bluuebunny/crossref_metadata_2025 huggingface dataset:
Plasmid-Encoded
asp
Operon Confers a Proton Motive Metabolic Cycle Catalyzed by an Aspartate-Alanine Exchange Reaction
Authors: Abe Keietsu , Ohnishi Fumito , Yagi Kyoko , Nakajima Tasuku , Higuchi Takeshi , Sano Motoaki , Machida Masayuki , Sarker Rafiquel I. , Maloney Peter C.
Published: July 2002

Tetragenococcus halophila
D10 catalyzes the decarboxylation of
l
-aspartate with nearly stoichiometric release of
l
-alanine and CO
2
. This trait is encoded on a 25-kb plasmid, pD1. We found in this plasmid a putative
asp
operon consisting of two genes, which we designated
aspD
and
aspT
, encoding an
l
-aspartate-β-decarboxylase (AspD) and an aspartate-alanine antiporter (AspT), respectively, and determined the nucleotide sequences. The sequence analysis revealed that the genes of the
asp
operon in pD1 were in the following order: promoter →
aspD
→
aspT
. The deduced amino acid sequence of AspD showed similarity to the sequences of two known
l
-aspartate-β-decarboxylases from
Pseudomonas dacunhae
and
Alcaligenes faecalis
. Hydropathy analyses suggested that the
aspT
gene product encodes a hydrophobic protein with multiple membrane-spanning regions. The operon was subcloned into the
Escherichia coli
expression vector pTrc99A, and the two genes were cotranscribed in the resulting plasmid, pTrcAsp. Expression of the
asp
operon in
E. coli
coincided with appearance of the capacity to catalyze the decarboxylation of aspartate to alanine. Histidine-tagged AspD (AspDHis) was also expressed in
E. coli
and purified from cell extracts. The purified AspDHis clearly exhibited activity of
l
-aspartate-β-decarboxylase. Recombinant AspT was solubilized from
E. coli
membranes and reconstituted in proteoliposomes. The reconstituted AspT catalyzed self-exchange of aspartate and electrogenic heterologous exchange of aspartate with alanine. Thus, the
asp
operon confers a proton motive metabolic cycle consisting of the electrogenic aspartate-alanine antiporter and the aspartate decarboxylase, which keeps intracellular levels of alanine, the countersubstrate for aspartate, high.
Agilent 2100 Bioanalyzer for Restriction Fragment Length Polymorphism Analysis of the
Campylobacter jejuni
Flagellin Gene
Authors: Nachamkin Irving , Panaro Nicholas J. , Li Ming , Ung Huong , Yuen Po Ki , Kricka Larry J. , Wilding Peter
Published: July 2002

The Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto, Calif.) utilizes capillary electrophoresis on a microchip device (LabChip 7500; Caliper Technologies, Mountain View, Calif.) that is capable of rapidly sizing small DNA fragments. To determine whether the system could replace conventional restriction fragment length polymorphism (RFLP) typing by agarose gel electrophoresis, we compared the analyzer with conventional flagellin RFLP for typing
Campylobacter jejuni
. Ninety-seven isolates representing 46 Fla types were initially analyzed. Correct Fla types were detected in 59% of the isolates. The major problem with the system was in resolving samples containing multiple DNA fragments differing from 8 to 20 bp. Overall, the bioanalyzer has the potential to replace conventional RFLP analysis by gel electrophoresis, but improvements in the chip separation are needed.
Chlamydial Serology: Comparative Diagnostic Value of Immunoblotting, Microimmunofluorescence Test, and Immunoassays Using Different Recombinant Proteins as Antigens
Authors: Bas S. , Muzzin P. , Ninet B. , Bornand J. E. , Scieux C. , Vischer T. L.
Published: July 2002

To improve the reliability of the serodiagnosis of
Chlamydia trachomatis
infections, an immunoblot analysis, a microimmunofluorescence titration, and different immunoassays using synthetic peptides derived from species-specific epitopes in variable domain IV of the major outer membrane protein or recombinant antigens (heat shock protein 70 [hsp70], hsp60, hsp10, polypeptide encoded by open reading frame 3 of the plasmid [pgp3], macrophage infectivity potentiator, and a fragment of the total lipopolysaccharide) were evaluated. Because cross-reactions between chlamydial species have been reported, the microimmunofluorescence tests were also performed with
Chlamydia pneumoniae
and
Chlamydia psittaci
used as antigens, and
C. pneumoniae
-specific antibodies were also determined by immunoassays. Since the presence of antimicrobial antibodies must be interpreted in light of their prevalence in the general population, responses obtained with serum samples from patients with well-defined infection (i.e., with positive urethral or endocervical
C. trachomatis
DNA amplification) were compared to those obtained with samples from healthy blood donors. The best sensitivity (86%) with a specificity of 81% was obtained for immunoblotting results, when the number of individuals with ≥10 immunoglobulin G (IgG) and/or ≥2 IgM responses to the different
C. trachomatis
antigens was considered. A 13-kDa antigen was recognized by most of the samples (86% for IgG) from patients with acute urogenital infection but rarely (3%) by those from healthy blood donors (
P
< 0.0001). The sensitivity and specificity results obtained for serum antibodies to peptides or recombinant antigens were slightly lower than those results obtained for the number of responses to whole
C. trachomatis
antigens, which were 76 and 77%, respectively, when IgG responses to both recombinant hsp60 and pgp3 were considered.
Human Babesiosis in Japan: Isolation of
Babesia microti
-Like Parasites from an Asymptomatic Transfusion Donor and from a Rodent from an Area Where Babesiosis Is Endemic
Authors: Wei Qiang , Tsuji Masayoshi , Zamoto Aya , Kohsaki Masatoshi , Matsui Toshimitsu , Shiota Tsunezo , Telford Sam R. , Ishihara Chiaki
Published: July 2002

To determine the source of infection for the Japanese index case of human babesiosis, we analyzed blood samples from an asymptomatic individual whose blood had been transfused into the patient. In addition, we surveyed rodents collected from near the donor's residence. Examination by microscopy and PCR failed to detect the parasite in the donor's blood obtained 8 months after the donation of the blood that was transfused. However, we were able to isolate
Babesia
parasites by inoculating the blood sample into SCID mice whose circulating red blood cells (RBCs) had been replaced with human RBCs. A
Babesia
parasite capable of propagating in human RBCs was also isolated from a field mouse (
Apodemus speciosus
) captured near the donor's residential area. Follow-up surveys over a 1-year period revealed that the donor continued to be asymptomatic but had consistently high immunoglobulin G (IgG) titers in serum and low levels of parasitemia which were microscopically undetectable yet which were repeatedly demonstrable by inoculation into animals. The index case patient's sera contained high titers of IgM and, subsequently, rising titers of IgG antibodies, both of which gradually diminished with the disappearance of the parasitemia. Analysis of the parasite's rRNA gene (rDNA) sequence and immunodominant antigens revealed the similarity between donor and patient isolates. The rodent isolate also had an rDNA sequence that was identical to that of the human isolates but that differed slightly from that of the human isolates by Western blot analysis. We conclude that the index case patient acquired infection by transfusion from a donor who became infected in Japan, that parasitemia in an asymptomatic carrier can persist for more than a year, and that
A. speciosus
serves as a reservoir of an agent of human babesiosis in Japan.
Identification of
Mycobacterium
spp. by Using a Commercial 16S Ribosomal DNA Sequencing Kit and Additional Sequencing Libraries
Authors: Cloud J. L. , Neal H. , Rosenberry R. , Turenne C. Y. , Jama M. , Hillyard D. R. , Carroll K. C.
Published: July 2002

Current methods for identification of
Mycobacterium
spp. rely upon time-consuming phenotypic tests, mycolic acid analysis, and narrow-spectrum nucleic acid probes. Newer approaches include PCR and sequencing technologies. We evaluated the MicroSeq 500 16S ribosomal DNA (rDNA) bacterial sequencing kit (Applied Biosystems, Foster City, Calif.) for its ability to identify
Mycobacterium
isolates. The kit is based on PCR and sequencing of the first 500 bp of the bacterial rRNA gene. One hundred nineteen mycobacterial isolates (94 clinical isolates and 25 reference strains) were identified using traditional phenotypic methods and the MicroSeq system in conjunction with separate databases. The sequencing system gave 87% (104 of 119) concordant results when compared with traditional phenotypic methods. An independent laboratory using a separate database analyzed the sequences of the 15 discordant samples and confirmed the results. The use of 16S rDNA sequencing technology for identification of
Mycobacterium
spp. provides more rapid and more accurate characterization than do phenotypic methods. The MicroSeq 500 system simplifies the sequencing process but, in its present form, requires use of additional databases such as the Ribosomal Differentiation of Medical Microorganisms (RIDOM) to precisely identify subtypes of type strains and species not currently in the MicroSeq library.
High Variability between Results of Different In-House Tests for Cytomegalovirus (CMV) Monitoring and a Standardized Quantitative Plasma CMV PCR Assay
Authors: von Müller Lutz , Hampl Walter , Hinz Joachim , Meisel Helga , Reip Angela , Engelmann Elisabeth , Heilbronn Regine , Gärtner Barbara , Krämer Oliver , Einsele Hermann , Hebart Holger , Ljubicic Tatjana , Löffler Jürgen , Mertens Thomas
Published: July 2002

A total of 2,718 blood samples were analyzed in five virological laboratories for the presence of cytomegalovirus (CMV) by in-house tests and one standardized plasma PCR assay. Results from in-house tests showed remarkable variability. Detection of CMV pp65 antigen or DNA from cells was more sensitive than that by plasma CMV PCR assay.
A Rev-Independent Human Immunodeficiency Virus Type 1 (HIV-1)-Based Vector That Exploits a Codon-Optimized HIV-1
gag-pol
Gene
Authors: Kotsopoulou Ekaterini , Kim V. Narry , Kingsman Alan J. , Kingsman Susan M. , Mitrophanous Kyriacos A.
Published: July 2002

The human immunodeficiency virus (HIV) genome is AU rich, and this imparts a codon bias that is quite different from the one used by human genes. The codon usage is particularly marked for the
gag
,
pol
, and
env
genes. Interestingly, the expression of these genes is dependent on the presence of the Rev/Rev-responsive element (RRE) regulatory system, even in contexts other than the HIV genome. The Rev dependency has been explained in part by the presence of RNA instability sequences residing in these coding regions. The requirement for Rev also places a limitation on the development of HIV-based vectors, because of the requirement to provide an accessory factor. We have now synthesized a complete codon-optimized HIV-1
gag-pol
gene. We show that expression levels are high and that expression is Rev independent. This effect is due to an increase in the amount of
gag-pol
mRNA. Provision of the RRE in
cis
did not lower protein or RNA levels or stimulate a Rev response. Furthermore we have used this synthetic
gag-pol
gene to produce HIV vectors that now lack all of the accessory proteins. These vectors should now be safer than murine leukemia virus-based vectors.
Kaposi's Sarcoma-Associated Herpesvirus Open Reading Frame 50/Rta Protein Activates the Entire Viral Lytic Cycle in the HH-B2 Primary Effusion Lymphoma Cell Line
Authors: Gradoville Lyndle , Gerlach Jennifer , Grogan Elizabeth , Shedd Duane , Nikiforow Sarah , Metroka Craig , Miller George
Published: July 2002

Rta, the gene product of Kaposi's sarcoma-associated herpesvirus (KSHV) encoded mainly in open reading frame 50 (ORF50), is capable of activating expression of viral lytic cycle genes. What was not demonstrated in previous studies was whether KSHV Rta was competent to initiate the entire viral lytic life cycle including lytic viral DNA replication, late-gene expression with appropriate kinetics, and virus release. In HH-B2, a newly established primary effusion lymphoma (PEL) cell line, KSHV ORF50 behaved as an immediate-early gene and autostimulated its own expression. Expression of late genes, ORF65, and K8.1 induced by KSHV Rta was eliminated by phosphonoacetic acid, an inhibitor of viral DNA polymerase. Transfection of KSHV Rta increased the production of encapsidated DNase-resistant viral DNA from HH-B2 cells. Thus, introduction of an ORF50 expression plasmid is sufficient to drive the lytic cycle to completion in cultured PEL cells.
The Fusion Glycoprotein of Human Respiratory Syncytial Virus Facilitates Virus Attachment and Infectivity via an Interaction with Cellular Heparan Sulfate
Authors: Feldman Steven A. , Audet Susette , Beeler Judy A.
Published: July 2002

Human respiratory syncytial virus (RSV) F glycoprotein (RSV-F) can independently interact with immobilized heparin and facilitate both attachment to and infection of cells via an interaction with cellular heparan sulfate. RSV-glycosaminoglycan (GAG) interactions were evaluated using heparin-agarose affinity chromatography. RSV-F from A2- and B1/
cp
-52 (
cp
-52)-infected cell lysates, RSV-F derived from a recombinant vaccinia virus, and affinity-purified F protein all bound to and were specifically eluted from heparin columns. In infectivity inhibition studies, soluble GAGs decreased the infectivity of RSV A2 and
cp
-52, with bovine lung heparin exhibiting the highest specific activity against both A2 (50% effective dose [ED
50
] = 0.28 ± 0.11 μg/ml) and
cp
-52 (ED
50
= 0.55 ± 0.14 μg/ml). Furthermore, enzymatic digestion of cell surface GAGs by heparin lyase I and heparin lyase III but not chondroitinase ABC resulted in a significant reduction in
cp
-52 infectivity. Moreover, bovine lung heparin inhibited radiolabeled A2 and
cp
-52 virus binding up to 90%. Taken together, these data suggest that RSV-F independently interacts with heparin/heparan sulfate and this type of interaction facilitates virus attachment and infectivity.
Differential Transcription Attenuation of Rabies Virus Genes by Intergenic Regions: Generation of Recombinant Viruses Overexpressing the Polymerase Gene
Authors: Finke Stefan , Cox James H. , Conzelmann Karl-Klaus
Published: July 2002

Gene expression of nonsegmented negative-sense RNA viruses involves sequential synthesis of monocistronic mRNAs and transcriptional attenuation at gene borders resulting in a transcript gradient. To address the role of the heterogeneous rabies virus (RV) intergenic regions (IGRs) in transcription attenuation, we constructed bicistronic model RNAs in which two reporter genes are separated by the RV N/P gene border. Replacement of the 2-nucleotide (nt) N/P IGR with the 5-nt IGRs from the P/M or M/G border resulted in attenuation of downstream gene transcription to 78 or 81%, respectively. A severe attenuation to 11% was observed for the 24-nt G/L border. This indicated that attenuation in RV is correlated with the length of the IGR, and, in particular, severe downregulation of the L (polymerase) gene by the 24 nt IGR. By reverse genetics, we recovered viable RVs in which the strongly attenuating G/L gene border of wild-type (wt) RV (SAD L16) was replaced with N/P-derived gene borders (SAD T and SAD T2). In these viruses, transcription of L mRNA was enhanced by factors of 1.8 and 5.1, respectively, resulting in exaggerated general gene expression, faster growth, higher virus titers, and induction of cytopathic effects in cell culture. The major role of the IGR in attenuation was further confirmed by reintroduction of the wt 24-nt IGR into SAD T, resulting in a ninefold drop of L mRNA. The ability to modulate RV gene expression by altering transcriptional attenuation is an advantage in the study of virus protein functions and in the development of gene delivery vectors.
Novel Class of Thiourea Compounds That Inhibit Herpes Simplex Virus Type 1 DNA Cleavage and Encapsidation: Resistance Maps to the UL6 Gene
Authors: van Zeijl Marja , Fairhurst Jeanette , Jones Thomas R. , Vernon Steven K. , Morin John , LaRocque James , Feld Boris , O'Hara Bryan , Bloom Jonathan D. , Johann Stephen V.
Published: July 2002

In our search for novel inhibitors of herpes simplex virus type 1 (HSV-1), a new class of thiourea inhibitors was discovered.
N
-{4-[3-(5-Chloro-2,4-dimethoxyphenyl)-thioureido]-phenyl}-acetamide and its 2-fluoro-benzamide derivative inhibited HSV-1 replication. HSV-2, human cytomegalovirus, and varicella-zoster virus were inhibited to a lesser extent. The compounds acted late in the replication cycle by impairing both the cleavage of concatameric viral DNA into progeny genome length and the packaging of the DNA into capsids, indicative of a defect in the encapsidation process. To uncover the molecular target of the inhibition, resistant HSV-1 isolates were generated, and the mutation responsible for the resistance was mapped using marker transfer techniques. Each of three independent isolates had point mutations in the UL6 gene which resulted in independent single-amino-acid changes. One mutation was located in the N terminus of the protein (E121D), while two were located close together in the C terminus (A618V and Q621R). Each of these point mutations was sufficient to confer drug resistance when introduced into wild-type virus. The UL6 gene is one of the seven HSV-1 genes known to play a role in DNA packaging. This novel class of inhibitors has provided a new tool for dissection of HSV-1 encapsidation mechanisms and has uncovered a new viable target for the treatment of herpesviral diseases.
The Late Lytic LMP-1 Protein of Epstein-Barr Virus Can Negatively Regulate LMP-1 Signaling
Authors: Erickson Kimberly D. , Martin Jennifer M.
Published: July 2002

The BNLF-1 open reading frame of Epstein-Barr virus (EBV) encodes two related proteins, latent membrane protein-1 (LMP-1) and lytic LMP-1 (lyLMP-1). LMP-1 is a latent protein required for immortalization of human B cells by EBV, whereas lyLMP-1 is expressed during the lytic cycle and is found in the EBV virion. We show here that, in contrast to LMP-1, lyLMP-1 is stable, with a half-life of >20 h in tetradecanoyl phorbol acetate- and butyrate-treated B95-8 cells. Although lyLMP-1 itself has negligible effects on NF-κB activity, it inhibits NF-κB activation by LMP-1 in a dose-dependent manner. The lyLMP-1 protein does not oligomerize with LMP-1, and the negative effect of lyLMP-1 on NF-κB activation by LMP-1 does not result from lyLMP-1-mediated disruption of LMP-1 oligomers. Modulation of LMP-1-activated signaling pathways is the first identified biological activity associated with lyLMP-1, and this activity may contribute to the progression of EBV's lytic cycle.
3′-Azido-3′-Deoxythymidine (AZT) and AZT-Resistant Reverse Transcriptase Can Increase the In Vivo Mutation Rate of Human Immunodeficiency Virus Type 1
Authors: Mansky Louis M. , Bernard Lisa C.
Published: July 2002

How antiretroviral drug resistance influences human immunodeficiency virus type 1 (HIV-1) evolution is not clear. This study tested the hypothesis that antiretroviral drugs such as 3′-azido-3′-deoxythymidine (AZT) can influence the in vivo mutation rate of HIV-1. It was observed that AZT can increase the rate of HIV-1 mutation by a factor of 7 in a single round of replication. In addition, (−)2′,3′-dideoxy-3′-thiacytidine (3TC) was also found to increase the mutation rate of HIV-1 by a factor of 3. It was also found that HIV-1 drug-resistant reverse transcriptase (RT) variants can influence the in vivo mutation rate. Replication of HIV-1 with AZT-resistant RTs increased the mutation rate by as much as a factor of 3, while replication of HIV-1 with a 3TC-resistant RT (M184V) had no significant effect on the mutation rate. It was observed that only high-level, AZT-resistant RT variants could influence the in vivo mutation rate (i.e., M41L/T215Y and M41L/D67N/K70R/T215Y). In total, these observations indicate that both antiretroviral drugs and drug resistance mutations can influence the in vivo mutation rate of HIV-1.
Dependence of Adenovirus Infectivity on Length of the Fiber Shaft Domain
Authors: Shayakhmetov Dmitry M. , Lieber André
Published: July 2002

One of the objectives in adenovirus (Ad) vector development is to target gene delivery to specific cell types. Major attention has been given to modification of the Ad fiber knob, which is thought to determine virus tropism. However, among the human Ad serotypes with different tissue tropisms, not only the knob but also the length of the fiber shaft domain varies significantly. In this study we attempted to delineate the role of fiber length in coxsackievirus-adenovirus receptor (CAR)- and non-CAR-mediated infection. A series of Ad serotype 5 (Ad5) capsid-based vectors containing long or short fibers with knob domains derived from Ad5, Ad9, or Ad35 was constructed and tested in adsorption, internalization, and transduction studies. For Ad5 or Ad9 knob-possessing vectors, a long-shafted fiber was critical for efficient adsorption/internalization and transduction of CAR/αv integrin-expressing cells. Ad5 capids containing short CAR-recognizing fibers were affected in cell adsorption and infection. In contrast, for the chimeric vectors possessing Ad35 knobs, which enter cells by a CAR/αv integrin-independent pathway, fiber shaft length had no significant influence on binding or infectibility on tested cells. The weak attachment of short-shafted Ad5 or Ad9 knob-possessing vectors seems to be causally associated with a charge-dependent repulsion between Ad5 capsid and acidic cell surface proteins. The differences between short- and long-shafted vectors in attachment or infection were abrogated by preincubation of cells with polycations. This study demonstrates that the fiber-CAR interaction is not the sole determinant for tropism of Ad vectors containing chimeric fibers. CAR- and αv integrin-mediated infections are influenced by other factors, including the length of the fiber shaft.
Latent Membrane Protein 2A-Mediated Effects on the Phosphatidylinositol 3-Kinase/Akt Pathway
Authors: Swart Rachel , Ruf Ingrid K. , Sample Jeffery , Longnecker Richard
Published: July 2002

Epstein-Barr virus (EBV) latent membrane protein 2A (LMP2A) is expressed on the membranes of B lymphocytes and blocks B-cell receptor (BCR) signaling in EBV-transformed B lymphocytes in vitro. The phosphotyrosine motifs at positions 74 or 85 and 112 within the LMP2A amino-terminal domain are essential for the LMP2A-mediated block of B-cell signal transduction. In vivo studies indicate that LMP2A allows B-cell survival in the absence of normal BCR signals. A possible role for Akt in the LMP2A-mediated B-cell survival was investigated. The protein kinase Akt is a crucial regulator of cell survival and is activated within B lymphocytes upon BCR cross-linking. LMP2A expression resulted in the constitutive phosphorylation of Akt, and this LMP2A effect is dependent on phosphatidylinositol 3-kinase activity. In addition, recruitment of Syk and Lyn protein tyrosine kinases (PTKs) to tyrosines 74 or 85 and 112, respectively, are critical for LMP2A-mediated Akt phosphorylation. However, the ability of LMP2A to mediate a survival phenotype downstream of Akt could not be detected in EBV-negative Akata cells. This would indicate that LMP2A is not responsible for EBV-dependent Burkitt's lymphoma cell survival.
Relative Replication Fitness of a High-Level 3′-Azido-3′-Deoxythymidine-Resistant Variant of Human Immunodeficiency Virus Type 1 Possessing an Amino Acid Deletion at Codon 67 and a Novel Substitution (Thr→Gly) at Codon 69
Authors: Imamichi Tomozumi , Berg Steve C. , Imamichi Hiromi , Lopez Juan C. , Metcalf Julia A. , Falloon Judith , Lane H. Clifford
Published: July 2002

The combination of an amino acid deletion at codon 67 (Δ67) and Thr-to-Gly change at codon 69 (T69G) in the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) is associated with high-level resistance to multiple RT inhibitors. To determine the relative contributions of the Δ67 and T69G mutations on viral fitness, we performed a series of studies of HIV replication using recombinant variants. A high-level 3′-azido-3′-deoxythymidine (AZT)-resistant variant containing Δ67 plus T69G/K70R/L74I/K103N/T215F/K219Q in RT replicated as efficiently as wild-type virus (Wt). In contrast, the construct without Δ67 exhibited impaired replication (23% of growth of Wt). A competitive fitness study failed to reveal any differences in replication rates between the Δ67+T69G/K70R/L74I/K103N/T215F/K219Q mutant and Wt. Evaluation of proviral DNA sequences over a 3-year period in a patient harboring the multiresistant HIV revealed that the T69G mutation emerged in the context of a D67N/K70R/T215F/K219Q mutant backbone prior to appearance of the Δ67 deletion. To assess the impact of this stepwise accumulation of mutations on viral replication, a series of recombinant variants was constructed and analyzed for replication competence. The T69G mutation was found to confer 2′,3′-dideoxyinosine resistance at the expense of fitness. Subsequently, the development of the Δ67 deletion led to a virus with improved replication and high-level AZT resistance.
Repair of Gaps in Retroviral DNA Integration Intermediates
Authors: Yoder Kristine E. , Bushman Frederic D.
Published: July 2002

Diverse mobile DNA elements are believed to pirate host cell enzymes to complete DNA transfer. Prominent examples are provided by retroviral cDNA integration and transposon insertion. These reactions initially involve the attachment of each element 3′ DNA end to staggered sites in the host DNA by element-encoded integrase or transposase enzymes. Unfolding of such intermediates yields DNA gaps at each junction. It has been widely assumed that host DNA repair enzymes complete attachment of the remaining DNA ends, but the enzymes involved have not been identified for any system. We have synthesized DNA substrates containing the expected gap and 5′ two-base flap structure present in retroviral integration intermediates and tested candidate enzymes for the ability to support repair in vitro. We find three required activities, two of which can be satisfied by multiple enzymes. These are a polymerase (polymerase beta, polymerase delta and its cofactor PCNA, or reverse transcriptase), a nuclease (flap endonuclease), and a ligase (ligase I, III, or IV and its cofactor XRCC4). A proposed pathway involving retroviral integrase and reverse transcriptase did not carry out repair under the conditions tested. In addition, prebinding of integrase protein to gapped DNA inhibited repair reactions, indicating that gap repair in vivo may require active disassembly of the integrase complex.
Accumulation of Terminally Deleted RNAs May Play a Role in Seoul Virus Persistence
Authors: Meyer Barbara J. , Schmaljohn Connie
Published: July 2002

Two independent, long-term infections were analyzed to determine whether changes in viral replication could contribute to the establishment and/or maintenance of persistent Seoul virus infections. Infected cell cultures initially contained high levels of infectious virus and intracellular viral RNA that peaked between approximately 7 to 16 days postinfection and then gradually declined until day 26. After day 26, the viral titers and the levels of the small (S), medium (M), and large (L) viral RNAs varied cyclically until the end of the studies. The changes in the concentrations of the RNAs and titer were similar in pattern and appeared to result from changes in the regulation of replication. Neither internal deletions nor an accumulation of nucleotide changes were found in the RNAs. However, fine mapping and sequence analysis revealed short deletions in some of the RNAs in the conserved complementary terminal sequences believed to contain the signals for initiation of replication and transcription. Deletions at the 3′ termini of S, M, and L virus-sense RNAs (vRNAs) accumulated during the acute phase of infection just before the time that the viral titer and the concentration of vRNAs and virus complementary-sense RNAs (cRNAs) began to decline. The absence of deletions at the 5′ termini of the S, M, and L cRNAs suggests that the 3′-deleted vRNAs may not be replication competent. Thus, as the percentage of 3′-deleted vRNAs increase in the population, they could potentially compete with standard virus and downregulate viral replication. Deletions at the 3′ L cRNA and 5′ L vRNA termini were also observed, and the proportion of these deleted RNAs varied cyclically during the infections. We propose a model in which terminal nucleotide deletions arise by nuclease activity of the viral polymerase. In addition, we speculate that cleaved terminal fragments might be used as primers during replication, resulting in the repair of some of the deleted RNAs.
Full Functional Rescue of a Complete Muscle (TA) in Dystrophic Hamsters by Adeno-Associated Virus Vector-Directed Gene Therapy
Authors: Xiao Xiao , Li Juan , Tsao Yeou-Ping , Dressman Devin , Hoffman Eric P. , Watchko Jon F.
Published: July 2002

Limb girdle muscular dystrophy (LGMD) 2F is caused by mutations in the δ-sarcoglycan (SG) gene. Previously, we have shown successful application of a recombinant adeno-associated virus (AAV) vector for genetic and biochemical rescue in the Bio14.6 hamster, a homologous animal model for LGMD 2F (J. Li et al., Gene Ther. 6:74–82, 1999). In this report, we show efficient and long-term δ-SG expression accompanied by nearly complete recovery of physiological function deficits after a single-dose AAV vector injection into the tibialis anterior muscle of the dystrophic hamsters. AAV vector treatment led to more than 97% recovery in muscle strength for both the specific twitch force and the specific tetanic force, when compared to the age-matched control. Vector treatment also prevented pathological muscle hypertrophy and resulted in normal muscle weight and size. Finally, vector-treated muscle showed substantial improvement of the histopathology. This is the first report of successful functional rescue of an entire muscle after AAV-mediated gene delivery. This report also demonstrates the feasibility of in vivo gene therapy for LGMD patients by using AAV vectors.
Biochemical Consequences of a Mutation That Controls the Cholesterol Dependence of Semliki Forest Virus Fusion
Authors: Chatterjee Prodyot K. , Vashishtha Malini , Kielian Margaret
Published: July 2002

The enveloped alphavirus Semliki Forest virus (SFV) infects cells via a low-pH-triggered membrane fusion reaction that requires cholesterol and sphingolipid in the target membrane. Cholesterol-depleted insect cells are highly resistant to alphavirus infection and were used to select
srf-3
, an SFV mutant that is ∼100-fold less cholesterol dependent for infection due to a single amino acid change in the E1 spike subunit, proline 226 to serine. Sensitive lipid-mixing assays here demonstrated that the in vitro fusion of
srf-3
and wild-type (wt) virus with cholesterol-containing liposomes had comparable kinetics, activation energies, and sphingolipid dependence. In contrast,
srf-3
fusion with sterol-free liposomes was significantly more efficient than that of wt virus. Thus, the
srf-3
mutation does not affect its general fusion properties with purified lipid bilayers but causes a marked and specific reduction in cholesterol dependence. Upon exposure to low pH, the E1 spike subunit undergoes distinct conformational changes, resulting in the exposure of an acid conformation-specific epitope and formation of an E1 homotrimer. These conformational changes were strongly cholesterol and sphingolipid dependent for wt SFV and strikingly less cholesterol dependent for
srf-3
. Our results thus demonstrate the functional importance of fusogenic E1 conformational changes in the control of SFV cholesterol dependence.
Proteolytic Processing of the Astrovirus Capsid
Authors: Bass D. M. , Qiu Shiqiang
Published: July 2002

To further characterize the nature of proteolytic processing of the astrovirus capsid, we infected Caco-2 cells with a high multiplicity of astrovirus without trypsin in the presence of 5 to 10% fetal calf serum. These infections were characterized by pulse-chase labeling with [
35
S]methionine, electron microscopy, gel electrophoresis of purified viral particles, and analysis of infectivity of such particles with and without added trypsin. Pulse-chase experiments showed that the astrovirus capsid protein was initially translated as an approximately 87-kDa protein. The 87-kDa capsid protein was rapidly converted intracellularly to a 79-kDa form which was found in smaller amounts in the cell supernatant. Purification by differential centrifugation yielded particles that appeared quite similar to trypsin-grown astrovirus particles by negatively stained electron microscopy. These particles were antigenically distinct from trypsin-treated virions as demonstrated by their various reactions with monoclonal antibodies in a solid-phase immunoassay. The purified trypsin-free particles were mainly composed of the 79-kDa capsid protein which was found to have an amino terminus at residue 71 of the entire open reading frame 2 (ORF2) product. The cleavage site was identified in a highly conserved region of the astrovirus ORF2 product. These trypsin-free particles were minimally infectious in cultured Caco-2 cells but became highly infectious (10
5
-fold increase) after trypsin but not chymotrypsin treatment. This trypsin-enhanced infectivity correlated with conversion of the 79-kDa capsid protein to three smaller peptides of approximately 34, 29, and 26 kDa.
Subcellular Localization, Stability, and
trans
-Cleavage Competence of the Hepatitis C Virus NS3-NS4A Complex Expressed in Tetracycline-Regulated Cell Lines
Authors: Wölk Benno , Sansonno Domenico , Kräusslich Hans-Georg , Dammacco Franco , Rice Charles M. , Blum Hubert E. , Moradpour Darius
Published: July 2002

A tetracycline-regulated gene expression system and a panel of novel monoclonal antibodies were used to examine the subcellular localization, stability, and
trans
-cleavage competence of the hepatitis C virus (HCV) NS3-NS4A complex in inducible cell lines. The NS3 serine protease domain and the full-length NS3 protein expressed in the absence of the NS4A cofactor were diffusely distributed in the cytoplasm and nucleus. Coexpression of NS4A, however, directed NS3 to the endoplasmic reticulum (ER) or an ER-like modified compartment, as demonstrated by colocalization with 3,3′-dihexyloxacarbocyanine iodide, protein disulfide isomerase, and calnexin, as well as subcellular fractionation analyses. In addition, coexpression with NS4A dramatically increased the intracellular stability of NS3 (mean protein half-life of 26 versus 3 h) and allowed for NS4A-dependent
trans
-cleavage at the NS4B-NS5A junction. Deletion analyses revealed that the hydrophobic amino-terminal domain of NS4A was required for ER targeting of NS3. These results demonstrate the importance of studying HCV proteins in their biological context and define a well-characterized cell culture system for further analyses of the NS3-NS4A complex and the evaluation of novel antiviral strategies against hepatitis C.
Human Immunodeficiency Virus Type 1 Vpr Induces Apoptosis through Caspase Activation
Authors: Stewart Sheila A. , Poon Betty , Song Joo Y. , Chen Irvin S. Y.
Published: July 2002

Human immunodeficiency virus type 1 (HIV-1) Vpr is a 96-amino-acid protein that is found associated with the HIV-1 virion. Vpr induces cell cycle arrest at the G2/M phase of the cell cycle, and this arrest is followed by apoptosis. We examined the mechanism of Vpr-induced apoptosis and found that HIV-1 Vpr-induced apoptosis requires the activation of a number of cellular cysteinyl aspartate-specific proteases (caspases). We demonstrate that ectopic expression of anti-apoptotic viral proteins, which inhibit caspase activity, and addition of synthetic peptides, which represent caspase cleavage sites, can inhibit Vpr-induced apoptosis. Finally, inhibition of caspase activity and subsequent inhibition of apoptosis results in increased viral expression, suggesting that therapeutic strategies aimed at reducing Vpr-induced apoptosis in vivo require careful consideration.
Exploitation of the Low Fidelity of Human Immunodeficiency Virus Type 1 (HIV-1) Reverse Transcriptase and the Nucleotide Composition Bias in the HIV-1 Genome To Alter the Drug Resistance Development of HIV
Authors: Balzarini Jan , Camarasa Maria-José , Pérez-Pérez Maria-Jesus , San-Félix Ana , Velázquez Sonsoles , Perno Carlo-Federico , De Clercq Erik , Anderson John N. , Karlsson Anna
Published: July 2002

The RNA genome of the lentivirus human immunodeficiency virus type 1 (HIV-1) is significantly richer in adenine nucleotides than the statistically equal distribution of the four different nucleotides that is expected. This compositional bias may be due to the guanine-to-adenine (G→A) nucleotide hypermutation of the HIV genome, which has been explained by dCTP pool imbalances during reverse transcription. The adenine nucleotide bias together with the poor fidelity of HIV-1 reverse transcriptase markedly enhances the genetic variation of HIV and may be responsible for the rapid emergence of drug-resistant HIV-1 strains. We have now attempted to counteract the normal mutational pattern of HIV-1 in response to anti-HIV-1 drugs by altering the endogenous deoxynucleoside triphosphate pool ratios with antimetabolites in virus-infected cell cultures. We showed that administration of these antimetabolic compounds resulted in an altered drug resistance pattern due to the reversal of the predominant mutational flow of HIV (G→A) to an adenine-to-guanine (A→G) nucleotide pattern in the intact HIV-1-infected lymphocyte cultures. Forcing the virus to change its inherent nucleotide bias may lead to better control of viral drug resistance development.
Genetic Retargeting of Adenovirus: Novel Strategy Employing “Deknobbing” of the Fiber
Authors: Magnusson Maria K. , Hong Saw See , Boulanger Pierre , Lindholm Leif
Published: July 2002

For efficient and versatile use of adenovirus (Ad) as an in vivo gene therapy vector, modulation of the viral tropism is highly desirable. In this study, a novel method to genetically alter the Ad fiber tropism is described. The knob and the last 15 shaft repeats of the fiber gene were deleted and replaced with an external trimerization motif and a new cell-binding ligand, in this case the integrin-binding motif RGD. The corresponding recombinant fiber retained the basic biological functions of the natural fiber, i.e., trimerization, nuclear import, penton formation, and ligand binding. The recombinant fiber bound to integrins but failed to react with antiknob antibody. For virus production, the recombinant fiber gene was rescued into the Ad genome at the exact position of the wild-type (WT) fiber to make use of the native regulation of fiber expression. The recombinant virus Ad5/FibR7-RGD yielded plaques on 293 cells, but the spread through the monolayer was two to three times delayed compared to WT, and the ratio of infectious to physical particles was 20 times lower. Studies on virus tropism showed that Ad5/FibR7-RGD was able to infect cells which did not express the coxsackie-adenovirus receptor (CAR), but did express integrins. Ad5/FibR7-RGD virus infectivity was unchanged in the presence of antiknob antibody, which neutralized the WT virus. Ad5/FibR7-RGD virus showed an expanded tropism, which is useful when gene transfer to cells not expressing CAR is needed. The described method should also make possible the construction of Ad genetically retargeted via ligands other than RGD.
Human Parainfluenza Virus Type 3 HN-Receptor Interaction: Effect of 4-Guanidino-Neu5Ac2en on a Neuraminidase-Deficient Variant
Authors: Porotto Matteo , Greengard Olga , Poltoratskaia Natalia , Horga Maria-Arantxa , Moscona Anne
Published: July 2002

The envelope of human parainfluenza virus type 3 (HPF3) contains two viral glycoproteins, the hemagglutinin-neuraminidase (HN) and the fusion protein (F). HN, which is responsible for receptor attachment and for promoting F-mediated fusion, also possesses neuraminidase (receptor-destroying) activity. We reported previously that 4-guanidino-neu5Ac2en (4-GU-DANA) and related sialic acid-based inhibitors of HPF3 neuraminidase activity also inhibit HN-mediated receptor binding and fusion processes not involving neuraminidase activity. We have now examined this mechanism, as well as neuraminidase's role in the viral life cycle, using a neuraminidase-deficient HPF3 variant (C28a) and stable cell lines expressing C28a or wild-type (wt) HN. C28a, which has a wt F sequence and two point mutations in the HN gene corresponding to two amino acid changes in the HN protein, is the first HPF3 variant with insignificant neuraminidase activity. Cells expressing C28a HN did not bind erythrocytes at 4°C unless pretreated with neuraminidase, but no such pretreatment was required for hemadsorption activity (HAD) at 22 or 37°C. HAD was blocked by 4-GU-DANA, attesting to the ability of this compound to inhibit HN's receptor-binding activity. C28a or wt plaque enlargement, a process that involves cell-cell fusion and does not depend on virion release, is diminished by the presence of 4-GU-DANA, confirming the inhibitory effect of 4-GU-DANA on the fusogenic function of C28a HN. In C28a-infected cell monolayers, virion release and thus multicycle replication are severely restricted. This defect was corrected by supplementation of exogenous neuraminidase and also by the addition of 4-GU-DANA; neuraminidase destroys the receptors whereby newly formed C28a virions would remain attached to the cell surface, whereas 4-GU-DANA prevents the attachment itself, obviating the need for receptor cleavage. In accord with the ability of 4-GU-DANA to prevent attachment, the neuraminidase inhibitory effect of 4-GU-DANA on wt HPF3 did not diminish virion release into the medium. Thus, it is by inhibition of viral entry and syncytium formation that sialic acid analogs like 4-GU-DANA may counteract wt HPF3 infection.
Phylogenetic Analysis of Enterovirus 71 Strains Isolated during Linked Epidemics in Malaysia, Singapore, and Western Australia
Authors: McMinn Peter , Lindsay Katie , Perera David , Chan Hung Ming , Chan Kwai Peng , Cardosa Mary Jane
Published: July 2002

Enterovirus 71 (EV71) is a frequent cause of hand, foot, and mouth disease (HFMD) epidemics associated with severe neurological sequelae in a small proportion of cases. There has been a significant increase in EV71 epidemic activity throughout the Asia-Pacific region since 1997. Recent HFMD epidemics in this region have been associated with a severe form of brainstem encephalitis associated with pulmonary edema and high case fatality rates. In this study, we show that four genetic lineages of EV71 have been prevalent in the Asia-Pacific region since 1997, including two previously undescribed genogroups (B3 and B4). Furthermore, we show that viruses belonging to genogroups B3 and B4 have circulated endemically in Southeast Asia during this period and have been the primary cause of several large HFMD or encephalitis epidemics in Malaysia, Singapore, and Western Australia.
Autonomous Role of 3′-Terminal CCCA in Directing Transcription of RNAs by Qβ Replicase
Authors: Tretheway David M. , Yoshinari Shigeo , Dreher Theo W.
Published: July 2002

We have studied transcription in vitro by Qβ replicase to deduce the minimal features needed for efficient end-to-end copying of an RNA template. Our studies have used templates ca. 30 nucleotides long that are expected to be free of secondary structure, permitting unambiguous analysis of the role of template sequence in directing transcription. A 3′-terminal CC
C
A (3′-CC
C
A) directs transcriptional initiation to opposite the underlined C; the amount of transcription is comparable between RNAs possessing upstream (CCA)

n

tracts, A-rich sequences, or a highly folded domain and is also comparable in single-round transcription assays to transcription of two amplifiable RNAs. Predominant initiation occurs within the 3′-CCCA initiation box when a wide variety of sequences is present immediately upstream, but CCA or a closely similar sequence in that position results in significant internal initiation. Removal of the 3′-A from the 3′-CCCA results in 5- to 10-fold-lower transcription, emphasizing the importance of the nontemplated addition of 3′-A by Qβ replicase during termination. In considering whether 3′-CCCA could provide sufficient specificity for viral transcription, and consequently amplification, in vivo, we note that tRNA
His
is the only stable
Escherichia coli
RNA with 3′-CCCA. In vitro-generated transcripts corresponding to tRNA
His
served as poor templates for Qβ replicase; this was shown to be due to the inaccessibility of the partially base-paired CCCA. These studies demonstrate that 3′-CCCA plays a major role in the control of transcription by Qβ replicase and that the abundant RNAs present in the host cell should not be efficient templates.
Altered Cellular mRNA Levels in Human Cytomegalovirus-Infected Fibroblasts: Viral Block to the Accumulation of Antiviral mRNAs
Authors: Browne Edward P. , Wing Bret , Coleman David , Shenk Thomas
Published: July 2002

The effect of human cytomegalovirus (HCMV) infection on cellular mRNA accumulation was analyzed by gene chip technology. During a 48-h time course after infection of human diploid fibroblasts, 1,425 cellular mRNAs were found to be up-regulated or down-regulated by threefold or greater in at least two consecutive time points. Several classes of genes were prominently affected, including interferon response genes, cell cycle regulators, apoptosis regulators, inflammatory pathway genes, and immune regulators. The number of mRNAs that were up-regulated or down-regulated were roughly equal over the complete time course. However, for the first 8 h after infection, the number of up-regulated mRNAs was significantly less than the number of down-regulated mRNAs. By analyzing the mRNA expression profile of cells infected in the presence of cycloheximide, it was found that a minimum of 25 mRNAs were modulated by HCMV in the absence of protein synthesis. These included mRNAs encoded by a small number of interferon-responsive genes, as well as beta interferon itself. Cellular mRNA levels in cytomegalovirus-infected cells were compared to the levels in cells infected with UV-inactivated virus. The inactivated virus caused the up-regulation of a much greater number of mRNAs, many of which encoded proteins with antiviral roles, such as interferon-responsive genes and proinflammatory cytokines. These data argue that one or more newly synthesized viral gene products block the induction of antiviral pathways that are triggered by HCMV binding and entry.
Polypeptide p41 of a Norwalk-Like Virus Is a Nucleic Acid-Independent Nucleoside Triphosphatase
Authors: Pfister Thomas , Wimmer Eckard
Published: July 2002

Southampton virus (SHV) is a member of the Norwalk-like viruses (NLVs), one of four genera of the family
Caliciviridae
. The genome of SHV contains three open reading frames (ORFs). ORF 1 encodes a polyprotein that is autocatalytically processed into six proteins, one of which is p41. p41 shares sequence motifs with protein 2C of picornaviruses and superfamily 3 helicases. We have expressed p41 of SHV in bacteria. Purified p41 exhibited nucleoside triphosphate (NTP)-binding and NTP hydrolysis activities. The NTPase activity was not stimulated by single-stranded nucleic acids. SHV p41 had no detectable helicase activity. Protein sequence comparison between the consensus sequences of NLV p41 and enterovirus protein 2C revealed regions of high similarity. According to secondary structure prediction, the conserved regions were located within a putative central domain of alpha helices and beta strands. This study reveals for the first time an NTPase activity associated with a calicivirus-encoded protein. Based on enzymatic properties and sequence information, a functional relationship between NLV p41 and enterovirus 2C is discussed in regard to the role of 2C-like proteins in virus replication.