bluuebunny/crossref_metadata_2025 huggingface dataset:
Transforming Growth Factor Beta 1 Stimulates Expression of the Epstein-Barr Virus BZLF1 Immediate-Early Gene Product ZEBRA by an Indirect Mechanism Which Requires the MAPK Kinase Pathway
Authors: Fahmi Hassan , Cochet Chantal , Hmama Zakariae , Opolon Paule , Joab Irene
Published: July 2002

Disruption of Epstein-Barr virus (EBV) latency is mediated by ZEBRA, the protein product of the immediate-early EBV gene, BZLF1. In vitro, phorbol 12-myristate 13-acetate (PMA), a potent activator of protein kinase C (PKC), induces reactivation of EBV. However, the physiological stimuli responsible for the disruption of viral latency are not well characterized. Transforming growth factor beta 1 (TGF-β1) has also been shown to trigger the reactivation of EBV in Burkitt lymphoma cell lines; however, the effect of TGF-β1 on ZEBRA expression has not been reported. To further understand this phenomenon, we have investigated the effect of TGF-β1 on ZEBRA expression. Our results indicate that the treatment of different EBV-positive Burkitt's lymphoma cell lines with TGF-β1 induces a time-dependent activation of BZLF1 transcription with a corresponding increase in the production of the protein ZEBRA. TGF-β1 has been shown to exert its effects through a wide range of intracellular routes; in the present study, we have explored these pathways. Transient expression of Smad proteins on their own had no effect on ZEBRA expression. A specific inhibitor of p38 mitogen-activated protein kinase (MAPK), SB203580, did not affect TGF-β1-induced ZEBRA expression, whereas treatment with the MAPK/ERK kinase inhibitors, PD98059 and U0126, dramatically decreased this induction. This suggests that TGF-β1 effect on BZLF1 expression requires the MAPK pathway. However, in Raji and B95-8 cells additional routes can be used, as (i) the inhibition of ZEBRA induction by PD98059 or U0126 was incomplete, whereas these inhibitors completely abolished PMA-induced ZEBRA expression, (ii) TGF-β1 induction of ZEBRA expression occurs in PKC-depleted cells, (iii) in Raji and in B95-8 cells, the effect of TGF-β1 and PMA are additive. Transient transfection of the EBV-negative B-cell line DG75 with a BZLF1 promoter-fusion construct (Zp-CAT) showed that under conditions where the BZLF1 promoter is activated by PMA treatment, TGF-β1 had no significant effect on the expression of the chloramphenicol acetyltransferase gene. Furthermore, TGF-β1 induction of BZLF1 transcripts is dependent on de novo protein synthesis, which suggests that TGF-β1 induces BZLF1 expression by an indirect mechanism.
Norwalk Virus Open Reading Frame 3 Encodes a Minor Structural Protein
Authors: Glass Pamela J. , White Laura J. , Ball Judith M. , Leparc-Goffart Isabelle , Hardy Michele E. , Estes Mary K.
Published: July 2002

Norwalk virus(NV) is a causative agent of acute epidemic nonbacterial gastroenteritis in humans. The inability to cultivate NV has required the use of molecular techniques to examine the genome organization and functions of the viral proteins. The function of the NV protein encoded by open reading frame 3 (ORF 3) has been unknown. In this paper, we report the characterization of the NV ORF 3 protein expressed in a cell-free translation system and in insect cells and show its association with recombinant virus-like particles (VLPs) and NV virions. Expression of the ORF 3 coding region in rabbit reticulocyte lysates resulted in the production of a single protein with an apparent molecular weight of 23,000 (23K protein), which is not modified by N-linked glycosylation. The ORF 3 protein was expressed in insect cells by using two different baculovirus recombinants; one recombinant contained the entire 3′ end of the genome beginning with the ORF 2 coding sequences (ORFs 2+3), and the second recombinant contained ORF 3 alone. Expression from the construct containing both ORF 2 and ORF 3 resulted in the expression of a single protein (23K protein) detected by Western blot analysis with ORF 3-specific peptide antisera. However, expression from a construct containing only the ORF 3 coding sequences resulted in the production of multiple forms of the ORF 3 protein ranging in size from 23,000 to 35,000. Indirect-immunofluorescence studies using an ORF 3 peptide antiserum showed that the ORF 3 protein is localized to the cytoplasm of infected insect cells. The 23K ORF 3 protein was consistently associated with recombinant VLPs purified from the media of insect cells infected with a baculovirus recombinant containing the entire 3′ end of the NV genome. Western blot analysis of NV purified from the stools of NV-infected volunteers revealed the presence of a 35K protein as well as multiple higher-molecular-weight bands specifically recognized by an ORF 3 peptide antiserum. These results indicate that the ORF 3 protein is a minor structural protein of the virion.
Ectodomain of Coxsackievirus and Adenovirus Receptor Genetically Fused to Epidermal Growth Factor Mediates Adenovirus Targeting to Epidermal Growth Factor Receptor-Positive Cells
Authors: Dmitriev Igor , Kashentseva Elena , Rogers Buck E. , Krasnykh Victor , Curiel David T.
Published: July 2002

Human adenovirus (Ad) is extensively used for a variety of gene therapy applications. However, the utility of Ad vectors is limited due to the low efficiency of Ad-mediated gene transfer to target cells expressing marginal levels of the Ad fiber receptor. Therefore, the present generation of Ad vectors could potentially be improved by modification of Ad tropism to target the virus to specific organs and tissues. The fact that coxsackievirus and adenovirus receptor (CAR) does not play any role in virus internalization, but functions merely as the virus attachment site, suggests that the extracellular part of CAR might be utilized to block the receptor recognition site on the Ad fiber knob domain. We proposed to design bispecific fusion proteins formed by a recombinant soluble form of truncated CAR (sCAR) and a targeting ligand. In this study, we derived sCAR genetically fused with human epidermal growth factor (EGF) and investigated its ability to target Ad infection to the EGF receptor (EGFR) overexpressed on cancer cell lines. We have demonstrated that sCAR-EGF protein is capable of binding to Ad virions and directing them to EGFR, thereby achieving targeted delivery of reporter gene. These results show that sCAR-EGF protein possesses the ability to effectively retarget Ad via a non-CAR pathway, with enhancement of gene transfer efficiency.
Reovirus-Induced Apoptosis Is Mediated by TRAIL
Authors: Clarke Penny , Meintzer Suzanne M. , Gibson Spencer , Widmann Christian , Garrington Timothy P. , Johnson Gary L. , Tyler Kenneth L.
Published: July 2002

Members of the tumor necrosis factor (TNF) receptor superfamily and their activating ligands transmit apoptotic signals in a variety of systems. We now show that the binding of TNF-related, apoptosis-inducing ligand (TRAIL) to its cellular receptors DR5 (TRAILR2) and DR4 (TRAILR1) mediates reovirus-induced apoptosis. Anti-TRAIL antibody and soluble TRAIL receptors block reovirus-induced apoptosis by preventing TRAIL-receptor binding. In addition, reovirus induces both TRAIL release and an increase in the expression of DR5 and DR4 in infected cells. Reovirus-induced apoptosis is also blocked following inhibition of the death receptor-associated, apoptosis-inducing molecules FADD (for FAS-associated death domain) and caspase 8. We propose that reovirus infection promotes apoptosis via the expression of DR5 and the release of TRAIL from infected cells. Virus-induced regulation of the TRAIL apoptotic pathway defines a novel mechanism for virus-induced apoptosis.
Bovine Respiratory Syncytial Virus Nonstructural Proteins NS1 and NS2 Cooperatively Antagonize Alpha/Beta Interferon-Induced Antiviral Response
Authors: Schlender Jörg , Bossert Birgit , Buchholz Ursula , Conzelmann Karl-Klaus
Published: July 2002

The functions of bovine respiratory syncytial virus (BRSV) nonstructural proteins NS1 and NS2 were studied by generation and analysis of recombinant BRSV carrying single and double gene deletions. Whereas in MDBK cells the lack of either or both NS genes resulted in a 5,000- to 10,000-fold reduction of virus titers, in Vero cells a moderate (10-fold) reduction was observed. Interestingly, cell culture supernatants from infected MDBK cells were able to restrain the growth of NS deletion mutants in Vero cells, suggesting the involvement of NS proteins in escape from cytokine-mediated host cell responses. The responsible factors in MDBK supernatants were identified as type I interferons by neutralization of the inhibitory effect with antibodies blocking the alpha interferon (IFN-α) receptor. Treatment of cells with recombinant universal IFN-α A/D or IFN-β revealed severe inhibition of single and double deletion mutants, whereas growth of full-length BRSV was not greatly affected. Surprisingly, all NS deletion mutants were equally repressed, indicating an obligatory cooperation of NS1 and NS2 in antagonizing IFN-mediated antiviral mechanisms. To verify this finding, we generated recombinant rabies virus (rRV) expressing either NS1 or NS2 and determined their IFN sensitivity. In cells coinfected with NS1- and NS2-expressing rRVs, virus replication was resistant to doses of IFN which caused a 1,000-fold reduction of replication in cells infected with wild-type RV or with each of the NS-expressing rRVs alone. Thus, BRSV NS proteins have the potential to cooperatively protect an unrelated virus from IFN-α/β mediated antiviral responses. Interestingly, BRSV NS proteins provided a more pronounced resistance to IFN in the bovine cell line MDBK than in cell lines of other origins, suggesting adaptation to host-specific antiviral responses. The findings described have a major impact on the design of live recombinant BRSV and HRSV vaccines.
Sensitivity of Human Immunodeficiency Virus Type 1 to the Fusion Inhibitor T-20 Is Modulated by Coreceptor Specificity Defined by the V3 Loop of gp120
Authors: Derdeyn Cynthia A. , Decker Julie M. , Sfakianos Jeffrey N. , Wu Xiaoyun , O'Brien William A. , Ratner Lee , Kappes John C. , Shaw George M. , Hunter Eric
Published: July 2002

T-20 is a synthetic peptide that potently inhibits replication of human immunodeficiency virus type 1 by interfering with the transition of the transmembrane protein, gp41, to a fusion active state following interactions of the surface glycoprotein, gp120, with CD4 and coreceptor molecules displayed on the target cell surface. Although T-20 is postulated to interact with an N-terminal heptad repeat within gp41 in a
trans
-dominant manner, we show here that sensitivity to T-20 is strongly influenced by coreceptor specificity. When 14 T-20-naive primary isolates were analyzed for sensitivity to T-20, the mean 50% inhibitory concentration (IC
50
) for isolates that utilize CCR5 for entry (R5 viruses) was 0.8 log
10
higher than the mean IC
50
for CXCR4 (X4) isolates (
P
= 0.0055). Using NL4.3-based envelope chimeras that contain combinations of envelope sequences derived from R5 and X4 viruses, we found that determinants of coreceptor specificity contained within the gp120 V3 loop modulate this sensitivity to T-20. The IC
50
for all chimeric envelope viruses containing R5 V3 sequences was 0.6 to 0.8 log
10
higher than that for viruses containing X4 V3 sequences. In addition, we confirmed that the N-terminal heptad repeat of gp41 determines the baseline sensitivity to T-20 and that the IC
50
for viruses containing GIV at amino acid residues 36 to 38 was 1.0 log
10
lower than the IC
50
for viruses containing a G-to-D substitution. The results of this study show that gp120-coreceptor interactions and the gp41 N-terminal heptad repeat independently contribute to sensitivity to T-20. These results have important implications for the therapeutic uses of T-20 as well as for unraveling the complex mechanisms of virus fusion and entry.
Relationship between Retroviral DNA Integration and Gene Expression
Authors: Weidhaas Joanne Barnes , Angelichio Elizabeth Lloyd , Fenner Sabine , Coffin John M.
Published: July 2002

Although retroviruses can integrate their DNA into a large number of sites in the host genome, factors controlling the specificity of integration remain controversial and poorly understood. To assess the effects of transcriptional activity on integration in vivo, we created quail cell clones containing a construct with a minigene cassette, whose expression is controlled by the papilloma virus E2 protein. From these clones we derived transcriptionally active subclones expressing the wild-type E2 protein and transcriptionally silent subclones expressing a mutant E2 protein that binds its target DNA but is unable to activate transcription. By infecting both clones and subclones with avian leukosis virus and using a PCR-based assay to determine viral DNA integration patterns, we were able to assess the effects of both protein binding and transcriptional activity on retroviral DNA integration. Contrary to the hypothesis that transcriptional activity enhances integration, we found an overall decrease in integration into our gene cassette in subclones expressing the wild-type E2 protein. We also found a decrease in integration into our gene cassette in subclones expressing the mutant E2 protein, but only into the protein binding region. Based on these findings, we propose that transcriptionally active DNA is not a preferred target for retroviral integration and that transcriptional activity may in fact be correlated with a decrease in integration.
Monoclonal Antibodies against the Adeno-Associated Virus Type 2 (AAV-2) Capsid: Epitope Mapping and Identification of Capsid Domains Involved in AAV-2–Cell Interaction and Neutralization of AAV-2 Infection
Authors: Wobus Christiane E. , Hügle-Dörr Barbara , Girod Anne , Petersen Gabriele , Hallek Michael , Kleinschmidt Jürgen A.
Published: July 2002

The previously characterized monoclonal antibodies (MAbs) A1, A69, B1, and A20 are directed against assembled or nonassembled adeno-associated virus type 2 (AAV-2) capsid proteins (A. Wistuba, A. Kern, S. Weger, D. Grimm, and J. A. Kleinschmidt, J. Virol. 71:1341–1352, 1997). Here we describe the linear epitopes of A1, A69, and B1 which reside in VP1, VP2, and VP3, respectively, using gene fragment phage display library, peptide scan, and peptide competition experiments. In addition, MAbs A20, C24-B, C37-B, and D3 directed against conformational epitopes on AAV-2 capsids were characterized. Epitope sequences on the capsid surface were identified by enzyme-linked immunoabsorbent assay using AAV-2 mutants and AAV serotypes, peptide scan, and peptide competition experiments. A20 neutralizes infection following receptor attachment by binding an epitope formed during AAV-2 capsid assembly. The newly isolated antibodies C24-B and C37-B inhibit AAV-2 binding to cells, probably by recognizing a loop region involved in binding of AAV-2 to the cellular receptor. In contrast, binding of D3 to a loop near the predicted threefold spike does not neutralize AAV-2 infection. The identified antigenic regions on the AAV-2 capsid surface are discussed with respect to their possible roles in different steps of the viral life cycle.
pRB-Dependent, J Domain-Independent Function of Simian Virus 40 Large T Antigen in Override of p53 Growth Suppression
Authors: Gjoerup Ole , Chao Herta , DeCaprio James A. , Roberts Thomas M.
Published: July 2002

Simian virus 40 (SV40) large T antigen (LT) can immortalize and transform many cell types. These activities are attributed in large part to the binding and functional inactivation by LT of two major tumor suppressors: p53 and the retinoblastoma protein, pRB. Most effects of LT on pRB have been shown to additionally require an intact J domain, which mediates binding to Hsc70. We show here that the J domain is not required for p53 override in full-length LT. Although LT binds p53, it was shown previously that overcoming a p53-induced cell cycle arrest requires binding to pRB family members (R. S. Quartin et al., J. Virol. 68:1334–1341). We demonstrate that an LT mutant defective for pRB family member binding (K1) can be complemented for efficient override of p53 arrest by a construct encoding the first 135 amino acids of LT with a J domain-inactivating mutation, H42Q. Hence, complementation does not require the J domain, and pRB binding by LT is important for more than dissociating pRB-E2F complexes, which is J dependent. In accordance with this notion, LT alleviates pRB small-pocket-mediated transcriptional repression independently of the J domain. The LT K1 mutant can also be complemented for p53 override by small t antigen (st) in a manner independent of its J domain. Our observations underscore the importance of multiple SV40 functions, two in LT and one in st, that act cooperatively to counteract p53 growth suppression.
The Leader Polypeptide of Theiler's Murine Encephalomyelitis Virus Is Required for the Assembly of Virions in Mouse L Cells
Authors: Badshah Cyrus , Calenoff Miriam A. , Rundell Kathleen
Published: July 2002

Deletion of the entire leader polypeptide of the GDVII strain of Theiler's murine encephalomyelitis virus (TMEV) results in the production of an attenuated virus that grows in baby hamster kidney (BHK) cells but cannot grow at all in mouse L-929 cells. This study examined the reasons for the failure of
dl
-L, the GDVII variant that lacks the leader polypeptide, to grow in mouse cells. At low multiplicities of infection, it was difficult to detect any viral proteins in mouse cells. However, levels of positive- and negative-strand RNA molecules were only moderately reduced in these infections. Viral RNA showed no major defect in translatability, as the mutant viral RNA was nearly as efficient as that of the wild-type (WT) virus in directing protein synthesis in vitro in assays using extracts prepared from mouse L cells. Viral protein synthesis was detected in
dl
-L-infected mouse cells as multiplicities of infection were increased and approached the levels observed in WT infections. Despite this, there was a total lack of virus production in high-multiplicity infections, and this was found to correlate with the failure of viral proteins and early virion precursors to assemble into virions in mouse cells. Thus, the inability of
dl
-L to grow in mouse cells reflects complex effects on various stages of the virus infection but is primarily a defect in virus assembly.
An In Vitro Rapid-Turnover Assay for Human Immunodeficiency Virus Type 1 Replication Selects for Cell-to-Cell Spread of Virus
Authors: Gummuluru Suryaram , Kinsey C. Mathew , Emerman Michael
Published: July 2002

We have developed a rapid-turnover culture system where the life span of a human immunodeficiency virus type 1-infected cell is controlled by periodic addition of a cytotoxic agent, mitomycin C. These mitomycin C-exposed cells are cocultured with a constant number of uninfected cells as new targets for the virus. Passage of the virus-infected cells under these conditions led to the emergence of a viral variant that was able to replicate efficiently in this culture system. After biologic and molecular cloning, we were able to identify a single frameshift mutation in thevpuopen reading frame that was sufficient for growth of the mutant virus in the rapid-turnover assay. This virus variant spread more efficiently by cell-to-cell transfer than the parental virus did. Electron micrographs of cells infected with the Δvpuvirus revealed a large number of mature viral capsids attached to the plasma membrane. The presence of these mature virus particles on the cell surface led to enhanced fusion and formation of giant syncytia with uninfected cells. Enhanced cell-to-cell transfer of the Δvpuvirus provides an explanation for the survival of this mutant virus in the rapid-turnover culture system. The in vitro rapid-turnover culture system is a good representation of the in vivo turnover kinetics of infected cells and their continual replacement by host lymphopoietic mechanisms.
A Structured RNA Motif Is Involved in Correct Placement of the tRNA
3
Lys
Primer onto the Human Immunodeficiency Virus Genome
Authors: Beerens Nancy , Klaver Bep , Berkhout Ben
Published: July 2002

Human immunodeficiency virus type 1 (HIV-1) reverse transcription is primed by the cellular tRNA
3
Lys
molecule that binds with its 3′-terminal 18 nucleotides to the fully complementary primer-binding site (PBS) on the viral RNA genome. Besides this complementarity, annealing of the primer may be stimulated by additional base-pairing interactions between other parts of the tRNA molecule and viral sequences flanking the PBS. According to the RNA secondary structure model of the HIV-1 leader region, part of the PBS sequence is involved in base pairing to form a small stem-loop structure, termed the U5-PBS hairpin. This hairpin may be involved in the process of reverse transcription. To study the role of the U5-PBS hairpin in the viral replication cycle, we introduced mutations in the U5 region that affect the stability of this structured RNA motif. Stabilization and destabilization of the hairpin significantly inhibited virus replication. Upon prolonged culturing of the virus mutant with the stabilized hairpin, revertant viruses were obtained with additional mutations that restore the thermodynamic stability of the U5-PBS hairpin. The thermodynamic stability of the U5-PBS hairpin apparently has to stay within narrow limits for efficient HIV-1 replication. Transient transfection experiments demonstrated that transcription of the proviral genomes, translation of the viral mRNAs, and assembly of the virions with a normal RNA content is not affected by the mutations within the U5-PBS hairpin. We show that stabilization of the hairpin reduced the amount of tRNA primer that is annealed to the PBS. Destabilization of the hairpin did not affect tRNA annealing, but the viral RNA-tRNA complex was less stable. These results suggest that the U5-PBS hairpin is involved in correct placement of the tRNA primer on the viral genome. The analysis of virus mutants and revertants and the RNA structure probing experiments presented in this study are consistent with the existence of the U5-PBS hairpin as predicted in the RNA secondary structure model.
A Previously Unrecognized H-2Db-Restricted Peptide Prominent in the Primary Influenza A Virus-Specific CD8+T-Cell Response Is Much Less Apparent following Secondary Challenge
Authors: Belz Gabrielle T. , Xie Weidong , Altman John D. , Doherty Peter C.
Published: July 2002

Respiratory challenge ofH-2bmice with an H3N2 influenza A virus causes an acute, transient pneumonitis characterized by the massive infiltration of CD8+T lymphocytes. The inflammatory process monitored by quantitative analysis of lymphocyte populations recovered by bronchoalveolar lavage is greatly enhanced by prior exposure to an H1N1 virus, with the recall of cross-reactive CD8+-T-cell memory leading to more rapid clearance of the infection from the lungs. The predominant epitope recognized by the influenza virus-specific CD8+set has long been thought to be a nucleoprotein (NP366–374) presented by H-2Db(DbNP366). This continues to be true for the secondary H3N2→H1N1 challenge but can no longer be considered the case for the primary response to either virus. Quantitative analysis based on intracellular staining for gamma interferon has shown that the polymerase 2 protein (PA224–233) provides a previously undetected epitope (DbPA224) that is at least as prominent as DbNP366during the first 10 days following primary exposure to either the H3N2 or H1N1 virus. The response to DbNP366seems to continue for longer, even when infectious virus can no longer be detected, but there is no obvious difference in the prevalence of memory T cells specific for DbNP366and DbPA224. The generalization that the magnitude of the functional memory T-cell pool is a direct consequence of the clonal burst size during the primary response may no longer be useful. Previous CD8+-T-cell immunodominance heirarchies defined largely by cytotoxic T-lymphocyte assays may need to be revised.
Functional Differences between the Long Terminal Repeat Transcriptional Promoters of Human Immunodeficiency Virus Type 1 Subtypes A through G
Authors: Jeeninga Rienk E. , Hoogenkamp Maarten , Armand-Ugon Mercedes , de Baar Michel , Verhoef Koen , Berkhout Ben
Published: July 2002

The current human immunodeficiency virus type 1 (HIV-1) shows an increasing number of distinct viral subtypes, as well as viruses that are recombinants of at least two subtypes. Although no biological differences have been described so far for viruses that belong to different subtypes, there is considerable sequence variation between the different HIV-1 subtypes. The HIV-1 long terminal repeat (LTR) encodes the transcriptional promoter, and the LTR of subtypes A through G was cloned and analyzed to test if there are subtype-specific differences in gene expression. Sequence analysis demonstrated a unique LTR enhancer-promoter configuration for each subtype. Transcription assays with luciferase reporter constructs showed that all subtype LTRs are functional promoters with a low basal transcriptional activity and a high activity in the presence of the viral Tat transcriptional activator protein. All subtype LTRs responded equally well to the Tat
trans
activator protein of subtype B. This result suggests that there are no major differences in the mechanism of Tat-mediated
trans
activation among the subtypes. Nevertheless, subtype-specific differences in the activity of the basal LTR promoter were measured in different cell types. Furthermore, we measured a differential response to tumor necrosis factor alpha treatment, and the induction level correlated with the number of NF-κB sites in the respective LTRs, which varies from one (subtype E) to three (subtype C). In general, subtype E was found to encode the most potent LTR, and we therefore inserted the core promoter elements of subtype E in the infectious molecular clone of the LAI isolate (subtype B). This recombinant LAI-E virus exhibited a profound replication advantage compared with the original LAI virus in the SupT1 T-cell line, indicating that subtle differences in LTR promoter activity can have a significant impact on viral replication kinetics. These results suggest that there may be considerable biological differences among the HIV-1 subtypes.
Variation in Adenovirus Transgene Expression between BALB/c and C57BL/6 Mice Is Associated with Differences in Interleukin-12 and Gamma Interferon Production and NK Cell Activation
Authors: Peng YuFeng , Falck-Pedersen Erik , Elkon Keith B.
Published: July 2002

The innate immune response against replication-defective adenoviruses (Ad) is poorly defined. We and others have previously observed striking differences in the rate at which the Ad vector itself or the virus encoding a variety of transgenes is eliminated in different mouse strains. Here, we report that Ad infection of BALB/ mice is associated with sixfold-higher levels of serum alanine aminotransferase and that Ad transgenes induce two- to threefold-higher levels of intrahepatic NK cells and NK activity compared to C57BL/6 mice. The increase in NK activation in BALB/c mice was associated with ∼4-fold higher level of mRNA expression of a newly described NKG2 receptor activator, H-60, as well as increased expression of interleukin-12 and gamma interferon mRNAs in BALB/c mice compared to C57BL/6 mice. NK depletion in BALB/c mice or defective NK function in C3H beige mice extended transgene expression compared to their appropriate controls, and attenuation of NK together with CD8 T-cell function had a synergistic effect. These findings indicate that there are intrinsic differences in the innate immune responses of different mouse strains to Ad and Ad transgenes and that NK cells, in cooperation with CD8 T cells, play a pivotal role in the early extinction of transgene expression in BALB/c mice.
Transcription Program of Human Herpesvirus 8 (Kaposi's Sarcoma-Associated Herpesvirus)
Authors: Paulose-Murphy Mini , Ha Nguyen-Khoi , Xiang Chunsheng , Chen Yidong , Gillim Laura , Yarchoan Robert , Meltzer Paul , Bittner Michael , Trent Jeffrey , Zeichner Steven
Published: July 2002

Human herpesvirus 8 (HHV-8), a gammaherpesvirus implicated in Kaposi's sarcoma, primary effusion lymphoma, and Castleman's disease, encodes several pathogenically important cellular homologs. To define the HHV-8 transcription program, RNA obtained from latently infected body cavity-based lymphoma 1 cells induced to undergo lytic replication was used to query a custom HHV-8 DNA microarray containing nearly every known viral open reading frame. The patterns of viral gene expression offer insights into the replication and pathogenic strategies of HHV-8.
Morbilliviruses Use Signaling Lymphocyte Activation Molecules (CD150) as Cellular Receptors
Authors: Tatsuo Hironobu , Ono Nobuyuki , Yanagi Yusuke
Published: July 2002

Morbilliviruses comprise measles virus, canine distemper virus, rinderpest virus, and several other viruses that cause devastating human and animal diseases accompanied by severe immunosuppression and lymphopenia. Recently, we have shown that human signaling lymphocyte activation molecule (SLAM) is a cellular receptor for measles virus. In this study, we examined whether canine distemper and rinderpest viruses also use canine and bovine SLAMs, respectively, as cellular receptors. The Onderstepoort vaccine strain and two B95a (marmoset B cell line)-isolated strains of canine distemper virus caused extensive cytopathic effects in normally resistant CHO (Chinese hamster ovary) cells after expression of canine SLAM. The Ako vaccine strain of rinderpest virus produced strong cytopathic effects in bovine SLAM-expressing CHO cells. The data on entry with vesicular stomatitis virus pseudotypes bearing measles, canine distemper, or rinderpest virus envelope proteins were consistent with development of cytopathic effects in SLAM-expressing CHO cell clones after infection with the respective viruses, confirming that SLAM acts at the virus entry step (as a cellular receptor). Furthermore, most measles, canine distemper, and rinderpest virus strains examined could any use of the human, canine, and bovine SLAMs to infect cells. Our findings suggest that the use of SLAM as a cellular receptor may be a property common to most, if not all, morbilliviruses and explain the lymphotropism and immunosuppressive nature of morbilliviruses.
Functional Analysis of the Disulfide-Bonded Loop/Chain Reversal Region of Human Immunodeficiency Virus Type 1 gp41 Reveals a Critical Role in gp120-gp41 Association
Authors: Maerz Anne L. , Drummer Heidi E. , Wilson Kirilee A. , Poumbourios Pantelis
Published: July 2002

Human immunodeficiency virus type 1 (HIV-1) entry into cells is mediated by the surface-exposed envelope protein (SU) gp120, which binds to cellular CD4 and chemokine receptors, triggering the membrane fusion activity of the transmembrane (TM) protein gp41. The core of gp41 comprises an N-terminal triple-stranded coiled coil and an antiparallel C-terminal helical segment which is packed against the exterior of the coiled coil and is thought to correspond to a fusion-activated conformation. The available gp41 crystal structures lack the conserved disulfide-bonded loop region which, in human T-lymphotropic virus type 1 (HTLV-1) and murine leukemia virus TM proteins, mediates a chain reversal, connecting the antiparallel N- and C-terminal regions. Mutations in the HTLV-1 TM protein gp21 disulfide-bonded loop/chain reversal region adversely affected fusion activity without abolishing SU-TM association (A. L. Maerz, R. J. Center, B. E. Kemp, B. Kobe, and P. Poumbourios, J. Virol. 74:6614–6621, 2000). We now report that in contrast to our findings with HTLV-1, conservative substitutions in the HIV-1 gp41 disulfide-bonded loop/chain reversal region abolished association with gp120. While the mutations affecting gp120-gp41 association also affected cell-cell fusion activity, HIV-1 glycoprotein maturation appeared normal. The mutant glycoproteins were processed, expressed at the cell surface, and efficiently immunoprecipitated by conformation-dependent monoclonal antibodies. The gp120 association site includes aromatic and hydrophobic residues on either side of the gp41 disulfide-bonded loop and a basic residue within the loop. The HIV-1 gp41 disulfide-bonded loop/chain reversal region is a critical gp120 contact site; therefore, it is also likely to play a central role in fusion activation by linking CD4 plus chemokine receptor-induced conformational changes in gp120 to gp41 fusogenicity. These gp120 contact residues are present in diverse primate lentiviruses, suggesting conservation of function.
Pocket Protein p130/Rb2 Is Required for Efficient Herpes Simplex Virus Type 1 Gene Expression and Viral Replication
Authors: Ehmann Ginger L. , Burnett Heather A. , Bachenheimer Steven L.
Published: July 2002

We have reported previously that herpes simplex virus type 1 (HSV-1) infection disrupts normal progression of the mammalian cell cycle, causing cells to enter a G
1
-like state. Infected cells were characterized by a decline in cyclin-dependent kinase 2 (CDK2) activities, loss of hyperphosphorylated retinoblastoma protein (pRb), accumulation of E2F-pocket protein complexes, and failure to initiate cellular DNA replication. In the present study, we investigated the role of the pocket proteins pRb, p107, and p130 in HSV-1-dependent cell cycle inhibition and cyclin kinase regulation by infecting murine 3T3 cells derived from wild-type (WT) mouse embryos or embryos with deletions of pRb (pRb
−/−
), p107 (p107
−/−
), p130 (p130
−/−
), or both p130 and p107 (p130
−/−
/p107
−/−
). With respect to CDK2 inhibition, viral protein accumulation, viral DNA replication, and progeny virus yield, WT, pRb
−/−
, and p107
−/−
cells were essentially identical. In contrast, after infection of p130
−/−
cells, we observed no inhibition of CDK2 activity, a 5- to 6-h delay in accumulation of viral proteins, an impaired ability to form viral DNA replication compartments, and reduced viral DNA synthesis. As a result, progeny virus yield was reduced 2 logs compared to that in WT cells. Notably, p130
−/−
/p107
−/−
double-knockout cells had a virus replication phenotype intermediate between those of the p107
−/−
and p130
−/−
cells. We conclude from these studies that p130 is a key factor in regulating aspects of cell cycle progression, as well as the timely expression of viral genes and replication of viral DNA.
Inhibition of Human Immunodeficiency Virus Type 1 Transcription by Chemical Cyclin-Dependent Kinase Inhibitors
Authors: Wang Dai , de la Fuente Cynthia , Deng Longwen , Wang Lai , Zilberman Irene , Eadie Carolyn , Healey Marlene , Stein Dana , Denny Thomas , Harrison Lawrence E. , Meijer Laurent , Kashanchi Fatah
Published: July 2002

Cyclin-dependent kinases (cdk's) have recently been suggested to regulate human immunodeficiency virus type 1 (HIV-1) transcription. Previously, we have shown that expression of one cdk inhibitor, p21/Waf1, is abrogated in HIV-1 latently infected cells. Based on this result, we investigated the transcription of HIV-1 in the presence of chemical drugs that specifically inhibited cdk activity and functionally mimicked p21/Waf1 activity. HIV-1 production in virally integrated lymphocytic and monocytic cell lines, such as ACH2, 8E5, and U1, as well as activated peripheral blood mononuclear cells infected with syncytium-inducing (SI) or non-syncytium-inducing (NSI) HIV-1 strains, were all inhibited by Roscovitine, a purine derivative that reversibly competes for the ATP binding site present in cdk's. The decrease in viral progeny in the HIV-1-infected cells was correlated with a decrease in the transcription of HIV-1 RNAs in cells treated with Roscovitine and not with the non-cdk general cell cycle inhibitors, such as hydroxyurea (G1/S blocker) or nocodazole (M-phase blocker). Cyclin A- and E-associated histone H1 kinases, as well as cdk 7 and 9 activities, were all inhibited in the presence of Roscovitine. The 50% inhibitory concentration of Roscovitine on cdk's 9 and 7 was determined to be ∼0.6 μM. Roscovitine could selectively sensitize HIV-1-infected cells to apoptosis at concentrations that did not impede the growth and proliferation of uninfected cells. Apoptosis induced by Roscovitine was found in both latent and activated infected cells, as evident by Annexin V staining and the cleavage of the PARP protein by caspase-3. More importantly, contrary to many apoptosis-inducing agents, where the apoptosis of HIV-1-infected cells accompanies production and release of infectious HIV-1 viral particles, Roscovitine treatment selectively killed HIV-1-infected cells without virion release. Collectively, our data suggest that cdk's are required for efficient HIV-1 transcription and, therefore, we propose specific cdk inhibitors as potential antiviral agents in the treatment of AIDS.
Centrosome Abnormalities and Genomic Instability by Episomal Expression of Human Papillomavirus Type 16 in Raft Cultures of Human Keratinocytes
Authors: Duensing Stefan , Duensing Anette , Flores Elsa R. , Do Anh , Lambert Paul F. , Münger Karl
Published: July 2002

Primary human keratinocytes with ectopic expression of high-risk human papillomavirus (HPV) E6 and E7 oncoproteins display abnormal centrosome numbers, multipolar mitoses, and aneusomy. However, it has not been explored whether these abnormalities can occur in cells containing HPV episomes where E6 and E7 expression is under viral transcriptional control. Here, we demonstrate that centrosome abnormalities and genomic instability occur in organotypic raft cultures of human keratinocytes with episomal HPV-16 even at low copy numbers. We conclude that HPV-16 DNA, when maintained as an episome, can disturb centrosome homeostasis and subvert genomic integrity of the host cell during early stages of the viral infection.
Construction of an Infectious cDNA Clone of Aichi Virus (a New Member of the Family
Picornaviridae
) and Mutational Analysis of a Stem-Loop Structure at the 5′ End of the Genome
Authors: Sasaki Jun , Kusuhara Yasuhiro , Maeno Yoshimasa , Kobayashi Nobumichi , Yamashita Teruo , Sakae Kenji , Takeda Naokazu , Taniguchi Koki
Published: July 2002

Aichi virus
is the type species of a new genus,
Kobuvirus
, of the family
Picornaviridae
. In this study, we constructed a full-length cDNA clone of Aichi virus whose in vitro transcripts were infectious to Vero cells. During construction of the infectious cDNA clone, a novel sequence of 32 nucleotides was identified at the 5′ end of the genome. Computer-assisted prediction of the secondary structure of the 5′ end of the genome, including the novel sequence, suggested the formation of a stable stem-loop structure consisting of 42 nucleotides. The function of this stem-loop in virus replication was investigated using various site-directed mutants derived from the infectious cDNA clone. Our data indicated that correct folding of the stem-loop at the 5′ end of the positive strand, but not at the 3′ end of the negative strand, is critical for viral RNA replication. The primary sequence in the lower part of the stem was also suggested to be crucial for RNA replication. In contrast, nucleotide changes in the loop segment did not so severely reduce the efficiency of virus replication. A double mutant, in which both nucleotide stretches of the middle part of the stem were replaced by their complementary nucleotides, had efficient RNA replication and translation abilities but was unable to produce viruses. These results indicate that the stem-loop at the 5′ end of the Aichi virus genome is an element involved in both viral RNA replication and production of infectious virus particles.
Expression of Immunoregulatory Cytokines by Recombinant Coxsackievirus B3 Variants Confers Protection against Virus-Caused Myocarditis
Authors: Henke Andreas , Zell Roland , Ehrlich Gunter , Stelzner Axel
Published: July 2002

Clinical and laboratory investigations have demonstrated the involvement of viruses and bacteria as potential causative agents in cardiovascular disease and have specifically found coxsackievirus B3 (CVB3) to be a leading cause. Experimental data indicate that cytokines are involved in controlling CVB3 replication. Therefore, recombinant CVB3 (CVB3rec) variants expressing the T-helper-1 (T
H
1)-specific gamma interferon (IFN-γ) or the T
H
2-specific interleukin-10 (IL-10) as well as the control virus CVB3(muIL-10), which produce only biologically inactive IL-10, were established. Coding regions of murine cytokines were cloned into the 5′ end of the CVB3 wild type (CVB3wt) open reading frame and were supplied with an artificial viral 3Cpro-specific Q-G cleavage site. Correct processing releases active cytokines, and the concentration of IFN-γ and IL-10 was analyzed by enzyme-linked immunosorbent assay and bioassays. In mice, CVB3wt was detectable in pancreas and heart tissue, causing massive destruction of the exocrine pancreas as well as myocardial inflammation and heart cell lysis. Most of the CVB3wt-infected mice revealed virus-associated symptoms, and some died within 28 days postinfection. In contrast, CVB3rec variants were present only in the pancreas of infected mice, causing local inflammation with subsequent healing. Four weeks after the first infection, surviving mice were challenged with the lethal CVB3H3 variant, causing casualties in the CVB3wt- and CVB3(muIL-10)-infected groups, whereas almost none of the CVB3(IFN-γ)- and CVB3(IL-10)-infected mice died and no pathological disorders were detectable. This study demonstrates that expression of immunoregulatory cytokines during CVB3 replication simultaneously protects mice against a lethal disease and prevents virus-caused tissue destruction.
Expression of Respiratory Syncytial Virus-Induced Chemokine Gene Networks in Lower Airway Epithelial Cells Revealed by cDNA Microarrays
Authors: Zhang Yuhong , Luxon Bruce A. , Casola Antonella , Garofalo Roberto P. , Jamaluddin Mohammad , Brasier Allan R.
Published: July 2002

TheParamyxovirusrespiratory syncytial virus (RSV) is the primary etiologic agent of serious epidemic lower respiratory tract disease in infants, immunosuppressed patients, and the elderly. Lower tract infection with RSV is characterized by a pronounced peribronchial mononuclear infiltrate, with eosinophilic and basophilic degranulation. Because RSV replication is restricted to airway epithelial cells, where RSV replication induces potent expression of chemokines, the epithelium is postulated to be a primary initiator of pulmonary inflammation in RSV infection. The spectrum of RSV-induced chemokines expressed by alveolar epithelial cells has not been fully investigated. In this report, we profile the kinetics and patterns of chemokine expression in RSV-infected lower airway epithelial cells (A549 and SAE). In A549 cells, membrane-based cDNA macroarrays and high-density oligonucleotide probe-based microarrays identified inducible expression of CC (I-309, Exodus-1, TARC, RANTES, MCP-1, MDC, and MIP-1α and -1β), CXC (GRO-α, -β, and -γ, ENA-78, interleukin-8 [IL-8], and I-TAC), and CX3C (Fractalkine) chemokines. Chemokines not previously known to be expressed by RSV-infected cells were independently confirmed by multiprobe RNase protection assay, Northern blotting, and reverse transcription-PCR. High-density microarrays performed on SAE cells confirmed a similar pattern of RSV-inducible expression of CC chemokines (Exodus-1, RANTES, and MIP-1α and -1β), CXC chemokines (I-TAC, GRO-α, -β, and -γ, and IL-8), and Fractalkine. In contrast, TARC, MCP-1, and MDC were not induced, suggesting the existence of distinct genetic responses for different types of airway-derived epithelial cells. Hierarchical clustering by agglomerative nesting and principal-component analyses were performed on A549-expressed chemokines; these analyses indicated that RSV-inducible chemokines are ordered into three related expression groups. These data profile the temporal changes in expression by RSV-infected lower airway epithelial cells of chemokines, chemotactic proteins which may be responsible for the complex cellular infiltrate in virus-induced respiratory inflammation.
Fv-4: Identification of the Defect in Env and the Mechanism of Resistance to Ecotropic Murine Leukemia Virus
Authors: Taylor Gwen M. , Gao Yi , Sanders David Avram
Published: July 2002

Mice expressing the
Fv-4
gene are resistant to infection by ecotropic murine leukemia viruses (MuLVs). The
Fv-4
gene encodes an envelope (Env) protein whose putative receptor-binding domain resembles that of ecotropic MuLV Env protein. Resistance to ecotropic MuLVs appears to result from viral interference involving binding of the endogenously expressed
Fv-4 env
-encoded protein to the ecotropic receptor, although the immune system also plays a role in resistance. The
Fv-4 env
-encoded protein is processed normally and can be incorporated into virus particles but is unable to promote viral entry. Among the many sequence variations between the transmembrane (TM) subunit of the
Fv-4 env
-encoded protein and the TM subunits of other MuLV Env proteins, there is a substitution of an arginine residue in the
Fv-4 env
-encoded protein for a glycine residue (gly-491 in Moloney MuLV Env) that is otherwise conserved in all of the other MuLVs. This residue is present in the MuLV TM fusion peptide sequence. In this study, gly-491 of Moloney MuLV Env has been replaced with other residues and a mutant Env bearing a substitution for gly-487 was also created. G491R recapitulates the
Fv-4
Env phenotype in cell culture, indicating that this substitution is sufficient for creation of an Env protein that can establish the interference-mediated resistance to ecotropic viruses produced by the
Fv-4
gene. Analysis of the mutant MuLV Env proteins also has implications for an understanding of the role of conserved glycine residues in fusion peptides and for the engineering of organismal resistance to retroviruses.
Expression of Endogenous Betaretroviruses in the Ovine Uterus: Effects of Neonatal Age, Estrous Cycle, Pregnancy, and Progesterone
Authors: Palmarini Massimo , Gray C. Allison , Carpenter Karen , Fan Hung , Bazer Fuller W. , Spencer Thomas E.
Published: July 2002

The ovine genome contains 15 to 20 copies of endogenous retroviruses (enJSRVs) highly related to the oncogenic jaagsiekte sheep retrovirus (JSRV) and enzootic nasal tumor virus. enJSRVs are highly expressed in the endometrial lumenal epithelia (LE) and glandular epithelia (GE) of the ovine uterus. The effects of neonatal age, estrous cycle, pregnancy, and progesterone on expression of enJSRVs in the ovine uterus were determined. Expression of enJSRV RNAs was absent from the uterus of ewes at birth, but enJSRV RNAs were expressed specifically in the LE and developing GE from postnatal day (PND) 7 to PND 56. In adult ewes, enJSRV RNAs were detected only in the epithelia of the uterine endometrium, as well as epithelia of the oviduct, cervix, and vagina. In cyclic ewes, endometrial enJSRV RNA abundance was lowest on day 1, increased 12-fold between days 1 and 13, and then decreased to day 15. In pregnant ewes, levels of endometrial enJSRV RNAs were high on day 11, increased to day 13, and then decreased to day 19. In day 17 and 19 conceptuses, enJSRV RNAs were also detected in binucleate cells of the trophectoderm. Immunoreactive JSRV capsid and envelope proteins were detected in the endometrial LE and GE, as well as in the binucleate cells of the conceptus. In transfection assays utilizing ovine endometrial LE cells, progesterone increased transcriptional activity of several enJSRV long terminal repeats. Collectively, these results indicate that transcription of enJSRVs in the endometrial epithelia of the ovine uterus is increased by progesterone and might support a role for enJSRVs in conceptus-endometrium interactions during the peri-implantation period and early placental morphogenesis.