bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of the Rhesus Macaque Rhadinovirus Lytic Origin of DNA Replication
Authors: Pari Gregory S. , AuCoin David , Colletti Kelly , Cei Sylvia A. , Kirchoff Veronica , Wong Scott W.
Published: July 2002

We have identified a lytic origin of DNA replication (oriLyt) for rhesus macaque rhadinovirus (RRV), the rhesus macaque homolog of human herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus. RRV oriLyt maps to the region of the genome between open reading frame 69 (ORF69) and ORF71 (vFLIP) and is composed of an upstream A+T-rich region followed by a short (300-bp) downstream G+C-rich DNA sequence. A set of overlapping cosmids corresponding to the entire genome of RRV was capable of complementing oriLyt-dependent DNA replication only when additional ORF50 was supplied as an expression plasmid in the transfection mixture, suggesting that the level of ORF50 protein originating from input cosmid DNA was insufficient. The requirement of RRV ORF50 in the cotransfection replication assay may also suggest a direct role for this protein in DNA replication. RRV oriLyt shares a high degree of nucleotide sequence and G+C base distribution with the corresponding loci in HHV-8.
Partial Restoration of Replication of Simian Immunodeficiency Virus by Point Mutations in either the Dimerization Initiation Site (DIS) or Gag Region after Deletion Mutagenesis within the DIS
Authors: Guan Yongjun , Diallo Karidia , Detorio Mervi , Whitney James B. , Liang Chen , Wainberg Mark A.
Published: July 2002

We used the simian immunodeficiency virus (SIV) molecular clone SIVmac239 to generate a deletion construct, termed SD2, in which we eliminated 22 nucleotides at positions +398 to +418 within the putative dimerization initiation site (DIS) stem. This SD2 deletion severely impaired viral replication, due to adverse effects on the packaging of viral genomic RNA, the processing of Gag proteins, and viral protein patterns. However, long-term culture of SD2 in either C8166 or CEMx174 cells resulted in restoration of replication capacity, due to two different sets of three compensatory point mutations, located within both the DIS and Gag regions. In the case of C8166 cells, both a K197R and a E49K mutation were identified within the capsid (CA) protein and the p6 protein of Gag, respectively, while the other point mutation (A423G) was found within the putative DIS loop. In the case of CEMx174 cells, two compensatory mutations were present within the viral nucleocapsid (NC) protein, E18G and Q31K, in addition to the same A423G substitution as observed with C8166 cells. A set of all three mutations was required in each case for restoration of replication capacity, and either set of mutations could be substituted for the other in both the C8166 and CEMx174 cell lines.
The Major Human Immunodeficiency Virus Type 2 (HIV-2) Packaging Signal Is Present on All HIV-2 RNA Species: Cotranslational RNA Encapsidation and Limitation of Gag Protein Confer Specificity
Authors: Griffin Stephen D. C. , Allen Jane F. , Lever Andrew M. L.
Published: July 2002

Deletion of a region of the human immunodeficiency virus type 2 (HIV-2) 5′ leader RNA reduces genomic RNA encapsidation to about 5% that of wild-type virus with no defect in viral protein production but severely limits virus spread in Jurkat T cells, indicating that this region contains a major
cis
-acting encapsidation signal, or psi (Ψ). Being upstream of the major splice donor, it is present on all viral transcripts. We have shown that HIV-2 selects its genomic RNA for encapsidation cotranslationally, rendering wild-type HIV-2 unable to encapsidate vector RNAs in
trans
. Virus with Ψ deleted, however, encapsidates an HIV-2 vector, demonstrating competition for Gag protein. HIV-2 overcomes the lack of packaging signal location specificity by two novel mechanisms, cotranslational packaging and competition for limiting Gag polyprotein.
Intact EAV-HP Endogenous Retrovirus in Sonnerat's Jungle Fowl
Authors: Sacco M. A. , Howes K. , Venugopal K.
Published: July 2002

The EAV-HP group of chicken endogenous retrovirus elements was previously shown to be defective, with large deletions of the
pol
gene. In this report, we demonstrate that genomes of other
Gallus
species also maintain EAV-HP elements with similar deletions. The chicken EAV-HP1 locus was detected in both red (
Gallus gallus gallus
) and Sonnerat's (
Gallus sonneratii
) jungle fowl with identical integration sites, indicating that these elements had integrated before separation of the
Gallus
species. Furthermore, we demonstrate for the first time that the
G. sonneratii
genome carries EAV-HP elements with intact
pol
regions.
Role for Human Immunodeficiency Virus Type 1 Tat Protein in Suppression of Viral Reverse Transcriptase Activity during Late Stages of Viral Replication
Authors: Kameoka Masanori , Rong Liwei , Götte Matthias , Liang Chen , Russell Rodney S. , Wainberg Mark A.
Published: July 2002

We have examined the role of the human immunodeficiency virus type 1 (HIV-1) Tat protein in the regulation of reverse transcription. We show that a two-exon but not a one-exon form of Tat markedly suppressed cell-free reverse transcriptase (RT) activity. Conversely, viruses expressing two-exon Tat (pNL43 and pNL101) showed rapid replication kinetics and more efficient endogenous RT activity compared with viruses expressing one-exon Tat (pM1ex). The pM1ex virions, as well as pM1ex-infected cells, also contained higher levels of viral DNA than did either the pNL43 or pNL101 viruses, indicating that reverse transcription might have continued during later stages of viral replication in the absence of the second Tat exon. Moreover, degradation of viral genomic RNA was more apparent in the pM1ex virions. Accordingly, we propose that the two-exon Tat may help augment viral infectivity by suppressing the reverse transcription reaction during late stages of viral synthesis and by preventing the synthesis of potentially deleterious viral DNA products.
Adenovirus Type 5 Viral Particles Pseudotyped with Mutagenized Fiber Proteins Show Diminished Infectivity of Coxsackie B-Adenovirus Receptor-Bearing Cells
Authors: Jakubczak John L. , Rollence Michele L. , Stewart David A. , Jafari Jonathon D. , Von Seggern Dan J. , Nemerow Glen R. , Stevenson Susan C. , Hallenbeck Paul L.
Published: July 2002

A major limitation of adenovirus type 5 (Ad5)-based gene therapy, the inability to target therapeutic genes to selected cell types, is attributable to the natural tropism of the virus for the widely expressed coxsackievirus-adenovirus receptor (CAR) protein. Modifications of the Ad5 fiber knob domain have been shown to alter the tropism of the virus. We have developed a novel system to rapidly evaluate the function of modified fiber proteins in their most relevant context, the adenoviral capsid. This transient transfection/infection system combines transfection of cells with plasmids that express high levels of the modified fiber protein and infection with Ad5.βgal.ΔF, an E1-, E3-, and fiber-deleted adenoviral vector encoding β-galactosidase. We have used this system to test the adenoviral transduction efficiency mediated by a panel of fiber protein mutants that were proposed to influence CAR interaction. A series of amino acid modifications were incorporated via mutagenesis into the fiber expression plasmid, and the resulting fiber proteins were subsequently incorporated onto adenoviral particles. Mutations located in the fiber knob AB and CD loops demonstrated the greatest reduction in fiber-mediated gene transfer in HeLa cells. We also observed effects on transduction efficiency with mutations in the FG loop, indicating that the binding site may extend to the adjacent monomer in the fiber trimer and in the HI loop. These studies support the concept that modification of the fiber knob domain to diminish or ablate CAR interaction should result in a detargeted adenoviral vector that can be combined simultaneously with novel ligands for the development of a systemically administered, targeted adenoviral vector.
Human Dendritic Cells Are Activated by Dengue Virus Infection: Enhancement by Gamma Interferon and Implications for Disease Pathogenesis
Authors: Libraty Daniel H. , Pichyangkul Sathit , Ajariyakhajorn Chuanpis , Endy Timothy P. , Ennis Francis A.
Published: July 2002

The ability of dendritic cells (DCs) to shape the adaptive immune response to viral infection is mediated largely by their maturation and activation state as determined by the surface expression of HLA molecules, costimulatory molecules, and cytokine production. Dengue is an emerging arboviral disease where the severity of illness is influenced by the adaptive immune response to the virus. In this report, we have demonstrated that dengue virus infects and replicates in immature human myeloid DCs. Exposure to live dengue virus led to maturation and activation of both the infected and surrounding, uninfected DCs and stimulated production of tumor necrosis factor alpha (TNF-α) and alpha interferon (IFN-α). Activation of the dengue virus-infected DCs was blunted compared to the surrounding, uninfected DCs, and dengue virus infection induced low-level release of interleukin-12 p70 (IL-12 p70), a key cytokine in the development of cell-mediated immunity (CMI). Upon the addition of IFN-γ, there was enhanced activation of dengue virus-infected DCs and enhanced dengue virus-induced IL-12 p70 release. The data suggest a model whereby DCs are the early, primary target of dengue virus in natural infection and the vigor of CMI is modulated by the relative presence or absence of IFN-γ in the microenvironment surrounding the virus-infected DCs. These findings are relevant to understanding the pathogenesis of dengue hemorrhagic fever and the design of new vaccination and therapeutic strategies.
Actin Rearrangement-Inducing Factor of Baculoviruses Is Tyrosine Phosphorylated and Colocalizes to F-Actin at the Plasma Membrane
Authors: Dreschers Stephan , Roncarati Renza , Knebel-Mörsdorf Dagmar
Published: July 2002

In previous studies we have identified actin rearrangement-inducing factor 1 as an early gene product of
Autographa californica
multicapsid nuclear polyhedrosis virus that is involved in the remodeling of the actin cytoskeleton. We have constructed viral recombinants with a mutated Arif-1 open reading frame that confirm the causal link of Arif-1 expression and the actin rearrangement observed as accumulation of F-actin at the plasma membrane at 3 to 7 h postinfection. Infection with Arif mutant viruses leads to the loss of actin accumulation at the plasma membrane in TN-368 cells, although in the course of infection, early actin cables and nuclear F-actin are observed as in wild-type-infected cells. By immunofluorescence studies, we have demonstrated the localization of Arif-1 at the plasma membrane, and confocal imaging reveals the colocalization to F-actin. Accordingly, the ∼47-kDa Arif-1 protein is observed exclusively in membrane fractions prepared at 4 to 48 h postinfection, with a decrease at 24 h postinfection. Phosphatase treatment suggests that Arif-1 is modified by phosphorylation. Antibodies against phosphotyrosine precipitate Arif-1 from membrane fractions, indicating that Arif-1 becomes tyrosine phosphorylated during the early and late phases of infection. In summary, our results indicate that functional Arif-1 is tyrosine phosphorylated and is located at the plasma membrane as a component of the actin rearrangement-inducing complex.
Sequence Requirements for Interaction of Human Herpesvirus 7 Origin Binding Protein with the Origin of Lytic Replication
Authors: Krug Laurie T. , Inoue Naoki , Pellett Philip E.
Published: July 2002

As do human herpesvirus 6 variants A and B (HHV-6A and -6B), HHV-7 encodes a homolog of the alphaherpesvirus origin binding protein (OBP), which binds at sites in the origin of lytic replication (
ori
Lyt) to initiate DNA replication. In this study, we sought to characterize the interaction of the HHV-7 OBP (OBP
H7
) with its cognate sites in the 600-bp HHV-7
ori
Lyt. We expressed the carboxyl-terminal domain of OBP
H7
and found that amino acids 484 to 787 of OBP
H7
were sufficient for DNA binding activity by electrophoretic mobility shift analysis. OBP
H7
has one high-affinity binding site (OBP-2) located on one flank of an AT-rich spacer element and a low-affinity site (OBP-1) on the other. This is in contrast to the HHV-6B OBP (OBP
H6B
), which binds with similar affinity to its two cognate OBP sites in the HHV-6B
ori
Lyt. The minimal recognition element of the OBP-2 site was mapped to a 14-bp sequence. The OBP
H7
consensus recognition sequence of the 9-bp core, BRT
YCWCC
T (where B is a T, G, or C; R is a G or A; Y is a T or C; and W is a T or A), overlaps with the OBP
H6B
consensus YGW
YCWCC
Y and establishes YCWCC as the roseolovirus OBP core recognition sequence. Heteroduplex analysis suggests that OBP
H7
interacts along one face of the DNA helix, with the major groove, as do OBP
H6B
and herpes simplex virus type 1 OBP. Together, these results illustrate both conserved and divergent DNA binding properties between OBP
H7
and OBP
H6B
.
Amino Acid Deletion at Codon 67 and Thr-to-Gly Change at Codon 69 of Human Immunodeficiency Virus Type 1 Reverse Transcriptase Confer Novel Drug Resistance Profiles
Authors: Imamichi Tomozumi , Murphy Michael A. , Imamichi Hiromi , Lane H. Clifford
Published: July 2002

The potential roles of an amino acid deletion at codon 67 (Δ67) and a Thr-to-Gly change at codon 69 (T69G) in the reverse transcriptase of human immunodeficiency virus (HIV) type 1 in drug sensitivity and relative replication fitness were studied. Our results suggest that the Δ67 and T69G changes can be categorized as mutations associated with multidrug resistance. The combination of both mutations with an L74I change (Δ67+T69G/L74I) leads to a novel 3′-azido-3′-deoxythymidine resistance motif and compensates for impaired HIV replication.
Functional Characterization of the N Termini of Murine Leukemia Virus Envelope Proteins
Authors: Lu Chi-Wei , Roth Monica J.
Published: July 2002

The function of the N terminus of the murine leukemia virus (MuLV) surface (SU) protein was examined. A series of five chimeric envelope proteins (Env) were generated in which the N terminus of amphotropic 4070A was replaced by equivalent sequences from ecotropic Moloney MuLV (M-MuLV). Viral titers of these chimeras indicate that exchange with homologous sequences could be tolerated, up to V17eco/T15ampho (crossover III). Constructs encoding the first 28 amino acids (aa) of ecotropic M-MuLV resulted in Env expression and binding to the receptor; however, the virus titer was reduced 5- to 45-fold, indicating a postbinding block. Additional exchange beyond the first 28 aa of ecotropic MuLV Env resulted in defective protein expression. These N-terminal chimeras were also introduced into the AE4 chimeric Env backbone containing the amphotropic receptor binding domain joined at the hinge region to the ecotropic SU C terminus. In this backbone, introduction of the first 17 aa of the ecotropic Env protein significantly increased the titer compared to that of its parental chimera AE4, implying a functional coordination between the N terminus of SU and the C terminus of the SU and/or transmembrane proteins. These data functionally dissect the N-terminal sequence of the MuLV Env protein and identify differential effects on receptor-mediated entry.
A Nucleocapsid Functionality Contained within the Amino Terminus of the Ty1 Protease That Is Distinct and Separable from Proteolytic Activity
Authors: Lawler Joseph F. , Merkulov Gennady V. , Boeke Jef D.
Published: July 2002

Ty1 is the most successful of the five endogenous yeast retrotransposons. The life cycle of Ty1 dictates that a number of nucleocapsid (NC)-facilitated events occur although the protein(s) responsible for these events has not been identified. The positioning of the NC peptide is conserved at the carboxy terminus of the Gag protein among most long terminal repeat (LTR)-containing retroelements. An analogous region of Ty1 that simultaneously encodes part of Gag, protease (PR), and the C-terminal p4 peptide was mutagenized. Some of these mutations result in smaller-than-normal virus-like particles (VLPs). The mutants were also found to impair an NC-like functionality contained within the amino terminus of the protease that is distinct and separable from its proteolytic activity. Remarkably, these mutants have distinct defects in reverse transcription.
The Small Viral Membrane-Associated Protein P32 Is Involved in Bacteriophage PRD1 DNA Entry
Authors: Grahn A. Marika , Daugelavičius Rimantas , Bamford Dennis H.
Published: July 2002

The lipid-containing bacteriophage PRD1 infects a variety of gram-negative cells by injecting its linear double-stranded DNA genome into the host cell cytoplasm, while the protein capsid is left outside. The virus membrane and several structural proteins are involved in phage DNA entry. In this work we identified a new infectivity protein of PRD1. Disruption of geneXXXIIresulted in a mutant phenotype defective in phage reproduction. The absence of the protein P32 did not compromise the particle assembly but led to a defect in phage DNA injection. In P32-deficient particles the phage membrane is unable to undergo a structural transformation from a spherical to a tubular form. Since P32−particles are able to increase the permeability of the host cell envelope to a degree comparable to that found with wild-type particles, we suggest that the tail-tube formation is needed to eject the DNA from the phage particle rather than to reach the host cell interior.
Expression of Transcription Factor AML-2 (RUNX3, CBFα-3) Is Induced by Epstein-Barr Virus EBNA-2 and Correlates with the B-Cell Activation Phenotype
Authors: Spender Lindsay C. , Cornish Georgina H. , Sullivan Alexandra , Farrell Paul J.
Published: July 2002

To identify cell proteins regulated by the Epstein-Barr virus (EBV) transcription factor EBNA-2, we analyzed a cell line with conditional EBNA-2 activity by using microarray expression profiling. This led to the identification of two novel target genes induced by EBNA-2. The first of these, interleukin-16, is an immunomodulatory cytokine involved in the regulation of CD4 T cells. The second, AML-2, is a member of the Runt domain family of transcription factors. Quiescent B cells initially expressed AML-1 but, 48 h after virus infection, the levels of AML-1 decreased dramatically, whereas the amount of AML-2 protein increased. Analysis of a panel of B-cell lines indicated that AML-2 expression is normally predominant in EBV latency III, whereas AML-1 is associated with EBV latency I or EBV-negative cells. The AML genes are the first example of cell transcription factors whose expression correlates with the latency I/III phenotype.
An NF-κB-Dependent Survival Pathway Protects against Cell Death Induced by TVB Receptors for Avian Leukosis Viruses
Authors: Chi Yuling , Diaz-Griffero Felipe , Wang Chenguang , Young John A. T. , Brojatsch Jürgen
Published: July 2002

TVB receptors are death receptors of the tumor necrosis factor receptor (TNFR) family and serve as cellular receptors for cytopathic subgroups B and D and noncytopathic subgroup E of the avian leukosis viruses (ALVs). Although TVB is essential for ALV-B-mediated cell death, binding of the ALV-B envelope protein to its cognate receptor TVB activates cell death only in the presence of protein biosynthesis inhibitors, which presumably block the expression of protective factors. In the case of TNFR-1, the main antiapoptotic pathway depends upon nuclear factor kappa B (NF-κB)-activated survival factors. Here we show that overexpression of TVB receptors in human 293 cells activates NF-κB via a mechanism involving the cytoplasmic death domains of these receptors. NF-κB is also activated upon binding of a soluble ALV-B or ALV-E surface envelope-immunoglobulin fusion protein to the cognate TVB receptors and by ALV-B infection of a chicken embryo fibroblast cell line (DF1). Importantly, the cycloheximide requirement for TVB-dependent cell death was overcome by the expression of a transdominant form of IκB-α, and downregulation of NF-κB by the immunomodulator pyrrolidinedithiocarbamate enhanced the cytopathogenicity of ALV-B. These results demonstrate that TVB receptors trigger NF-κB-dependent gene expression and that NF-κB-regulated survival factors can protect against virus-induced cell death.
Expansion of a CD28-Intermediate Subset among CD8 T Cells in Patients with Infectious Mononucleosis
Authors: Uda Hiroshi , Mima Toru , Yamaguchi Norihiko , Katada Yoshinori , Fukuda Masayuki , Fujii Naohiko , Nakamura Kojiro , Saiki Osamu
Published: July 2002

Infectious mononucleosis (IM) is an acute sporadic infection that usually affects young adults, and during infection a massive expansion of CD8 T cells is generally considered to occur. However, CD28 expression of the expanded cells has not been characterized. When peripheral blood mononuclear cells of acute IM (AIM) patients were analyzed by flow cytometry, a continuous spectrum of CD28 intensity ranging from negative to high, which could be separated into CD28 negative, intermediate (int), and positive, was seen for CD8 T cells. We studied 26 IM patients who were diagnosed on the basis of standard methods and found that all patients had the continuous CD28 spectrum. CD28 is a costimulatory molecule on T cells, and its expression is associated with the subdivision of CD8 cells into cytotoxic (CD28-positive) and suppressor (CD28-negative) T cells. After 24 h of ex vivo culturing, however, the continuous spectrum was found to consist of only CD28-positive and CD28-negative CD8 T cells, because the CD28-int cells had disappeared due to apoptosis. The CD28-int T cells have several cytotoxic functions, suggesting that CD28-int T cells are effectors. Examination of other costimulatory markers in AIM patients showed that CD80 and CD152 were not affected. In patients with other viral infections, such as measles or rubella, however, the continuous spectrum was not detected. These results suggest that there is an unusual CD28 expression pattern in patients with AIM, namely, the presence of a functional CD28-int subset among CD8 T cells. These findings are of special importance for clarifying the defense mechanism against Epstein-Barr virus infection, and the role of CD28 molecules in humans and should also be helpful for the diagnosis of AIM.
Novel Design Architecture for Genetic Stability of Recombinant Poliovirus: the Manipulation of G/C Contents and Their Distribution Patterns Increases the Genetic Stability of Inserts in a Poliovirus-Based RPS-Vax Vector System
Authors: Lee Sang-Gu , Kim Dae-You , Hyun Byung-Hwa , Bae Yong-Soo
Published: July 2002

Poliovirus has been studied as a live recombinant vaccine vector because of its attractive characteristics. The genetic instability, however, has hampered recombinant polioviruses (PVs) from being developed as an appropriate vaccine. A variety of different foreign inserts were cloned directly into our poliovirus Sabin 1-based RPS-Vax vector system, resulting in the production of recombinant PVs. The genetic stability of each recombinant PV was examined during 12 rounds of consecutive passage. It was found that the genetic stability of the recombinants was not well correlated with their insert size. Instead, elevated stability was frequently observed in recombinants with inserts of high G/C contents. Furthermore, a comparative study using different constructs of the human immunodeficiency virus
env
gene revealed that the internal deletion of the unstable insert was seemingly caused by the presence of the adjacent A/T-rich region. The instability of these inserts was completely remedied by (i) increasing the G/C contents and (ii) replacing the local A/T-rich region with the G/C-rich codon without a change of the amino acid. This means that stability is closely associated with the G/C content and the G/C distribution pattern. To see whether these findings can be applied to the design of genetically stable recombinant PV, we have reconstructed the heteromultimeric insert based on our design architecture, including the above-mentioned G/C rules and the template/ligation-free PCR protocol. The heteromultimeric insert was very unstable, as expected, but the manipulated insert with the same amino acid sequence showed complete genetic stability, not only in vitro, but also in vivo. Even though this guideline was established with our RPS-Vax vector system, to some extent, it can also be applied to other live viral vaccine vectors.
Alteration of Sendai Virus Morphogenesis and Nucleocapsid Incorporation due to Mutation of Cysteine Residues of the Matrix Protein
Authors: Sakaguchi Takemasa , Uchiyama Tsuneo , Huang Cheng , Fukuhara Noriko , Kiyotani Katsuhiro , Nagai Yoshiyuki , Yoshida Tetsuya
Published: July 2002

The matrix (M) protein of Sendai virus (SeV) has five cysteine residues, at positions 83, 106, 158, 251, and 295. To determine the roles of the cysteine residues in viral assembly, we generated mutant M cDNA possessing a substitution to serine at one of the cysteine residues or at all of the cysteine residues. Some mutant M proteins were unstable when expressed in cultured cells, suggesting that cysteine residues affect protein stability, probably by disrupting the proper conformation. In an attempt to generate virus from cDNA, SeV M-C
83
S, SeV M-C
106
S, and SeV M-C
295
S were successfully recovered from cDNA, while recombinant SeVs possessing other mutations were not. SeV M-C
83
S and SeV M-C
106
S had smaller virus particles than did the wild-type SeV, whereas SeV M-C
295
S had larger and heterogeneously sized particles. Furthermore, SeV M-C
106
S had a significant amount of empty particles lacking nucleocapsids. These results indicate that a single-point mutation at a cysteine residue of the M protein affects virus morphology and nucleocapsid incorporation, showing direct involvement of the M protein in SeV assembly. Cysteine-dependent conformation of the M protein was not due to disulfide bond formation, since the cysteines were shown to be free throughout the viral life cycle.
Ubiquitination of both Adeno-Associated Virus Type 2 and 5 Capsid Proteins Affects the Transduction Efficiency of Recombinant Vectors
Authors: Yan Ziying , Zak Roman , Luxton G. W. Gant , Ritchie Teresa C. , Bantel-Schaal Ursula , Engelhardt John F.
Published: July 2002

In the presence of complementing adeno-associated virus type 2 (AAV-2) Rep proteins, AAV-2 genomes can be pseudotyped with the AAV-5 capsid to assemble infectious virions. Using this pseudotyping strategy, the involvement of the ubiquitin-proteasome system in AAV-5 and AAV-2 capsid-mediated infections was compared. A recombinant AAV-2 (rAAV-2) proviral luciferase construct was packaged into both AAV-2 and AAV-5 capsid particles, and transduction efficiencies in a number of cell lines were compared. Using luciferase expression as the end point, we demonstrated that coadministration of the viruses with proteasome inhibitors not only increased the transduction efficiency of rAAV-2, as previously reported, but also augmented rAAV-5-mediated gene transfer. Increased transgene expression was independent of viral genome stability, since there was no significant difference in the amounts of internalized viral DNA in the presence or absence of proteasome inhibitors. Western blot assays of immunoprecipitated viral capsid proteins from infected HeLa cell lysates and in vitro reconstitution experiments revealed evidence for ubiquitin conjugation of both AAV-2 and AAV-5 capsids. Interestingly, heat-denatured virus particles were preferential substrates for in vitro ubiquitination, suggesting that endosomal processing of the viral capsid proteins is a prelude to ubiquitination. Furthermore, ubiquitination may be a signal for processing of the capsid at the time of virion disassembly. These studies suggest that the previously reported influences of the ubiquitin-proteasome system on rAAV-2 transduction are also active for rAAV-5 and provide a clearer mechanistic framework for understanding the functional significance of ubiquitination.
Distinct Glycoprotein O Complexes Arise in a Post-Golgi Compartment of Cytomegalovirus-Infected Cells
Authors: Theiler Regan N. , Compton Teresa
Published: July 2002

Human cytomegalovirus (CMV) glycoproteins H, L, and O (gH, gL, and gO, respectively) form a heterotrimeric disulfide-bonded complex that participates in the fusion of the viral envelope with the host cell membrane. During virus maturation, this complex undergoes a series of intracellular assembly and processing events which are not entirely defined (M. T. Huber and T. Compton, J. Virol. 73:3886-3892, 1999). Here, we demonstrate that gO does not undergo the same posttranslational processing in transfected cells as it does in infected cells. We further determined that gO is modified by O-linked glycosylation and that this terminally processed form is highly enriched in virions. However, during studies of gO processing, novel gO complexes were discovered in CMV virions. The newly identified gO complexes, including gO-gL heterodimers, were not readily detected in CMV-infected cells. Further characterization of the trafficking of gO through the secretory pathway of infected cells localized gH, gL, and gO primarily to the Golgi apparatus and trans-Golgi network, supporting the conclusion that the novel virion-associated gO complexes arise in a post-Golgi compartment of infected cells.
Construction of a Human Immunodeficiency Virus Type 1 (HIV-1) Library Containing Random Combinations of Amino Acid Substitutions in the HIV-1 Protease due to Resistance by Protease Inhibitors
Authors: Yusa Keisuke , Song Wei , Bartelmann Matthias , Harada Shinji
Published: July 2002

Human immunodeficiency virus type 1 (HIV-1) heterogeneity contributes to the emergence of drug-resistant virus, escape from host defense systems, and/or conversion of the cellular tropism. To establish an in vitro system to address a heterogeneous virus population, we constructed a library of HIV-1 molecular clones containing a set of random combinations of zero to 11 amino acid substitutions associated with resistance to protease inhibitors by the HIV-1 protease. The complexity (2.1 × 10
5
) of the HIV-1 library pNG-PRL was large enough to cover all of the possible combinations of zero to 11 amino acid substitutions (a total of 4,096 substitutions possible). The T-cell line MT-2 was infected with the HIV-1 library, and resistant viruses were selected after treatment by the protease inhibitor ritonavir (0.03 to 0.30 μM). The viruses that contained three to eight amino acid substitutions could be selected within 2 weeks. These results demonstrate that this HIV-1 library could serve as an alternative in vitro system to analyze the emergence of drug resistance and to evaluate the antiviral activity of novel compounds against multidrug-resistant viruses.
Ty5
gag
Mutations Increase Retrotransposition and Suggest a Role for Hydrogen Bonding in the Function of the Nucleocapsid Zinc Finger
Authors: Gao Xiang , Rowley Daniel J. , Gai Xiaowu , Voytas Daniel F.
Published: July 2002

The Ty5 retrotransposon of
Saccharomyces paradoxus
transposes in
Saccharomyces cerevisiae
at frequencies 1,000-fold lower than do the native Ty1 elements. The low transposition activity of Ty5 could be due to differences in cellular environments between these yeast species or to naturally occurring mutations in Ty5. By screening of a Ty5 mutant library, two single mutants (D252N and Y68C) were each found to increase transposition approximately sixfold. When combined, transposition increased 36-fold, implying that the two mutations act independently. Neither mutation affected Ty5 protein synthesis, processing, cDNA recombination, or target site choice. However, cDNA levels in both single mutants and the double mutant were significantly higher than in the wild type. The D252N mutation resides in the zinc finger of nucleocapsid and increases the potential for hydrogen bonding with nucleic acids. We generated other mutations that increase the hydrogen bonding potential (i.e., D252R and D252K) and found that they similarly increased transposition. This suggests that hydrogen bonding within the zinc finger motif is important for cDNA production and builds upon previous studies implicating basic amino acids flanking the zinc finger as important for zinc finger function. Although NCp zinc fingers differ from the zinc finger motifs of cellular enzymes, the requirement for efficient hydrogen bonding is likely universal.
Replication Fidelity of the
supF
Gene Integrated in the Thymidine Kinase Locus of Herpes Simplex Virus Type 1
Authors: Hwang Ying T. , Liu Bu-Yuan , Hwang Charles B. C.
Published: July 2002

Recombinant viruses were constructed to have an
Escherichia coli
replicon containing a mutagenesis marker, the
supF
gene, integrated within the thymidine kinase locus (
tk
) of herpes simplex virus type 1. These viruses expressed either wild-type or mutant DNA polymerase (Pol) and were tested in a mutagenesis assay for the fidelity of their replication of the
supF
gene. A mutation frequency of approximately 10
−4
was observed for wild-type strain KOS-derived recombinants in their replication of the
supF
gene. However, recombinants derived from the PAA
r
5 Pol mutant, which has been demonstrated to have an antimutator phenotype in replicating the
tk
gene, had three- to fourfold increases in
supF
mutation frequency (
P
< 0.01), a result similar to that exhibited when the
supF
gene was induced to replicate as episomal DNA (Y. T. Hwang, B.-Y. Liu, C.-Y. Hong, E. J. Shillitoe, and C. B. C. Hwang, J. Virol. 73:5326-5332, 1999). Thus, the PAA
r
5 Pol mutant had an antimutator function in replicating the
tk
gene and was less accurate in replicating the
supF
gene than was the wild-type strain. The spectra of mutations and distributions of substituted bases within the
supF
genes that replicated as genomic DNA were different from those in the genes that replicated as episomal DNA. Therefore, the differences in sequence contents between the two target genes influenced the accuracy of the Pol during viral replication. Furthermore, the replication mode of the target gene also affected the mutational spectrum.
Oligomerization and Cooperative RNA Synthesis Activity of Hepatitis C Virus RNA-Dependent RNA Polymerase
Authors: Wang Q. May , Hockman Michelle A. , Staschke Kirk , Johnson Robert B. , Case Katharine A. , Lu Jirong , Parsons Steve , Zhang Faming , Rathnachalam Radhakrishnan , Kirkegaard Karla , Colacino Joseph M.
Published: July 2002

The NS5B RNA-dependent RNA polymerase encoded by hepatitis C virus (HCV) plays a key role in viral replication. Reported here is evidence that HCV NS5B polymerase acts as a functional oligomer. Oligomerization of HCV NS5B protein was demonstrated by gel filtration, chemical cross-linking, temperature sensitivity, and yeast cell two-hybrid analysis. Mutagenesis studies showed that the C-terminal hydrophobic region of the protein was not essential for its oligomerization. Importantly, HCV NS5B polymerase exhibited cooperative RNA synthesis activity with a dissociation constant,

K
d

, of ≈22 nM, suggesting a role for the polymerase-polymerase interaction in the regulation of HCV replicase activity. Further functional evidence includes the inhibition of the wild-type NS5B polymerase activity by a catalytically inactive form of NS5B. Finally, the X-ray crystal structure of HCV NS5B polymerase was solved at 2.9 Å. Two extensive interfaces have been identified from the packing of the NS5B molecules in the crystal lattice, suggesting a higher-order structure that is consistent with the biochemical data.
Selective STAT Protein Degradation Induced by Paramyxoviruses Requires both STAT1 and STAT2 but Is Independent of Alpha/Beta Interferon Signal Transduction
Authors: Parisien Jean-Patrick , Lau Joe F. , Rodriguez Jason J. , Ulane Christina M. , Horvath Curt M.
Published: July 2002

The alpha/beta interferon (IFN-α/β)-induced STAT signal transduction pathway leading to activation of the ISGF3 transcription complex and subsequent antiviral responses is the target of viral pathogenesis strategies. Members of the
Rubulavirus
genus of the Paramyxovirus family of RNA viruses have acquired the ability to specifically target either STAT1 or STAT2 for proteolytic degradation as a countermeasure for evading IFN responses. While type II human parainfluenza virus induces STAT2 degradation, simian virus 5 induces STAT1 degradation. The components of the IFN signaling system that are required for STAT protein degradation by these paramyxoviruses have been investigated in a series of human somatic cell lines deficient in IFN signaling proteins. Results indicate that neither the IFN-α/β receptor, the tyrosine kinases Jak1 or Tyk2, nor the ISGF3 DNA-binding subunit, IFN regulatory factor 9 (IRF9), is required for STAT protein degradation induced by either virus. Nonetheless, both STAT1 and STAT2 are strictly required in the host cell to establish a degradation-permissive environment enabling both viruses to target their respective STAT protein. Complementation studies reveal that STAT protein-activating tyrosine phosphorylation and functional src homology 2 (SH2) domains are dispensable for creating a permissive STAT degradation environment in degradation-incompetent cells, but the N terminus of the missing STAT protein is essential. Protein-protein interaction analysis indicates that V and STAT proteins interact physically in vitro and in vivo. These results constitute genetic and biochemical evidence supporting a virus-induced, IFN-independent STAT protein degradation complex that contains at least STAT1 and STAT2.
Certain Problems with the Application of Stochastic Diffusion Processes for the Description of Chemical Engineering Phenomena. Formulation of Boundary Conditions in Flow Systems
Authors: Kudrna Vladimír , Hasal Pavel , Vejmola Libor
Published: July 2002

Problems associated with the formulation of the boundary conditions for diffusion equations describing flow-through chemical-engineering systems from the point of view of stochastic process theory are discussed. An approach to modelling such systems is presented, allowing the one-dimensional diffusion (dispersion) model of a continuous flow mixer, commonly used in chemical engineering, to be reassessed from a rather general point of view.
Pervaporation of Ethanol and Water Through Polymeric Membranes with Incorporated N-(Halogenophenyl)maleimide Units
Authors: Lokaj Jan , Bílá Jitka
Published: July 2002

Styrene N-(chloro- or bromophenyl)maleimide and methyl methacrylate N-(chloro- or bromophenyl)maleimide copolymers were synthesized. Membranes casted from the copolymer solutions were used for the pervaporation of ethanol water mixtures at 35 °C. The membranes were characterized by the separation factor related to the preferentially transported water and by the permeate flux. The pervaporation characteristics were dependent on the hydrophilicity or polarity of the polymer chains, on the kind and position of the halogen and on the composition of the mixture to be separated. Membranes made from the copolymers of styrene with N-(2-chlorophenyl)maleimide or N-(2-bromophenyl)maleimide show the highest separation factors in the separation of concentrated ethanol solutions. The permeate flux through membranes made from the copolymers of methyl methacrylate with N-(halogenophenyl)maleimide was by one order of magnitude higher than through membranes made from similar styrene copolymers.
Conformations of Insoluble Blocks in Swollen Micellar Cores of Multimolecular Block Copolymer Micelles Studied by Monte Carlo Simulation Technique
Authors: Procházka Karel , Limpouchová Zuzana
Published: July 2002

Monte Carlo simulations of chain conformations in restricted spherical volumes with an increasing radius were performed on a tetrahedral lattice (ca 2 700 to 9 200 lattice sites) at relatively high densities of the occupied lattice sites. A simultaneous self-avoiding walk together with the equilibration algorithm similar to that of Siepmann and Frenkel were used to create the equilibrated multi-chain conformations. (a) A series of simulations was carried out for a constant average segment density,  = 0.52, together with the three values of the radius of the sphere, R = 10 l, 12.5 l and 15 l (l is the lattice distance), and various numbers of chains, N ∈ <15, 86>, and chain lengths, L ∈ <31, 163>. The results give information on the system behavior and on the effects of: (i) multi-chain conformational correlations, which depend both on N and L, (ii) the L-dependent chain flexibility, and (iii) R-dependent external geometrical constraints. Another two series of data: (b) for a constant average segment density,  = 0.36, a constant N = 21, and L proportional to R3, and (c) for  = 0.36, L = 47 and N proportional to R3, are shown to give a supplementary detailed information on conformational behavior of individual chains. Various physical quantities (e.g. the densities of chain free ends, gF(r), or distributions of the tethered end-to-the free end distances, ρTF(rTF), etc.) were calculated in the course of computer simulations and their shapes and physical significance is discussed with respect to the changing values of N, L and R.
Kinetics of Substitution of 4-Methoxyphenylazo Groups of 2,6-Dioxo-5(3)-(4-methoxyphenylazo)-3(5)-(4-methoxyphenylhydrazono)-1,2,3,6-tetrahydropyridine-4-carboxylic Acid by Reaction with 4-Nitrobenzenediazonium Cation
Authors: Horáčková Jaroslava , Štěrba Vojeslav
Published: July 2002

Kinetics have been studied of gradual replacement of 4-methoxyphenylazo groups in 2,6-dioxo-5(3)-(4-methoxyphenylazo)-3(5)-(4-methoxyphenylhydrazono)-1,2,3,6-tetrahydropyridine-4-carboxylic acid (IIIa) by 4-nitrophenylazo groups using the reaction with 4-nitrobenzenediazonium cation (IIc) in acetate and phosphate buffers. The rate constant of replacement of the second methoxyphenylazo group is lower by a factor of ca 60. From the experimentally found pKa values of the corresponding azohydrazone compounds with methoxy, chloro, or nitro substituent at 4-position (IIIa - IIIf) it has been concluded that the 5(3)-(4-methoxyphenylazo)-3(5)-(4-nitrophenylhydrazono) derivative is formed in the first step.
Excess Volume of Mixtures of Oxaalkanes and Branched Alkanes
Authors: Trejo Luis Miguel , Costas Miguel , Andreoli-Ball Lina , Patterson Donald
Published: July 2002

Equimolar values of the excess volume, VE, have been obtained at 25 °C for a series of oxaalkanes, 2,5-dioxahexane, 2,5,8-trioxanonane, 2,5,8,11-tetraoxadodecane, and 2,5,8,11,14-pentaoxapentadecane, mixed with a series of highly-branched alkanes: 2,2-dimethylbutane, 2,2,4-trimethylpentane, 2,2,4,6,6-pentamethylheptane, and 2,2,4,4,6,8,8-heptamethylnonane. The Prigogine-Flory thermodynamic theory predicts the large variations of VE with the chain-lengths of the molecules in the two series.
A Return to the Plesek Reaction and Some Useful Variations. Carbon-Substituted Methyl and Phenyl Derivatives of 5,6-Dicarba-nido-decaborane(12), nido-5,6-C2B8H12
Authors: Štíbr Bohumil , Holub Josef , Jelínek Tomáš , Grüner Bohumír , Fusek Jiří , Plzák Zbyněk , Teixidor Francesc , Viñas Clara , Kennedy John D.
Published: July 2002

A modification of the Plesek reaction has been employed to improve the yield in the synthesis of the parent nido ten-vertex dicarbaborane 5,6-C2B8H12, and to synthesize some of its 5- and 6-substituted derivatives. The method is based on the oxidation of the anions [7,8-R1R2-nido-5,6-C2B9H10]- with acidic aqueous of [Fe(H2O)6]Cl3 to produce the series of substituted dicarbaboranes 5,6-R1R2-nido-5,6-C2B8H10, where R1, R2 = H, H; H, Me; Me, H; Me, Me; H, Ph; Ph, H, and Ph, Ph. The characteristic reaction mode is an oxidative elimination of one boron vertex that is adjacent to the carbon vertex in the open pentagonal face of the starting eleven-vertex nido dicarbaborane anion. The products are characterized by mass spectrometry and 1H and 11B NMR spectroscopy including [11B-11B]-COSY and 1H-{11B(selective)} measurements that permit complete assignments of all the observed resonances to individual cluster {BH} units.
Syntheses of Insect Sex Pheromones. A Review of the Literature 1990-1998
Authors: Koutek Bohumír , Streinz Ludvík , Romaňuk Miroslav
Published: July 2002

The term "insect sex pheromones" denotes compounds used for chemical communication between sexual partners in the insect kingdom. The aim of this review covering the literature 1990-1998 is to survey papers on their syntheses. A review with 201 references.
The "Cyclohexane-Like" Structure of (MeOBF2)3 and Other Species Arising from Mixing (MeO)3B and BF3. An ab initio/IGLO/NMR Investigation
Authors: Rasul Golam , Williams Robert E.
Published: July 2002

An ab initio/IGLO/NMR investigation shows that (MeOBF2)3, 3, is the thermodynamically most stable product when (MeO)3B and BF3 are mixed in a 1 : 2 ratio. Compound 3 has a symmetrical, "cyclohexane-like", structure with C3v symmetry. The ab initio calculated structures of less stable intermediates, i.e. the monomeric MeOBF2, 1, the dimeric (MeOBF2)2, 2, the "diadduct" MeOB(O(BF3)Me)O(BF3)Me, 4, and the "monoadduct" (MeO)2BO(BF3)Me, 5, are also discussed.
3-(Tributylstannyl)allyl Alcohols: Useful Building Blocks for Solid-Phase Synthesis of Skipped Dienes and Trienes
Authors: Havránek Miroslav , Dvořák Dalimil
Published: July 2002

Repeated Stille coupling of 3-substituted 3-(tributylstannyl)allyl alcohols 2 on a solid support was used to synthesize a 21 × 21 library of skipped dienes and a 21 × 21 × 21 library of skipped trienes. Starting 3-(tributylstannyl)allyl alcohols were prepared by Pd-catalyzed hydrostannation of substituted prop-2-yn-1-ols, by hydroalumination by LiAlH4 followed with transmetallation to tin using tributyltin methoxide, or by substitution of chlorine in (Z)-6-chloro-3-(tributylstannyl)hex-2-en-1-ol with appropriate nucleophile. Synthesized libraries were tested for the activity to endorphin receptors, but with negative results.
Reaction of Aromatic Radical Cations with RuCl3·3H2O
Authors: Michman Michael , Oron Miriam , Schaefer Hans J.
Published: July 2002

Interactions of RuCl3·3H2O with radical cations of aromatic ethers, 1,4-di-tert-butyl- 2,5-dimethoxybenzene, 3,5-di-tert-butyl-1,2-dimethoxybenzene and 2-tert-butyl-1,4-di- methoxybenzene, and with aromatic amines, 2,4,6-tri-tert-butylaniline and N,N,N',N'-tetra- methyl-1,4-phenylenediamine, were observed by voltammetry. ESR and UV spectroscopies were used for the study of the the first two ethers. The effect of RuCl3·3H2O was also examined by controlled potential electrolysis.
Analytic Energy Derivatives for the Direct Iterative Approach to the Generalized Bloch Equation
Authors: Meissner Holger , Paldus Josef
Published: July 2002

A general formalism for the analytic energy derivatives in the context of the recently developed state-selective version of the direct iterative approach to the generalized Bloch equation is presented. An explicit formalism is developed for both the gradients and the Hessian by exploiting the so-called Z-vector method. A procedure for the development of the corresponding algorithm for higher than the second-order properties is also briefly outlined.