bluuebunny/crossref_metadata_2025 huggingface dataset:
The Domains of Glycoprotein D Required To Block Apoptosis Depend on Whether Glycoprotein D Is Present in the Virions Carrying Herpes Simplex Virus 1 Genome Lacking the Gene Encoding the Glycoprotein
Authors: Zhou Guoying , Roizman Bernard
Published: July 2002

An earlier report showed that viruses lacking the open reading frames encoding glycoproteins J and D but containing the glycoprotein D in their envelopes (gD−/+ stocks) and viruses lacking both the open reading frames and the glycoproteins in their envelopes (gD−/− stocks) induce apoptosis (G. Zhou, V. Galvan, G. Campadelli-Fiume, and B. Roizman, J. Virol. 74:11782–11791, 2000). Furthermore, apoptosis was blocked by delivery in
trans
of genes expressing glycoprotein D or J. Whereas gD−/− stocks attach but cannot initiate productive infection, gD−/+ stocks infect cells and produce gD−/− progeny virus. The difference in the infectivity of these two stocks suggested the possibility that the requirements for blocking apoptosis may be different. To test this hypothesis, we cloned into baculoviruses the entire wild-type glycoprotein D (Bac-gD-WT), the ectodomain only (Bac-gD-A), the ectodomain and the transmembrane domain (Bac-gD-B), the ectodomain and the cytoplasmic domain without the transmembrane domain (Bac-gD-C), or the transmembrane domain and the carboxyl-terminal cytoplasmic domain (Bac-gD-D). We report the following. Apoptosis induced by gD−/+ stocks was blocked by delivery in
trans
of recombinant baculovirus Bac-gD-WT, Bac-gD-A, Bac-gD-B, or Bac-gD-C but not of Bac-gD. Apoptosis induced by gD−/− stocks was blocked by Bac-gD-WT or by a mixture of Bac-gD-B and Bac-gD-D but not by any baculoviruses expressing truncated glycoprotein D alone or by the mixture of Bac-gD-A and Bac-gD-D. We conclude that the requirements to block apoptosis induced by the two virus stocks are different. The gD ectodomain is sufficient to block apoptosis induced by gD, whereas both the ectodomain and the cytoplasmic domain are required to block apoptosis induced by gD−/− stocks. The results indicate that in the case of gD−/− stocks, the transmembrane domain is required either to deliver the ectodomain to the appropriate intracellular compartment or to form multimeric constructs which virtually reconstitute gD through the interaction of transmembrane domains.
Hybrid Vectors Based on Adeno-Associated Virus Serotypes 2 and 5 for Muscle-Directed Gene Transfer
Authors: Hildinger Markus , Auricchio Alberto , Gao Guangping , Wang Lili , Chirmule Narendra , Wilson James M.
Published: July 2002

Vectors based on hybrids consisting of adeno-associated virus types 2 (ITRs and Rep) and 5 (Cap) were evaluated for muscle-directed gene transfer (called AAV2/5). Evaluation in immune-competent mice revealed greater transduction efficacy with AAV2/5 than with AAV2 and no cross-neutralization between AAV2/5 and AAV2. Interestingly, we saw no immunologic evidence of previous exposure to AAV5 capsids in a large population of healthy human subjects.
Enhancement of Muscle Gene Delivery with Pseudotyped Adeno-Associated Virus Type 5 Correlates with Myoblast Differentiation
Authors: Duan Dongsheng , Yan Ziying , Yue Yongping , Ding Wei , Engelhardt John F.
Published: July 2002

Adeno-associated virus (AAV)-based muscle gene therapy has achieved tremendous success in numerous animal models of human diseases. Recent clinical trials with this vector have also demonstrated great promise. However, to achieve therapeutic benefit in patients, large inocula of virus will likely be necessary to establish the required level of transgene expression. For these reasons, efforts aimed at increasing the efficacy of AAV-mediated gene delivery to muscle have the potential for improving the safety and therapeutic benefit in clinical trials. In the present study, we compared the efficiency of gene delivery to mouse muscle cells for recombinant AAV type 2 (rAAV-2) and rAAV-2cap5 (AAV-2 genomes pseudo-packaged into AAV-5 capsids). Despite similar levels of transduction by these two vectors in undifferentiated myoblasts, pseudotyped rAAV-2cap5 demonstrated dramatically enhanced transduction in differentiated myocytes in vitro (>500-fold) and in skeletal muscle in vivo (>200-fold) compared to rAAV-2. Serotype-specific differences in transduction efficiency did not directly correlate with viral binding to muscle cells but rather appeared to involve endocytic or intracellular barriers to infection. Furthermore, application of this pseudotyped virus in a mouse model of Duchenne's muscular dystrophy also demonstrated significantly improved transduction efficiency. These findings should have a significant impact on improving rAAV-mediated gene therapy in muscle.
Differential Regulation of Hepatitis B Virus Gene Expression by the Sp1 Transcription Factor
Authors: Li Jie , Ou Jing-hsiung
Published: July 2002

The expression of hepatitis B virus (HBV) genes is regulated by a number of transcription factors. One such factor, Sp1, has two binding sites in the core promoter and one in its upstream regulatory element, which is also known as the ENII enhancer. In this study, we have analyzed the effects of these three Sp1 binding sites on the expression of HBV genes. Our results indicate that both Sp1 binding sites in the core promoter are important for the transcription of the core RNA and the precore RNA. Moreover, while the downstream Sp1 site (the Sp1-1 site) in the core promoter did not affect the transcription of the S gene and the X gene, the upstream Sp1 site (the Sp1-2 site) in the core promoter was found to negatively regulate the transcription of the S gene and the X gene, as removal of the latter led to enhancement of transcription of these two genes. The Sp1 binding site in the ENII enhancer (the Sp1-3 site) positively regulates the expression of all of the HBV genes, as its removal by mutation suppressed the expression of all of the HBV genes. However, the suppressive effect of the Sp1-3 site mutation on the expression of the S gene and the X gene was abolished if the two Sp1 sites in the core promoter were also mutated. These results indicate that Sp1 can serve both as a positive regulator and as a negative regulator for the expression of HBV genes. This dual activity may be important for the differential regulation of HBV gene expression.
Hemagglutinin-Neuraminidase-Independent Fusion Activity of Simian Virus 5 Fusion (F) Protein: Difference in Conformation between Fusogenic and Nonfusogenic F Proteins on the Cell Surface
Authors: Tsurudome Masato , Ito Morihiro , Nishio Machiko , Kawano Mitsuo , Komada Hiroshi , Ito Yasuhiko
Published: July 2002

The fusion (F) protein of simian virus 5 (SV5) strain W3A is known to induce cell fusion in the absence of hemagglutinin-neuraminidase (HN) protein. In contrast, the F protein of SV5 strain WR induces cell fusion only when coexpressed with the HN protein, the same as do other paramyxovirus F proteins. When Leu-22 in the subunit F2 of the WR F protein is replaced with the counterpart (Pro) in the W3A F protein, the resulting mutant L22P induces extensive cell fusion by itself. In the present study, we obtained anti-L22P monoclonal antibodies (MAbs) 21-1 and 6-7, whose epitopes were located in the middle (amino acids [aa] 227 to 320) of subunit F1. The amino-terminal region (aa 20 to 47) of subunit F2 was also involved in the formation of MAb 21-1 epitope. Flow cytometric analysis revealed that both the MAbs reacted very faintly with native WR F protein that was expressed on the cell surface whereas they reacted efficiently with native L22P irrespective of whether it is cleaved into F1 and F2. However, by heating the cells at 47°C after mild formaldehyde fixation, the epitopes for MAb 6-7 and mAb 21-1 in the WR F protein were exposed and the reactivity of the MAbs with the WR F protein became comparable to their reactivity with L22P. Thus, the two MAbs seem to distinguish the difference in native conformation between fusogenic mutant L22P and its parental nonfusogenic WR F protein. The native conformation of L22P may represent an intermediate between native and postfusion conformations of a typical paramyxovirus F protein.
Analysis of Mason-Pfizer Monkey Virus Gag Particles by Scanning Transmission Electron Microscopy
Authors: Parker Scott D. , Wall Joseph S. , Hunter Eric
Published: July 2002

Mason-Pfizer monkey virus immature capsids selected from the cytoplasm of baculovirus-infected cells were imaged by scanning transmission electron microscopy. The masses of individual selected Gag particles were measured, and the average mass corresponded to 1,900 to 2,100 Gag polyproteins per particle. A large variation in Gag particle mass was observed within each population measured.
Herpes Simplex Virus gE/gI Sorts Nascent Virions to Epithelial Cell Junctions, Promoting Virus Spread
Authors: Johnson David C. , Webb Mike , Wisner Todd W. , Brunetti Craig
Published: July 2002

Alphaherpesviruses spread rapidly through dermal tissues and within synaptically connected neuronal circuitry. Spread of virus particles in epithelial tissues involves movement across cell junctions. Herpes simplex virus (HSV), varicella-zoster virus (VZV), and pseudorabies virus (PRV) all utilize a complex of two glycoproteins, gE and gI, to move from cell to cell. HSV gE/gI appears to function primarily, if not exclusively, in polarized cells such as epithelial cells and neurons and not in nonpolarized cells or cells that form less extensive cell junctions. Here, we show that HSV particles are specifically sorted to cell junctions and few virions reach the apical surfaces of polarized epithelial cells. gE/gI participates in this sorting. Mutant HSV virions lacking gE or just the cytoplasmic domain of gE were rarely found at cell junctions; instead, they were found on apical surfaces and in cell culture fluids and accumulated in the cytoplasm. A component of the AP-1 clathrin adapter complexes, μ1B, that is involved in sorting of proteins to basolateral surfaces was involved in targeting of PRV particles to lateral surfaces. These results are related to recent observations that (i) HSV gE/gI localizes specifically to the trans-Golgi network (TGN) during early phases of infection but moves out to cell junctions at intermediate to late times (T. McMillan and D. C. Johnson, J. Virol., in press) and (ii) PRV gE/gI participates in envelopment of nucleocapsids into cytoplasmic membrane vesicles (A. R. Brack, B. G. Klupp, H. Granzow, R. Tirabassi, L. W. Enquist, and T. C. Mettenleiter, J. Virol. 74:4004–4016, 2000). Therefore, interactions between the cytoplasmic domains of gE/gI and the AP-1 cellular sorting machinery cause glycoprotein accumulation and envelopment into specific TGN compartments that are sorted to lateral cell surfaces. Delivery of virus particles to cell junctions would be expected to enhance virus spread and enable viruses to avoid host immune defenses.
PE2 Cleavage Mutants of Sindbis Virus: Correlation between Viral Infectivity and pH-Dependent Membrane Fusion Activation of the Spike Heterodimer
Authors: Smit Jolanda M. , Klimstra William B. , Ryman Kate D. , Bittman Robert , Johnston Robert E. , Wilschut Jan
Published: July 2002

The spike glycoprotein E2 of Sindbis virus (SIN) is synthesized in the infected cell as a PE2 precursor protein, which matures through cleavage by a cellular furin-like protease. Previous work has shown that SIN mutants impaired in PE2 cleavage are noninfectious on BHK-21 cells, the block in infection being localized at a step after virus-receptor interaction but prior to RNA replication. Here, we studied the membrane fusion properties of SIN PE2 cleavage mutants and observed that these viruses are impaired in their ability to form an E1 homotrimer and to fuse with liposomes at a mildly acidic pH. The block in spike rearrangement and fusion could be overridden by exposure of the mutant viruses to very low pH (<4.5). Cleavage mutants with second-site resuscitating mutations in PE2 were highly infectious for BHK-21 cells. The ability of these viruses to form E1 homotrimers and to fuse at a mildly acidic pH was completely restored despite a sustained lack of PE2 cleavage.
Vaccinia Virus Infection Disarms the Mitochondrion-Mediated Pathway of the Apoptotic Cascade by Modulating the Permeability Transition Pore
Authors: Wasilenko Shawn T. , Meyers Adrienne F. A. , Vander Helm Kathleen , Barry Michele
Published: July 2002

Many viruses have evolved strategies that target crucial components within the apoptotic cascade. One of the best studied is the caspase 8 inhibitor, crmA/Spi-2, encoded by members of the poxvirus family. Since many proapoptotic stimuli induce apoptosis through a mitochondrion-dependent, caspase 8-independent pathway, we hypothesized that vaccinia virus would encode a mechanism to directly modulate the mitochondrial apoptotic pathway. In support of this, we observed that Jurkat cells, which undergo Fas-mediated apoptosis exclusively through the mitochondrial route, were resistant to Fas-induced death following infection with a crmA/Spi-2-deficient strain of vaccinia virus. In addition, vaccinia virus-infected cells subjected to the proapoptotic stimulus staurosporine exhibited decreased levels of both cytochromecreleased from the mitochondria and caspase 3 activation. In all cases we found that the loss of the mitochondrial membrane potential, which occurs as a result of opening the multimeric permeability transition pore complex, was prevented in vaccinia virus-infected cells. Moreover, vaccinia virus infection specifically inhibited opening of the permeability transition pore following treatment with the permeability transition pore ligand atractyloside andt-butylhydroperoxide. These studies indicate that vaccinia virus infection directly impacts the mitochondrial apoptotic cascade by influencing the permeability transition pore.
Differential Regulation of the Overlapping Kaposi's Sarcoma-Associated Herpesvirus vGCR (orf74) and LANA (orf73) Promoters
Authors: Jeong Joseph , Papin James , Dittmer Dirk
Published: July 2002

Similar to that of other herpesviruses, Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) lytic replication destroys the host cell, while the virus can persist in a latent state in synchrony with the host. During latency only a few genes are transcribed, and the question becomes one of what determines latent versus lytic gene expression. Here we undertake a detailed analysis of the latency-associated nuclear antigen (LANA [orf73]) promoter (LANAp). We characterized a minimal region that is necessary and sufficient to maintain high-level transcription in all tissues tested, including primary endothelial cells and B cells, which are the suspected natural host for KSHV. We show that in transient-transfection assays LANAp mimics the expression pattern observed for the authentic promoter in the context of the KSHV episome. Unlike other KSHV promoters tested thus far, LANAp is not affected by tetradecanoyl phorbol acetate or viral lytic cycle functions. It is, however, subject to control by LANA itself and cellular regulatory factors, such as p53. This is in contrast to the K14/vGCR (orf74) promoter, which overlaps LANAp and directs transcription on the opposite strand. We isolated a minimal
cis
-regulatory region sufficient for K14/vGCR promoter activity and show that it, too, mimics the regulation observed for the authentic viral promoter. In particular, we demonstrate that its activity is absolutely dependent on the immediate-early transactivator orf50, the KSHV homolog of the Epstein-Barr virus Rta transactivator.
Conservation of Human Immunodeficiency Virus Type 1 gp120 Inner-Domain Sequences in Lentivirus and Type A and B Retrovirus Envelope Surface Glycoproteins
Authors: Hötzel Isidro , Cheevers William P.
Published: July 2002

We recently described a sequence similarity between the small ruminant lentivirus surface unit glycoprotein (SU) gp135 and the second conserved region (C2) of the primate lentivirus gp120 which indicates a structural similarity between gp135 and the inner proximal domain of the human immunodeficiency virus type 1 gp120 (I. Hötzel and W. P. Cheevers, Virus Res. 69:47–54, 2000). Here we found that the seven-amino-acid sequence of the gp120 strand β25 in the C5 region, which is also part of the inner proximal domain, was conserved in the SU of all lentiviruses in similar or identical positions relative to the carboxy terminus of SU. Sequences conforming to the gp135-gp120 consensus for β-strand 5 in the C2 region, which is antiparallel to β25, were then sought in the SU of other lentiviruses and retroviruses. Except for the feline immunodeficiency virus, sequences similar to the gp120-gp135 consensus for β5 and part of the preceding strand β4 were present in the SU of all lentiviruses. This motif was highly conserved among strains of each lentivirus and included a strictly conserved cysteine residue in β4. In addition, the β4/β5 consensus motif was also present in the conserved carboxy-terminal region of all type A and B retroviral envelope surface glycoproteins analyzed. Thus, the antiparallel β-strands 5 and 25 of gp120 form an SU surface highly conserved among the lentiviruses and at least partially conserved in the type A and B retroviral envelope glycoproteins.
Self-Association and Mapping of the Interaction Domain of Hepatitis E Virus ORF3 Protein
Authors: Tyagi Shweta , Jameel Shahid , Lal Sunil K.
Published: July 2002

Hepatitis E virus (HEV) is a major human pathogen in the developing world. In the absence of an in vitro culture system, very little information on the basic biology of the virus exists. A small protein (∼13.5 kDa) of unknown function, pORF3, is encoded by the third open reading frame of HEV. The N-terminal region of pORF3 is associated with the cytoskeleton using one of its hydrophobic domains. The C-terminal half of pORF3 is rich in proline residues and contains a putative
src
homology 3 (SH3) binding domain and a mitogen-activated protein kinase phosphorylation site. In this study, we demonstrate that pORF3 can homodimerize in vivo, using the yeast two-hybrid system. We have isolated a 43-amino-acid interaction domain of pORF3 which is capable of self-association in vivo and in vitro. The overlap of the dimerization domain with the SH3 binding and phosphorylation domains suggests that pORF3 may have a dimerization-dependent regulatory role to play in the signal transduction pathway.
Adaptation of Reovirus to Growth in the Presence of Protease Inhibitor E64 Segregates with a Mutation in the Carboxy Terminus of Viral Outer-Capsid Protein ς3
Authors: Ebert Daniel H. , Wetzel J. Denise , Brumbaugh David E. , Chance Stacey R. , Stobie Laura E. , Baer Geoffrey S. , Dermody Terence S.
Published: July 2002

Reovirus virions are internalized into cells by receptor-mediated endocytosis. Within the endocytic compartment, the viral outer capsid undergoes acid-dependent proteolysis leading to degradation of ς3 protein and proteolytic cleavage of μ1/μ1C protein. E64 is a specific inhibitor of cysteine-containing proteases that blocks disassembly of reovirus virions. To identify domains in reovirus proteins that influence susceptibility to E64-mediated inhibition of disassembly, we selected variant viruses by serial passage of strain type 3 Dearing (T3D) in murine L929 cells treated with E64. E64-adapted variant viruses (D-EA viruses) produced 7- to 17-fold-greater yields than T3D did after infection of cells treated with 100 μM E64. Viral genes that segregate with growth of D-EA viruses in the presence of E64 were identified by using reassortant viruses isolated from independent crosses of E64-sensitive strain type 1 Lang and two prototype D-EA viruses. Growth of reassortant viruses in the presence of E64 segregated with the S4 gene, which encodes outer-capsid protein ς3. Sequence analysis of S4 genes of three D-EA viruses isolated from independent passage series revealed a common tyrosine-to-histidine mutation at amino acid 354 in the deduced amino acid sequence of ς3. Proteolysis of D-EA virions by endocytic protease cathepsin L occurred with faster kinetics than proteolysis of wild-type T3D virions. Treatment of D-EA virions, but not T3D virions, with cathepsin D resulted in proteolysis of ς3, a property that also was found to segregate with the D-EA S4 gene. These results indicate that a region in ς3 protein containing amino acid 354 influences susceptibility of ς3 to proteolysis during reovirus disassembly.
Human Immunodeficiency Virus Seroconversion and Evolution of the Hepatitis C Virus Quasispecies
Authors: Mao Qing , Ray Stuart C. , Laeyendecker Oliver , Ticehurst John R. , Strathdee Steffanie A. , Vlahov David , Thomas David L.
Published: July 2002

When chronic hepatitis C virus (HCV) infections are complicated by acquisition of human immunodeficiency virus (HIV), liver disease appears to accelerate and serum levels of HCV RNA may rise. We hypothesized that HIV might affect the HCV quasispecies by decreasing both complexity (if HIV-induced immunosuppression lessens pressure for selecting HCV substitutions) and the ratio of nonsynonymous (

d
N

) to synonymous (

d
S

) substitutions, because

d
N

may be lower (if there is less selective pressure). To test this hypothesis, we studied the evolution of HCV sequences in 10 persons with chronic HCV infection who seroconverted to HIV and, over the next 3 years, had slow or rapid progression of HIV-associated disease. From each subject, four serum specimens were selected with reference to HIV seroconversion: (i) more than 2 years prior, (ii) less than 2 years prior, (iii) less than 2 years after, and (iv) more than 2 years after. The HCV quasispecies in these specimens was characterized by generating clones containing 1 kb of cDNA that spanned the E1 gene and the E2 hypervariable region 1 (HVR1), followed by analysis of clonal frequencies (via electrophoretic migration) and nucleotide sequences. We examined 1,320 cDNA clones (33 per time point) and 287 sequences (median of 7 per time point). We observed a trend toward lower

d
N
/d
S

after HIV seroconversion in 7 of 10 subjects and lower

d
N
/d
S

in those with rapid HIV disease progression. However, the magnitude of these differences was small. These results are consistent with the hypothesis that HIV infection alters the HCV quasispecies, but the number of subjects and observation time may be too low to characterize the full effect.
Detection of Direct Binding of Human Herpesvirus 8-Encoded Interleukin-6 (vIL-6) to both gp130 and IL-6 Receptor (IL-6R) and Identification of Amino Acid Residues of vIL-6 Important for IL-6R-Dependent and -Independent Signaling
Authors: Li Hong , Wang Hailin , Nicholas John
Published: July 2002

Human herpesvirus 8 (HHV-8) is associated with Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease; in all of these diseases, interleukin-6 (IL-6) has been implicated as a likely mitogenic and/or angiogenic factor. HHV-8 encodes a homologue of IL-6 (viral IL-6 [vIL-6]) that has been shown to be biologically active in several assays and whose activities mirror those of its mammalian counterparts. Like these proteins, vIL-6 mediates its effects through the gp130 signal transducer, but signaling is not dependent on the structurally related IL-6 receptor (IL-6R; gp80) subunit of the receptor-signal transducer complex. However, as we have shown previously, IL-6R can enhance vIL-6 signal transduction and can enable signaling through a gp130 variant (gp130.PM5) that is itself unable to support vIL-6 activity, indicating that IL-6R can form part of the signaling complex. Also, our analysis of a panel of vIL-6 mutants in transfection experiments in Hep3B cells (that express IL-6R and gp130) showed that most were able to function normally in this system. Here, we have used in vitro vIL-6–receptor binding assays to demonstrate direct binding of vIL-6 to both gp130 and IL-6R and vIL-6-induced gp130–IL-6R complex formation, and we have extended our functional analyses of the vIL-6 variants to identify residues important for IL-6R-independent and IL-6R-dependent signaling through native gp130 and gp130.PM5, respectively. These studies have identified residues in vIL-6 that are important for IL-6R-independent and IL-6R-mediated functional complex formation between vIL-6 and gp130 and that may be involved directly in binding to gp130 and IL-6R.
Replication of Naturally Occurring Woodchuck Hepatitis Virus Deletion Mutants in Primary Hepatocyte Cultures and after Transmission to Naive Woodchucks
Authors: Lu Mengji , Hilken Gero , Yang Dongliang , Kemper Thekla , Roggendorf Michael
Published: July 2002

Woodchuck hepatitis virus (WHV) mutants with core internal deletions (CID) occur naturally in chronically WHV-infected woodchucks, as do hepatitis B virus mutants in humans. We studied the replication of WHV deletion mutants in primary woodchuck hepatocyte cultures and in vivo after transmission to naive woodchucks. By screening 14 wild-caught, chronically WHV-infected woodchucks, two woodchucks, WH69 and WH70, were found to harbor WHV CID mutants. Consistent with previous results, WHV CID mutants from both animals had deletions of variable lengths (90 to 135 bp) within the middle of the WHV core gene. In woodchuck WH69, WHV CID mutants represented a predominant fraction of the viral population in sera, normal liver tissues, and to a lesser extent, in liver tumor tissues. In primary hepatocytes of WH69, the replication of wild-type WHV and CID mutants was maintained at least for 7 days. Although WHV CID mutants were predominant in fractions of cellular WHV replicative intermediates, mutant covalently closed circular DNAs (cccDNAs) appeared to be a small part of cccDNA-enriched fractions. Analysis of cccDNA-enriched fractions from liver tissues of other woodchucks confirmed that mutant cccDNA represents only a small fraction of the total cccDNA pool. Four naive woodchucks were inoculated with sera from woodchuck WH69 or WH70 containing WHV CID mutants. All four woodchucks developed viremia after 3 to 4 weeks postinoculation (p.i.). They developed anti-WHV core antigen (WHcAg) antibody, lymphoproliferative response to WHcAg, and anti-WHV surface antigen. Only wild-type WHV, but no CID mutant, was found in sera from these woodchucks. The WHV CID mutant was also not identified in liver tissue from one woodchuck sacrificed in week 7 p.i. Three remaining woodchucks cleared WHV. Thus, the presence of WHV CID mutants in the inocula did not significantly change the course of acute self-limiting WHV infection. Our results indicate that the replication of WHV CID mutants might require some specific selective conditions. Further investigations on WHV CID mutants will allow us to have more insight into hepadnavirus replication.
Human Neuron-Committed Teratocarcinoma NT2 Cell Line Has Abnormal ND10 Structures and Is Poorly Infected by Herpes Simplex Virus Type 1
Authors: Hsu Wei-Li , Everett Roger D.
Published: July 2002

Herpes simplex virus type 1 (HSV-1) immediate-early regulatory protein ICP0 stimulates the initiation of lytic infection and reactivation from quiescence in human fibroblast cells. These functions correlate with its ability to localize to and disrupt centromeres and specific subnuclear structures known as ND10, PML nuclear bodies, or promyelocytic oncogenic domains. Since the natural site of herpesvirus latency is in neurons, we investigated the status of ND10 and centromeres in uninfected and infected human cells with neuronal characteristics. We found that NT2 cells, a neuronally committed human teratocarcinoma cell line, have abnormal ND10 characterized by low expression of the major ND10 component PML and no detectable expression of another major ND10 antigen, Sp100. In addition, PML is less extensively modified by the ubiquitin-like protein SUMO-1 in NT2 cells compared to fibroblasts. After treatment with retinoic acid, NT2 cells differentiate into neuron-like hNT cells which express very high levels of both PML and Sp100. Infection of both NT2 and hNT cells by HSV-1 was poor compared to human fibroblasts, and after low-multiplicity infection yields of virus were reduced by 2 to 3 orders of magnitude. ICP0-deficient mutants were also disabled in the neuron-related cell lines, and cells quiescently infected with an ICP0-null virus could be established. These results correlated with less-efficient disruption of ND10 and centromeres induced by ICP0 in NT2 and hNT cells. Furthermore, the ability of ICP0 to activate gene expression in transfection assays in NT2 cells was poor compared to Vero cells. These results suggest that a contributory factor in the reduced HSV-1 replication in the neuron-related cells is inefficient ICP0 function; it is possible that this is pertinent to the establishment of latent infection in neurons in vivo.
Retrieval of Human Cytomegalovirus Glycoprotein B from Cell Surface Is Not Required for Virus Envelopment in Astrocytoma Cells
Authors: Jarvis Michael A. , Fish Kenneth N. , Söderberg-Naucler Cecilia , Streblow Daniel N. , Meyers Heather L. , Thomas Gary , Nelson Jay A.
Published: July 2002

Human cytomegalovirus (HCMV) is a prototypic member of the betaherpesvirus family. The HCMV virion is composed of a large DNA genome encapsidated within a nucleocapsid, which is wrapped within an inner proteinaceous tegument and an outer lipid envelope containing viral glycoproteins. Although genome encapsidation clearly occurs in the nucleus, the subsequent steps in the virion assembly process are unclear. HCMV glycoprotein B (gB) is a major component of the virion envelope that plays a critical role in virus entry and is essential for the production of infectious virus progeny. The aim of our present study was to identify the secretory compartment to which HCMV gB was localized and to investigate the role of endocytosis in mediating gB localization and HCMV biogenesis. We show that HCMV gB is localized to the
trans
-Golgi network (TGN) in HCMV-infected cells and that gB contains all of the trafficking information necessary for TGN localization. Endocytosis of gB was shown to play a role in mediating TGN localization of gB and in targeting of the protein to the site of virus envelopment. However, inhibition of endocytosis with a dominant-negative dynamin I molecule did not affect the production of infectious virus. These observations indicate that, although endocytosis is involved in the trafficking of gB to the site of glycoprotein accumulation in the TGN, endocytosis of gB is not required for the production of infectious HCMV.
Inefficient Enhancement of Viral Infectivity and CD4 Downregulation by Human Immunodeficiency Virus Type 1 Nef from Japanese Long-Term Nonprogressors
Authors: Tobiume Minoru , Takahoko Mikako , Yamada Takeshi , Tatsumi Masashi , Iwamoto Aikichi , Matsuda Michiyuki
Published: July 2002

It has been reported that patients infected withnef-defective human immunodeficiency virus type 1 (HIV-1) do not progress to AIDS; however, mutations that abrogate Nef expression are not common in long-term nonprogressors (LTNPs). We postulated that Nef function might be impaired in LTNPs, irrespective of the presence or absence of detectable amino acid sequence anomalies. To challenge this hypothesis we compared in vitro function ofnefalleles that were derived from three groups of Japanese patients: LTNPs, progressors, and asymptomatic carriers (ACs). The patient-derivednefalleles were subcloned into anef-defective infectious HIV-1 molecular clone and an expression vector. We first examined Nef-dependent enhancement of infection in a single-round infectivity assay by the use of MAGNEF cells, in which Nef is required more strictly for the infection than in the parent MAGI cells. Allnefalleles from LTNPs showed reduced enhancement in the infectivity ofnef-defective HIV-1 mutants compared to thenefalleles of progressors or ACs. Second, we found thatnefalleles from LTNPs were less efficient in CD4 downregulation than those of progressors or ACs. Third, allnefalleles from LTNPs, progressors, and ACs reduced the cell surface expression of major histocompatibility complex class I to a similar level. Last, there was no correlation between Hck-binding activity of Nef and clinical grouping. In conclusion, we detected inefficient enhancement of HIV-1 infectivity and CD4 downregulation by HIV-1nefalleles of LTNPs. It awaits further study to conclude that these characteristics ofnefalleles are the cause or the consequence of the long-term nonprogression after HIV-1 infection.
Evidence for Human Immunodeficiency Virus Type 1 Replication In Vivo in CD14
+
Monocytes and Its Potential Role as a Source of Virus in Patients on Highly Active Antiretroviral Therapy
Authors: Zhu Tuofu , Muthui David , Holte Sarah , Nickle David , Feng Feng , Brodie Scott , Hwangbo Yon , Mullins James I. , Corey Lawrence
Published: July 2002

In vitro studies show that human immunodeficiency virus type 1 (HIV-1) does not replicate in freshly isolated monocytes unless monocytes differentiate to monocyte-derived macrophages. Similarly, HIV-1 may replicate in macrophages in vivo, whereas it is unclear whether blood monocytes are permissive to productive infection with HIV-1. We investigated HIV-1 replication in CD14
+
monocytes and resting and activated CD4
+
T cells by measuring the levels of cell-associated viral DNA and mRNA and the genetic evolution of HIV-1 in seven acutely infected patients whose plasma viremia had been <100 copies/ml for 803 to 1,544 days during highly active antiretroviral therapy (HAART). HIV-1 DNA was detected in CD14
+
monocytes as well as in activated and resting CD4
+
T cells throughout the course of study. While significant variation in the decay slopes of HIV-1 DNA was seen among individual patients, viral decay in CD14
+
monocytes was on average slower than that in activated and resting CD4
+
T cells. Measurements of HIV-1 sequence evolution and the concentrations of unspliced and multiply spliced mRNA provided evidence of ongoing HIV-1 replication, more pronounced in CD14
+
monocytes than in resting CD4
+
T cells. Phylogenetic analyses of HIV-1 sequences indicated that after prolonged HAART, viral populations related or identical to those found only in CD14
+
monocytes were seen in plasma from three of the seven patients. In the other four patients, HIV-1 sequences in plasma and the three cell populations were identical. CD14
+
monocytes appear to be one of the potential in vivo sources of HIV-1 in patients receiving HAART.
The VP1 N-Terminal Sequence of Canine Parvovirus Affects Nuclear Transport of Capsids and Efficient Cell Infection
Authors: Vihinen-Ranta Maija , Wang Dai , Weichert Wendy S. , Parrish Colin R.
Published: July 2002

The unique N-terminal region of the parvovirus VP1 capsid protein is required for infectivity by the capsids but is not required for capsid assembly. The VP1 N terminus contains a number of groups of basic amino acids which resemble classical nuclear localization sequences, including a conserved sequence near the N terminus comprised of four basic amino acids, which in a peptide can act to transport other proteins into the cell nucleus. Testing with a monoclonal antibody recognizing residues 2 to 13 of VP1 (anti-VP1-2-13) and with a rabbit polyclonal serum against the entire VP1 unique region showed that the VP1 unique region was not exposed on purified capsids but that it became exposed after treatment of the capsids with heat (55 to 75°C), or urea (3 to 5 M). A high concentration of anti-VP1-2-13 neutralized canine parvovirus (CPV) when it was incubated with the virus prior to inoculation of cells. Both antibodies blocked infection when injected into cells prior to virus inoculation, but neither prevented infection by coinjected infectious plasmid DNA. The VP1 unique region could be detected 4 and 8 h after the virus capsids were injected into cells, and that sequence exposure appeared to be correlated with nuclear transport of the capsids. To examine the role of the VP1 N terminus in infection, we altered that sequence in CPV, and some of those changes made the capsids inefficient at cell infection.
Inhibition of Duck Hepatitis B Virus Infection by a Myristoylated Pre-S Peptide of the Large Viral Surface Protein
Authors: Urban Stephan , Gripon Philippe
Published: July 2002

We have used the duck hepatitis B virus (DHBV) model to study the interference with infection by a myristoylated peptide representing an N-terminal pre-S subdomain of the large viral envelope protein. Although lacking the essential part of the carboxypeptidase D (formerly called gp180) receptor binding site, the peptide binds hepatocytes and subsequently blocks DHBV infection. Since its activity requires an amino acid sequence involved in host discrimination between DHBV and the related heron HBV (T. Ishikawa and D. Ganem, Proc. Natl. Acad. Sci. USA 92:6259-6263, 1995), we suggest that it is related to the postulated host-discriminating cofactor of infection.
A Novel Expression Cassette of Lyssavirus Shows that the Distantly Related Mokola Virus Can Rescue a Defective Rabies Virus Genome
Authors: Le Mercier Philippe , Jacob Yves , Tanner Kyle , Tordo Noël
Published: July 2002

By comparing three expression vectors for the rabies virus (Rv) minigenome, we show that the characteristic of the Rv RNA is important for efficient rescue despite its not being crucial for replication. Moreover, we show that the coexpression of the viral proteins from helper Rv and Mokola virus could rescue the Rv minigenome while Rv-related European bat lyssavirus 1 could not, suggesting that the signals controlling transcription and replication are conserved in the distantly related Rv and Mokola virus.
Safety and Immunogenicity of ALVAC vCP1452 and Recombinant gp160 in Newly Human Immunodeficiency Virus Type 1-Infected Patients Treated with Prolonged Highly Active Antiretroviral Therapy
Authors: Jin Xia , Ramanathan, Murugappan , Barsoum Shady , Deschenes Geoffrey R. , Ba Lei , Binley James , Schiller Daryl , Bauer Daniel E. , Chen Donald C. , Hurley Arlene , Gebuhrer Lucette , El Habib Raphaelle , Caudrelier Pierre , Klein Michel , Zhang Linqi , Ho David D. , Markowitz Martin
Published: July 2002

In order to boost immune responses in persons in whom highly active antiretroviral therapy (HAART) was initiated within 120 days of the onset of symptoms of newly acquired human immunodeficiency virus type 1 (HIV-1) infection, we administered vaccines containing a canarypox virus vector, vCP1452, with HIV-1 genes encoding multiple HIV-1 proteins, and recombinant gp160. Fifteen HIV-1-infected subjects who achieved sustained suppression of plasma viremia for at least 2 years were enrolled. While continuing antiretroviral therapy, each subject received at least four intramuscular injections of the vaccines on days 0, 30, 90, and 180. Adverse events were mild, with the most common being transient tenderness at the vCP1452 injection site. Of the 14 patients who completed vaccination, 13 had significant increases in anti-gp120 or anti-p24 antibody titers, and 9 had transient augmentation of their T-cell proliferation responses to gp160 and/or p24. HIV-1-specific CD8
+
T cells were quantified using an intracellular gamma interferon staining assay. Among 11 patients who had increased CD8
+
T-cell responses, seven had responses to more than one HIV-1 antigen. In summary, vaccination with vCP1452 and recombinant gp160 appears safe and immunogenic in newly HIV-1-infected patients on HAART.
Efficient Human Immunodeficiency Virus Replication Requires a Fine-Tuned Level of Transcription
Authors: Marzio Giuseppe , Vink Monique , Verhoef Koen , de Ronde Anthony , Berkhout Ben
Published: July 2002

Transcription represents a crucial step in the life cycle of human immunodeficiency virus (HIV) and is highly regulated. Here we show that the strength of the viral long terminal repeat (LTR) promoter is optimized for efficient replication. Artificially increasing the rate of LTR-driven transcription was strongly detrimental for viral fitness, and HIV was able to regain replication capacity by selecting for variants with a weaker LTR. Strikingly, the strength of the evolved promoter was equivalent to that of the wild-type LTR.
Vesicular Stomatitis Virus Glycoprotein Does Not Determine the Site of Virus Release in Polarized Epithelial Cells
Authors: Zimmer Gert , Zimmer Klaus-Peter , Trotz Ina , Herrler Georg
Published: July 2002

In polarized epithelial cells, the vesicular stomatitis virus glycoprotein is segregated to the basolateral plasma membrane, where budding of the virus takes place. We have generated recombinant viruses expressing mutant glycoproteins without the basolateral-membrane-targeting signal in the cytoplasmic domain. Though about 50% of the mutant glycoproteins were found at the apical plasma membranes of infected MDCK cells, the virus was still predominantly released at the basolateral membranes, indicating that factors other than the glycoprotein determine the site of virus budding.
Characterization of the Protease of a Fish Retrovirus, Walleye Dermal Sarcoma Virus
Authors: Fodor Sharon K. , Vogt Volker M.
Published: July 2002

Three fish retroviruses infecting walleyes constitute the recently recognized genus called epsilonretrovirus. The founding member of this group, walleye dermal sarcoma virus (WDSV), induces benign skin tumors in the infected fish and replicates near 4°C. While the viral genomic sequence is known, biochemical characterization of the virus has been limited to the identification of the mature structural and envelope proteins present in virions. We undertook this study to determine the cleavage sites in the WDSV Pro and Pol proteins and to characterize the viral protease (PR) in vitro. A recombinant PR was expressed in and purified from
Escherichia coli
as a larger fusion with additional nucleocapsid and reverse transcriptase residues flanking the PR domain. Autocleavage produced a functional, mature PR. Autocleavage as well as cleavage of peptides and of Gag protein by the mature PR occurred at a pH optimum of 7.0, higher than that of other retroviral proteases. Analysis of the cleavage sites identified a glutamine residue in the P2 position of all WDSV sites, both in Gag and in Pol. Amino acid sequence alignments of Gag-Pro-Pol from WDSV, walleye epidermal hyperplasia virus type 1, and walleye epidermal hyperplasia virus type 2 showed the P2 glutamine to be conserved in all cleavage sites in these three viruses. Such conservation is unprecedented in other retroviruses.
Cinnamoylacetonitrile Arylhydrazones in Heterocyclic Synthesis: Simple Synthesis of Pyridazines and their Condensed Derivatives
Authors: Nawwar Galal A. M. , Swellem Randa H. , Chabaka Laila M.
Published: July 2002

A new route for the synthesis of the title ring systems starting with the cinnamoylacetonitrile arylhydrazones (Ia, Ib) is described. It involves the self-cyclization of I which was found to be pH dependent.
3,5-Di-tert-butyl-1,2-benzoquinone Cleaves a CC-bond in Vicinal Aminobenzyl Alcohols
Authors: Horák Václav , Mermersky Yonco , Guirguis Dalal B.
Published: July 2002

Two vicinal aminobenzyl alcohols, L-threo-1-(4-methylthiophenyl)-2-amino-1,3-propanediol (IIa) and 1-phenyl-2-amino-1,3-propanediol (IIb), underwent, under mild conditions, CC-bond cleavage with 3,5-di-tert-butyl-1,2-benzoquinone (I) producing in high yields 4-methylthiobenzaldehyde (Va) and benzaldehyde (Vb), respectively, and 2-hydroxymethyl-4,6-di-tert-butylbenzoxazole (VII). Ethanolamine (VIII) under identical conditions produced benzoxazole VII. The reported reaction is a second case in which quinone I mimics reactions of pyridoxal.
Synthesis of 1-(3-Azido-2,3-dideoxy-β-D-ribo-hexofuranosyl)-, 1-(2,3-Dideoxy-β-D-erythro-hexofuranosyl)- and 1-(2,3-Dideoxy-β-D-erythro-hex-2-enofuranosyl)pyrimidine Nucleosides
Authors: Hřebabecký Hubert , Holý Antonín
Published: July 2002

1-(3-Azido-2,3-dideoxy-β-D-ribo-hexofuranosyl)uracil (XXII) and 1-(2,3-dideoxy-β-D-erythro-hex-2-enofuranosyl)uracil (VIII) were prepared starting from 1-(2-O-acetyl-3,5,6-tri-O-benzoyl-β-D-glucofuranosyl)uracil (I) by a procedure described previously for thymine analogs. 1-(2,3-Dideoxy-β-D-erythro-hexofuranosyl)uracil (XIII) was obtained by catalytic hydrogenation of 1-(5,6-di-O-benzoyl-2,3-dideoxy-β-D-erythro-hex-2-enofuranosyl)uracil (VII) and subsequent methanolysis. Reaction of dibenzoyl derivative VII, 1-(5,6-di-O-benzoyl-2,3-dideoxy-β-D-erythro-hexofuranosyl)uracil (XII) and the diacetate prepared by acetylation of azido derivative XXII with Lawesson's reagent, followed by methanolysis, afforded 1-(2,3-dideoxy-β-D-erythro-hex-2-enofuranosyl)-4-thiouracil (X), 1-(2,3-dideoxy-β-D-erythro-hexofuranosyl)-4-thiouracil (XV) and 1-(3-azido-2,3-dideoxy-β-D-ribo-hexofuranosyl)-4-thiouracil (XXIII), respectively. Heating of thio derivatives X, XV and XXIII with methanolic ammonia at 100 °C gave 1-(2,3-dideoxy-β-D-erythro-hex-2-enofuranosyl)cytosine (XI), 1-(2,3-dideoxy-β-D-erythro-hexofuranosyl)cytosine (XVI) and 1-(3-azido-2,3-dideoxy-β-D-ribo-hexofuranosyl)cytosine (XXIV).
Synthesis and Antiviral Activity of Acyclic Nucleoside Analogues of 6-Methyluracil and 4-Alkylamino-6-methyl-2(1H)-pyrimidinones
Authors: Bhat Sunita
Published: July 2002

Reaction of 2,4-Dimethoxy-6-methylpyrimidine (I) with allyl bromide or benzyl bromide afforded 1-substituted 4-methoxy-6-methyl-2(1H)-pyrimidinone intermediates III, X. Oxidation of the compound III afforded racemic cis diol VI. O-Demethylation and nucleophilic displacement of the intermediates III, VI and X gave 1-substituted 6-methyluracils IV, VII, IX and 1-substituted 4-alkylamino-6-methyl-2(1H)-pyrimidinones V, VIII, XII in good yields. The compounds II - XII were evaluated against Ranikhet disease virus (RDV); compounds Vb, VII, X, XIIb - XIId showed 100, 43, 44, 75, 72 and 100% inhibition, respectively.
A Study of Effects of Temperature and Medium on Reaction of Triethylamine with Ethyl Bromide
Authors: Nevěčná Taťjana , Bekárek Vojtěch , Pytela Oldřich
Published: July 2002

The rate constants of reaction of triethylamine with ethyl bromide have been measured in 13 solvents at the temperatures of 293, 313, 333, and 373 K. The activation entropies in the individual solvents increase when going from nonpolar to dipolar aprotic and polar protic solvents, which is explained by dominant solvation of the basic triethylamine and by formation of highly ordered associates without solvent in the activated complex in nonpolar solvent media. No isokinetic relationship has been found between the activation entropy and activation enthalpy, which indicates different solvent effects on the two quantities. The activation enthalpy and entropy of the reaction investigated are close to those of the reaction of triethylamine with ethyl iodide. Three methods have been used to evaluate the effect of medium at all the temperatures, their success being decreased in the order: Pytela's method - Kamlet-Taft - Koppel-Palm. Irrespective of the temperature, all the methods indicate that the reaction is accelerated by the solvent polarity, the significance of other effects being reflected differently depending on the temperature and the correlation equation used. A complex evaluation involving also the interpretation of the entropy and enthalpy components by means of empiric solvent parameters has shown that the resulting Gibbs energy represents a superposition of different effects of solvents on the two thermodynamic quantities, the solvent effect upon the activation entropy being predominant at the higher temperatures.
X-Ray Absolute Structure of Nicergoline (Form I). Quantitative Analysis of Nicergoline Phase Mixture: Form I/Form II
Authors: Hušák Michal , Had Jiří , Kratochvíl Bohumil , Cvak Ladislav , Stuchlík Josef , Jegorov Alexandr
Published: July 2002

The structure of semisynthetic ergot derivative nicergoline (form I) was determined from X-ray single crystal diffraction data. The title alkaloid (10α-methoxy-1,6-dimethylergoline-8β-methanol-5'-bromonicotinate, C24H26BrN3O3) crystallizes in the triclinic space group P1, with a = 7.729(7), b = 8.695(4), c = 17.030(8) Å, α = 100.90(4), β = 98.67(5), γ = 93.04(5)°, Z = 2. A structural comparison of nicergoline forms I and II was made. The quantitative analyses of some model phase mixtures form I / form II were carried out using both Rietveld full profile fitting method and linear regression of the peak intensity concentration dependence. Results obtained are in good mutual agreement. The smallest limit of detection of form II in binary mixture with form I is about 3% as found from powder diffraction study.