, a phenomenon which is not observed in the CD4-dependent HIV-1 NDK parental strain. This down-regulation of CXCR4 was demonstrated by fluorescence-activated cell sorter analysis and was confirmed by the absence of CXCR4 functionality in m7NDK-infected cells, independently of the presence of CD4 protein. Furthermore, a drastic reduction of the intracellular level of CXCR4 protein was also observed. Reduced levels of CXCR4 mRNA transcripts were found in m7NDK-infected HeLa and CEM cells, reduced levels that could not be attributed to a reduced stability of CXCR4 mRNA. Down-regulation of CXCR4 on m7NDK-infected cells may thus be explained by transcriptional regulation.
Herpes Simplex Virus Type 1 Corneal Infection Results in Periocular Disease by Zosteriform Spread
Authors: Summers Bretton C. , Margolis Todd P. , Leib David A.
Published: July 2002

In humans and animal models of herpes simplex virus infection, zosteriform skin lesions have been described which result from anterograde spread of the virus following invasion of the nervous system. Such routes of viral spread have not been fully examined following corneal infection, and the possible pathologic consequences of such spread are unknown. To investigate this, recombinant viruses expressing reporter genes were generated to quantify and correlate gene expression with replication in eyes, trigeminal ganglia, and periocular tissue. Reporter activity peaked in eyes 24 h postinfection and rapidly fell to background levels by 48 h despite the continued presence of viral titers. Reporter activity rose in the trigeminal ganglia at 60 h and peaked at 72 h, concomitant with the appearance and persistence of infectious virus. Virus was present in the periocular skin from 24 h despite the lack of significant reporter activity until 84 h postinfection. This detection of reporter activity was followed by the onset of periocular disease on day 4. Corneal infection with a thymidine kinase-deleted reporter virus displayed a similar profile of reporter activity and viral titer in the eyes, but little or no detectable activity was observed in trigeminal ganglia or periocular tissue. In addition, no periocular disease symptoms were observed. These findings demonstrate that viral infection of periocular tissue and subsequent disease development occurs by zosteriform spread from the cornea to the periocular tissue via the trigeminal ganglion rather than by direct spread from cornea to the periocular skin. Furthermore, clinical evidence is discussed suggesting that a similar mode of spreading and disease occurs in humans following primary ocular infection.
JC Virus Regulatory Region Tandem Repeats in Plasma and Central Nervous System Isolates Correlate with Poor Clinical Outcome in Patients with Progressive Multifocal Leukoencephalopathy
Authors: Pfister Luz-Andrea , Letvin Norman L. , Koralnik Igor J.
Published: July 2002

JC virus (JCV), the causative agent of progressive multifocal leukoencephalopathy (PML), has a hypervariable regulatory region (JCV RR). A conserved archetype form is found in the urines of healthy and immunocompromised individuals, whereas forms with tandem repeats and deletions are found in the brains of PML patients. Type I JCV RR, seen in MAD-1, the first sequenced strain of JCV, contains two 98-bp tandem repeats each containing a TATA box. Type II JCV RR has additional 23-bp and 66-bp inserts or fragments thereof and only one TATA box. We cloned and sequenced JCV RR from different anatomic compartments of PML patients and controls and correlated our findings with the patients' clinical outcome. Twenty-three different sequences were defined in 198 clones obtained from 16 patients. All 104 clones with tandem repeats were type II JCV RR. Patients with poor clinical outcome had high proportions of JCV RR clones with both tandem repeats in plasma (54%) and brain or cerebrospinal fluid (85%). In those who became survivors of PML, archetype sequences predominated in these anatomic compartments (75 and 100%, respectively). In patients with advanced human immunodeficiency virus infection without PML, only 8% of JCV RR clones obtained in the plasma contained tandem repeats. These data suggest that the presence of tandem repeats in plasma and CNS JCV RR clones is associated with poor clinical outcome in patients with PML.
Role of the 3′-Untranslated Regions of Alfalfa Mosaic Virus RNAs in the Formation of a Transiently Expressed Replicase in Plants and in the Assembly of Virions
Authors: Vlot A. Corina , Neeleman Lyda , Linthorst Huub J. M. , Bol John F.
Published: July 2002

Alfalfa mosaic virus (AMV) RNAs 1 and 2 encode the replicase proteins P1 and P2, respectively, whereas RNA 3 encodes the movement protein and the coat protein (CP). When RNAs 1 and 2 were transiently expressed from a T-DNA vector (R12 construct) by agroinfiltration of
Nicotiana benthamiana
, the infiltrated leaves accumulated minus-strand RNAs 1 and 2 and relatively small amounts of plus-strand RNAs. In addition, RNA-dependent RNA polymerase (RdRp) activity could be detected in extracts of the infiltrated leaves. After transient expression of RNAs 1 and 2 with the 3′-untranslated regions (UTRs) of both RNAs deleted (R1Δ/2Δ construct), no replication of RNAs 1 and 2 was observed, while the infiltrated leaves supported replication of RNA 3 after inoculation of the leaves with RNA 3 or expression of RNA 3 from a T-DNA vector (R3 construct). No RdRp activity could be isolated from leaves infiltrated with the R1Δ/2Δ construct, although P1 and P2 sedimented in a region of a glycerol gradient where active RdRp was found in plants infiltrated with R12. RdRp activity could be isolated from leaves infiltrated with constructs R1Δ/2 (3′-UTR of RNA 1 deleted), R1/2Δ (3′-UTR of RNA 2 deleted), or R1Δ/2Δ plus R3. This demonstrates that the 3′-UTR of AMV RNAs is required for the formation of a complex with in vitro enzyme activity. RNAs 1 and 2 with the 3′-UTRs deleted were encapsidated into virions by CP expressed from RNA 3. This shows that the high-affinity binding site for CP at the 3′-termini of AMV RNAs is not required for assembly of virus particles.
Inhibition of TRAIL-Induced Apoptosis and Forced Internalization of TRAIL Receptor 1 by Adenovirus Proteins
Authors: Tollefson Ann E. , Toth Karoly , Doronin Konstantin , Kuppuswamy Mohan , Doronina Oksana A. , Lichtenstein Drew L. , Hermiston Terry W. , Smith Craig A. , Wold William S. M.
Published: July 2002

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) induces apoptosis through two receptors, TRAIL-R1 (also known as death receptor 4) and TRAIL-R2 (also known as death receptor 5), that are members of the TNF receptor superfamily of death domain-containing receptors. We show that human adenovirus type 5 encodes three proteins, named RID (previously named E3-10.4K/14.5K), E3-14.7K, and E1B-19K, that independently inhibit TRAIL-induced apoptosis of infected human cells. This conclusion was derived from studies using wild-type adenovirus, adenovirus replication-competent mutants that lack one or more of theRID,E3-14.7K, andE1B-19Kgenes, and adenovirus E1-minus replication-defective vectors that express all E3 genes, RID plus E3-14.7K only, RID only, or E3-14.7K only. RID inhibits TRAIL-induced apoptosis when cells are sensitized to TRAIL either by adenovirus infection or treatment with cycloheximide. RID induces the internalization of TRAIL-R1 from the cell surface, as shown by flow cytometry and indirect immunofluorescence for TRAIL-R1. TRAIL-R1 was internalized in distinct vesicles which are very likely to be endosomes and lysosomes. TRAIL-R1 is degraded, as indicated by the disappearance of the TRAIL-R1 immunofluorescence signal. Degradation was inhibited by bafilomycin A1, a drug that prevents acidification of vesicles and the sorting of receptors from late endosomes to lysosomes, implying that degradation occurs in lysosomes. RID was also shown previously to internalize and degrade another death domain receptor, Fas, and to prevent apoptosis through Fas and the TNF receptor. RID was shown previously to force the internalization and degradation of the epidermal growth factor receptor. E1B-19K was shown previously to block apoptosis through Fas, and both E1B-19K and E3-14.7K were found to prevent apoptosis through the TNF receptor. These findings suggest that the receptors for TRAIL, Fas ligand, and TNF play a role in limiting virus infections. The ability of adenovirus to inhibit killing through these receptors may prolong acute and persistent infections.
Three Herpes Simplex Virus Type 1 Latency-Associated Transcript Mutants with Distinct and Asymmetric Effects on Virulence in Mice Compared with Rabbits
Authors: Perng Guey-Chuen , Esmaili Daniel , Slanina Susan M. , Yukht Ada , Ghiasi Homayon , Osorio Nelson , Mott Kevin R. , Maguen Barak , Jin Ling , Nesburn Anthony B. , Wechsler Steven L.
Published: July 2002

Herpes simplex virus type 1 latency-associated transcript (LAT)-null mutants have decreased reactivation but normal virulence in rabbits and mice. We report here on
d
LAT1.5, a mutant with LAT nucleotides 76 to 1667 deleted. Following ocular infection of rabbits,
d
LAT1.5 reactivated at a lower rate than its wild-type parent McKrae (6.1 versus 11.8%;
P
= 0.0025 [chi-square test]). Reactivation was restored in the marker-rescued virus
d
LAT1.5R (12.6%;
P
= 0.53 versus wild type), confirming the importance of the deleted region in spontaneous reactivation. Compared with wild-type or marker-rescued virus,
d
LAT1.5 had similar or slightly reduced virulence in rabbits (based on survival following ocular infection). In contrast, in mice,
d
LAT1.5 had increased virulence (
P
< 0.0001). Thus, deletion of LAT nucleotides 76 to 1667 increased viral virulence in mice but not in rabbits. In contrast, we also report here that LAT2.9A, a LAT mutant that we previously reported to have increased virulence in rabbits (G. C. Perng, S. M. Slanina, A. Yuhkt, B. S. Drolet, W. J. Keleher, H. Ghiasi, A. B. Nesburn, and S. L. Wechsler, J. Virol. 73:920–929, 1999), had decreased virulence in mice (
P
= 0.03). In addition, we also found that
d
LAT371, a LAT mutant that we previously reported to have wild-type virulence in rabbits (G. C. Perng, S. M. Slanina, H. Ghiasi, A. B. Nesburn, and S. L. Wechsler, J. Virol. 70:2014–2018, 1996), had decreased virulence in mice (
P
< 0.05). Thus, these three mutants, each of which encodes a different LAT RNA, have different virulence phenotypes.
d
LAT1.5 had wild-type virulence in rabbits but increased virulence in mice. In contrast, LAT2.9A had increased virulence in rabbits but decreased virulence in mice, and
d
LAT371 had wild-type virulence in rabbits but decreased virulence in mice. Taken together, these results suggest that (i) the 5′ end of LAT and/or a gene that overlaps part of this region is involved in viral virulence, (ii) this virulence appears to have species-specific effects, and (iii) regulation of this virulence may be complex.
Role of CD28/CD80-86 and CD40/CD154 Costimulatory Interactions in Host Defense to Primary Herpes Simplex Virus Infection
Authors: Edelmann Kurt H. , Wilson Christopher B.
Published: July 2002

Dependence of the primary antiviral immune response on costimulatory interactions between CD28/CD80-86 and between CD40/CD154 (CD40 ligand) has been correlated with the extent of viral replication in two models of systemic infection, lymphocytic choriomeningitis virus and vesicular stomatitis virus. To determine the role of these costimulatory interactions in the context of an acute cytolytic, but locally replicating viral infection, herpes simplex virus (HSV) infection was assessed in mice that had the CD28/CD80-86 or CD40/CD154 interactions disrupted either genetically or with blocking reagents (CTLA4Ig and MR1, respectively). CTLA4Ig treatment greatly reduced paralysis-free survival during primary acute HSV infection. This reflected an almost total ablation of the anti-HSV CD4+and CD8+T-cell responses due to anergy and reduced cell numbers, respectively. Disruption of CD40/CD154 interactions impaired survival, but the effect was less severe than that observed in CTLA4Ig-treated mice, with reductions observed in the CD4+T-cell but not CD8+T-cell responses. These two costimulatory pathways functioned in part independently, since disruption of both further impaired survival. The dependence on these costimulatory interactions for the control of primary HSV infection may represent a more widespread paradigm for nonsystemic viruses, which have restricted sites of replication and which employ immunoevasive measures.
Overexpression of Simian Virus 40 Small-T Antigen Blocks Centrosome Function and Mitotic Progression in Human Fibroblasts
Authors: Gaillard Stéphanie , Fahrbach Kelly M. , Parkati Rajini , Rundell Kathleen
Published: July 2002

Recombinant adenoviruses that express high levels of the simian virus 40 (SV40) small-t (ST) antigen have been used to study the requirement for ST to drive cell cycle proliferation of confluent human diploid fibroblasts. This occurs when either large-T (LT) antigen or serum is added to provide a second signal. While cells readily completed S phase in these experiments, they were found to accumulate with 4N DNA content. Cellular and nuclear morphology, as well as the biochemical status of cyclin B complexes, showed that these cells entered mitosis but were blocked prior to mitotic metaphase. The defect appears to reflect an inability of cells overexpressing ST to form organized centrosomes that duplicate and separate normally during the cell cycle and, therefore, the absence of a mitotic spindle. The ability of ST to bind protein phosphatase 2A was required for this pattern, suggesting that altered phosphorylation of key centrosomal components may occur when ST is overexpressed. Although the possible significance of ST effects on the centrosome cycle is not fully understood, these findings suggest that ST could influence chromosomal instability patterns that are a hallmark of SV40-transformed cells and LT expression.
Glycoproteins E and I of Marek's Disease Virus Serotype 1 Are Essential for Virus Growth in Cultured Cells
Authors: Schumacher Daniel , Tischer B. Karsten , Reddy Sanjay M. , Osterrieder Nikolaus
Published: July 2002

The role of glycoprotein E (gE) and gI of Marek's disease virus serotype 1 (MDV-1) for growth in cultured cells was investigated. MDV-1 mutants lacking either gE (20ΔgE), gI (20ΔgI), or both gE and gI (20ΔgEI) were constructed by recE/T-mediated mutagenesis of a recently established infectious bacterial artificial chromosome (BAC) clone of MDV-1 (D. Schumacher, B. K. Tischer, W. Fuchs, and N. Osterrieder, J. Virol. 74:11088–11098, 2000). Deletion of either gE or gI, which form a complex in MDV-1-infected cells, resulted in the production of virus progeny that were unable to spread from cell to cell in either chicken embryo fibroblasts or quail muscle cells. This was reflected by the absence of virus plaques and the detection of only single infected cells after transfection, even after coseeding of transfected cells with uninfected cells. In contrast, growth of rescuant viruses, in which the deleted glycoprotein genes were reinserted by homologous recombination, was indistinguishable from that of parental BAC20 virus. In addition, the 20ΔgE mutant virus was able to spread from cell to cell when cotransfected into chicken embryo fibroblasts with an expression plasmid encoding MDV-1 gE, and the 20ΔgI mutant virus exhibited cell-to-cell spread capability after cotransfection with a gI expression plasmid. The 20ΔgEI mutant virus, however, was not able to spread in the presence of either a gE or gI expression plasmid, and only single infected cells were detected by indirect immunofluorescence. The results reported here demonstrate for the first time that both gE and gI are absolutely essential for cell-to-cell spread of a member of the
Alphaherpesvirinae
.
A Cyclic Dodecapeptide–Multiple-Antigen Peptide Conjugate from the Undecapeptidyl Arch (from Arg
168
to Cys
178
) of Extracellular Loop 2 in CCR5 as a Novel Human Immunodeficiency Virus Type 1 Vaccine
Authors: Misumi Shogo , Nakajima Reina , Takamune Nobutoki , Shoji Shozo
Published: July 2002

A cyclic closed-chain dodecapeptide (cDDR5) mimicking the conformation-specific domain of CCR5 was prepared in which Gly-Asp, as a dipeptide forming a spacer arm, links the amino and carboxyl termini of the decapeptidyl linear chain (Arg
168
to Thr
177
) derived from the undecapeptidyl arch (UPA; Arg
168
to Cys
178
) of extracellular loop 2 (ECL2) in CCR5. Novel monoclonal antibodies were raised against cDDR5 conjugated with a multiple-antigen peptide (cDDR5-MAP), and the purified antibody [KB8C12, immunoglobulin M(κ)] reacted with cDDR5, but not with linear DDR5, in real-time biomolecular interaction analysis using surface plasmon resonance. The antibody also reacted with cells expressing CCR5, but not with cells expressing CXCR4, and the immunoreaction was competed by cDDR5-MAP. The antibody significantly interfered with chemotaxis induced by macrophage inflammatory protein, 1β, and at a concentration of 1.67 nM it almost completely inhibited infection by human immunodeficiency virus type 1 (HIV-1) R5, but not by HIV-1 X4, as observed by use of a new phenotypic assay for drug susceptibility of HIV-1 using the CCR5-expressing HeLa CD4
+
cell clone 1-10 (MAGIC-5). Furthermore, cDDR5-MAP suppressed infection by HIV-1 R5 at relatively high concentrations (50 to 400 μM) in a dose-dependent manner but did not suppress infection by HIV-1 X4. Taken together, these results indicate that the antibody is conformation specific and recognizes the conformation-specific domain of the UPA of ECL2. Moreover, both the antibody and its immunogen, the cDDR5-MAP conjugate, may be useful in developing a new candidate vaccine for HIV therapy.
Two Point Mutations Produce Infectious Retrovirus Bearing a Green Fluorescent Protein-SU Fusion Protein
Authors: Kizhatil Krishnakumar , Gromley Adam , Albritton Lorraine M.
Published: July 2002

Two second-site mutations in Moloney murine leukemia virus envelope surface protein (SU) were previously shown to rescue infection of two different SU mutants, a fusion-defective point mutant and a fusion-defective modified SU that exhibits weak subunit association. We report here that they also rescue infection of a third defective SU, one modified by insertion of the green fluorescent protein (GFP) between serine 6 and proline 7. GFP-SU assembled into virions and showed a strong association with the transmembrane protein (TM). However, these virions were noninfectious. GFP-SU expression was not maintained within cells, suggesting that the protein was toxic. Addition of the second-site mutations rendered the GFP-SU virus infectious and resulted in prolonged expression of the modified envelope protein. This virus showed a slight reduction in receptor binding but not in envelope protein processing, suggesting that addition of the GFP sequences results in subtle structural changes. Extrapolating these data, we see that the fundamental problem with the GFP-SU envelope protein appears to be a folding problem, suggesting that the second-site mutations rescue GFP-SU primarily by a mechanism that involves stabilizing the envelope protein structure.
Identification of Sequences in Brome Mosaic Virus Replicase Protein 1a That Mediate Association with Endoplasmic Reticulum Membranes
Authors: den Boon Johan A. , Chen Jianbo , Ahlquist Paul
Published: July 2002

RNA replication of all positive-strand RNA viruses is closely associated with intracellular membranes. Brome mosaic virus (BMV) RNA replication occurs on the perinuclear region of the endoplasmic reticulum (ER), both in its natural plant host and in the yeast
Saccharomyces cerevisiae
. The only viral component in the BMV RNA replication complex that localizes independently to the ER is 1a, a multifunctional protein with an N-terminal RNA capping domain and a C-terminal helicase-like domain. The other viral replication components, the RNA polymerase-like protein 2a and the RNA template, depend on 1a for recruitment to the ER. We show here that, in membrane extracts, 1a is fully susceptible to proteolytic digestion in the absence of detergent and thus, a finding consistent with its roles in RNA replication, is wholly or predominantly on the cytoplasmic face of the ER with no detectable lumenal protrusions. Nevertheless, 1a association with membranes is resistant to high-salt and high-pH treatments that release most peripheral membrane proteins. Membrane flotation gradient analysis of 1a deletion variants and 1a segments fused to green fluorescent protein (GFP) showed that sequences in the N-terminal RNA capping module of 1a mediate membrane association. In particular, a region C-terminal to the core methyltransferase homology was sufficient for high-affinity ER membrane association. Confocal immunofluorescence microscopy showed that even though these determinants mediate ER localization, they fail to localize GFP to the narrow region of the perinuclear ER, where full-length 1a normally resides. Instead, they mediate a more globular or convoluted distribution of ER markers. Thus, additional sequences in 1a that are distinct from the primary membrane association determinants contribute to 1a's normal subcellular distribution, possibly through effects on 1a conformation, orientation, or multimerization on the membrane.