bluuebunny/crossref_metadata_2025 huggingface dataset:
Methionine Regeneration and Aspartate Aminotransferase in Parasitic Protozoa
Authors: Berger Louise C. , Wilson Judith , Wood Pamela , Berger Bradley J.
Published: July 2002

Aspartate aminotransferases have been cloned and expressed from
Crithidia fasciculata
,
Trypanosoma brucei brucei
,
Giardia intestinalis
, and
Plasmodium falciparum
and have been found to play a role in the final step of methionine regeneration from methylthioadenosine. All five enzymes contain sequence motifs consistent with membership in the Ia subfamily of aminotransferases; the crithidial and giardial enzymes and one trypanosomal enzyme were identified as cytoplasmic aspartate aminotransferases, and the second trypanosomal enzyme was identified as a mitochondrial aspartate aminotransferase. The plasmodial enzyme contained unique sequence substitutions and appears to be highly divergent from the existing members of the Ia subfamily. In addition, the
P. falciparum
enzyme is the first aminotransferase found to lack the invariant residue G197 (P. K. Mehta, T. I. Hale, and P. Christen, Eur. J. Biochem
.
214:549–561, 1993), a feature shared by sequences discovered in
P. vivax
and
P. berghei
. All five enzymes were able to catalyze aspartate-ketoglutarate, tyrosine-ketoglutarate, and amino acid-ketomethiobutyrate aminotransfer reactions. In the latter, glutamate, phenylalanine, tyrosine, tryptophan, and histidine were all found to be effective amino donors. The crithidial and trypanosomal cytosolic aminotransferases were also able to catalyze alanine-ketoglutarate and glutamine-ketoglutarate aminotransfer reactions and, in common with the giardial aminotransferase, were able to catalyze the leucine-ketomethiobutyrate aminotransfer reaction. In all cases, the kinetic constants were broadly similar, with the exception of that of the plasmodial enzyme, which catalyzed the transamination of ketomethiobutyrate significantly more slowly than aspartate-ketoglutarate aminotransfer. This result obtained with the recombinant
P. falciparum
aminotransferase parallels the results seen for total ketomethiobutyrate transamination in malarial homogenates; activity in the latter was much lower than that in homogenates from other organisms. Total ketomethiobutyrate transamination in
Trichomonas vaginalis
and
G. intestinalis
homogenates was extensive and involved lysine-ketomethiobutyrate enzyme activity in addition to the aspartate aminotransferase activity. The methionine production in these two species could be inhibited by the amino-oxy compounds canaline and carboxymethoxylamine. Canaline was also found to be an uncompetitive inhibitor of the plasmodial aspartate aminotransferase, with a

K
i

of 27 μM.
DNA Inversion in the Tail Fiber Gene Alters the Host Range Specificity of Carotovoricin Er, a Phage-Tail-Like Bacteriocin of Phytopathogenic
Erwinia carotovora
subsp.
carotovora
Er
Authors: Nguyen Hoa Anh , Tomita Toshio , Hirota Morihiko , Kaneko Jun , Hayashi Tetsuya , Kamio Yoshiyuki
Published: July 2002

Carotovoricin Er is a phage-tail-like bacteriocin produced by
Erwinia carotovora
subsp.
carotovora
strain Er, a causative agent for soft rot disease in plants. Here we studied binding and killing spectra of carotovoricin Er preparations for various strains of the bacterium (strains 645Ar, EC-2, N786, and P7) and found that the preparations contain two types of carotovoricin Er with different host specificities; carotovoricin Era possessing a tail fiber protein of 68 kDa killed strains 645Ar and EC-2, while carotovoricin Erb with a tail fiber protein of 76 kDa killed strains N786 and P7. The tail fiber proteins of 68 and 76 kDa had identical N-terminal amino acid sequences for at least 11 residues. A search of the carotovoricin Er region in the chromosome of strain Er indicated the occurrence of a DNA inversion system for the tail fiber protein consisting of (i) two 26-bp inverted repeats inside and downstream of the tail fiber gene that flank a 790-bp fragment and (ii) a putative DNA invertase gene with a 90-bp recombinational enhancer sequence. In fact, when a 1,400-bp region containing the 790-bp fragment was amplified by a PCR using the chromosomal DNA of strain Er as the template, both the forward and the reverse nucleotide sequences of the 790-bp fragment were detected. DNA inversion of the 790-bp fragment also occurred in
Escherichia coli
DH5α when two compatible plasmids carrying either the 790-bp fragment or the invertase gene were cotransformed into the bacterium. Furthermore, hybrid carotovoricin CGE possessing the tail fiber protein of 68 or 76 kDa exhibited a host range specificity corresponding to that of carotovoricin Era or Erb, respectively. Thus, a DNA inversion altered the C-terminal part of the tail fiber protein of carotovoricin Er, altering the host range specificity of the bacteriocin.
The Pyrimidine Operon
pyrRPB-carA
from
Lactococcus lactis
Authors: Martinussen Jan , Schallert Jette , Andersen Birgit , Hammer Karin
Published: July 2002

The four genes
pyrR, pyrP, pyrB
, and
carA
were found to constitute an operon in
Lactococcus lactis
subsp.
lactis
MG1363. The functions of the different genes were established by mutational analysis. The first gene in the operon is the pyrimidine regulatory gene,
pyrR
, which is responsible for the regulation of the expression of the pyrimidine biosynthetic genes leading to UMP formation. The second gene encodes a membrane-bound high-affinity uracil permease, required for utilization of exogenous uracil. The last two genes in the operon,
pyrB
and
carA
, encode pyrimidine biosynthetic enzymes; aspartate transcarbamoylase (
pyrB
) is the second enzyme in the pathway, whereas carbamoyl-phosphate synthetase subunit A (
carA
) is the small subunit of a heterodimeric enzyme, catalyzing the formation of carbamoyl phosphate. The
carA
gene product is shown to be required for both pyrimidine and arginine biosynthesis. The expression of the pyrimidine biosynthetic genes including the
pyrRPB-carA
operon is subject to control at the transcriptional level, most probably by an attenuator mechanism in which PyrR acts as the regulatory protein.
Expression of the Putative Siderophore Receptor Gene
bfrZ
Is Controlled by the Extracytoplasmic-Function Sigma Factor BupI in
Bordetella bronchiseptica
Authors: Pradel Elizabeth , Locht Camille
Published: July 2002

A new gene from
Bordetella bronchiseptica, bfrZ
encoding a putative siderophore receptor, was identified in a Fur-repressor titration assay. A
bfrZ
null mutant was constructed by allelic exchange. The protein profile of this mutant is similar to that of the wild-type parent strain. The BfrZ
−
-BfrZ
+
isogenic pair was tested for utilization of 132 different siderophores as iron sources. None of these iron sources acted as a ligand for BfrZ. Translational
bfrZ
::
phoA
and transcriptional
bfrZ
::
lacZ
fusions were introduced into the
B. bronchiseptica bfrZ
locus. No alkaline phosphatase or β-galactosidase activity was detected. Sequence analysis of the
bfrZ
upstream region revealed the presence of two tightly linked genes,
bupI
and
bupR
. Both of these genes are located downstream from a Fur-binding sequence. BupI is homologous to
Escherichia coli
FecI and
Pseudomonas putida
PupI and belongs to the family of extracytoplasmic-function sigma factors involved in transcription of genes with extracytoplasmic functions. BupR is homologous to the FecR and PupR antisigma factors and is predicted to be localized in the inner membrane. Similar to the surface signaling receptors FecA and PupB, BfrZ bears an N-terminal extension. We found that
bfrZ
is not transcribed when
bupI
and
bupR
are expressed at the same level. However, overexpression of
bupI
from a multicopy plasmid triggers
bfrZ
transcription, and under these conditions BfrZ was detected in membrane fractions. By analogy with the FecI-FecR-FecA and PupI-PupR-PupB systems, our data suggest that
bfrZ
expression is inducible by binding of the cognate ligand to BfrZ and transduction of a signal through the envelope.
Biological Properties and Cell Tropism of Chp2, a Bacteriophage of the Obligate Intracellular BacteriumChlamydophila abortus
Authors: Everson J. S. , Garner S. A. , Fane B. , Liu B.-L. , Lambden P. R. , Clarke I. N.
Published: July 2002

A number of bacteriophages belonging to theMicroviridaehave been described infecting chlamydiae. Phylogenetic studies divide theChlamydiaceaeinto two distinct genera,ChlamydiaandChlamydophila, containing three and six different species, respectively. In this work we investigated the biological properties and host range of the recently described bacteriophage Chp2 that was originally discovered inChlamydophila abortus. The obligate intracellular development cycle of chlamydiae has precluded the development of quantitative approaches to assay bacteriophage infectivity. Thus, we prepared hybridomas secreting monoclonal antibodies (monoclonal antibodies 40 and 55) that were specific for Chp2. We demonstrated that Chp2 binds bothC. abortuselementary bodies and reticulate bodies in an enzyme-linked immunosorbent assay. Monoclonal antibodies 40 and 55 also detected bacteriophage Chp2 antigens in chlamydia-infected eukaryotic cells. We used these monoclonal antibodies to monitor the ability of Chp2 to infect all nine species of chlamydiae. Chp2 does not infect members of the genusChlamydia(C. trachomatis, C. suis, orC. muridarum). Chp2 can infectC. abortus, C. felis, andC. pecorumbut is unable to infect other members of this genus, includingC. caviaeandC. pneumoniae, despite the fact that these chlamydial species support the replication of very closely related bacteriophages.
Contribution of Membrane-Binding and Enzymatic Domains of Penicillin Binding Protein 5 to Maintenance of Uniform Cellular Morphology of
Escherichia coli
Authors: Nelson David E. , Ghosh Anindya S. , Paulson Avery L. , Young Kevin D.
Published: July 2002

Four low-molecular-weight penicillin binding proteins (LMW PBPs) of
Escherichia coli
are closely related and have similar
dd
-carboxypeptidase activities (PBPs 4, 5, and 6 and DacD). However, only one, PBP 5, has a demonstrated physiological function. In its absence, certain mutants of
E. coli
have altered diameters and lose their uniform outer contour, resulting in morphologically aberrant cells. To determine what differentiates the activities of these LMW PBPs, we constructed fusion proteins combining portions of PBP 5 with fragments of other
dd
-carboxypeptidases to see which hybrids restored normal morphology to a strain lacking PBP 5. Functional complementation occurred when truncated PBP 5 was combined with the terminal membrane anchor sequences of PBP 6 or DacD. However, complementation was not restored by the putative carboxy-terminal anchor of PBP 4 or by a transmembrane region of the osmosensor protein ProW, even though these hybrids were membrane bound. Site-directed mutagenesis of the carboxy terminus of PBP 5 indicated that complementation required a generalized amphipathic membrane anchor but that no specific residues in this region seemed to be required. A functional fusion protein was produced by combining the N-terminal enzymatic domain of PBP 5 with the C-terminal β-sheet domain of PBP 6. In contrast, the opposite hybrid of PBP 6 to PBP 5 was not functional. The results suggest that the mode of PBP 5 membrane anchoring is important, that the mechanism entails more than a simple mechanical tethering of the enzyme to the outer face of the inner membrane, and that the physiological differences among the LMW PBPs arise from structural differences in the
dd
-carboxypeptidase enzymatic core.
Regulation ofnapGene Expression and Periplasmic Nitrate Reductase Activity in the Phototrophic BacteriumRhodobacter sphaeroidesDSM158
Authors: Gavira Mónica , Roldán M. Dolores , Castillo Francisco , Moreno-Vivián Conrado
Published: July 2002

Bacterial periplasmic nitrate reductases (Nap) can play different physiological roles and are expressed under different conditions depending on the organism.Rhodobacter sphaeroidesDSM158 has a Nap system, encoded by thenapKEFDABCgene cluster, but nitrite formed is not further reduced because this strain lacks nitrite reductase. Nap activity increases in the presence of nitrate and oxygen but is unaffected by ammonium. Reverse transcription-PCR and Northern blots demonstrated that thenapKEFDABCgenes constitute an operon transcribed as a single 5.5-kb product. Northern blots andnap-lacZfusions revealed thatnapexpression is threefold higher under aerobic conditions but is regulated by neither nitrate nor ammonium, although it is weakly induced by nitrite. On the other hand, nitrate but not nitrite causes a rapid enzyme activation, explaining the higher Nap activity found in nitrate-grown cells. Translationalnap′-′lacZfusions reveal that thenapKandnapDgenes are not efficiently translated, probably due to mRNA secondary structures occluding the translation initiation sites of these genes. Neither butyrate nor caproate increasesnapexpression, although cells growing phototrophically on these reduced substrates show a very high Nap activity in vivo (nitrite accumulation is sevenfold higher than in medium with malate). Phototrophic growth on butyrate or caproate medium is severely reduced in the NapA−mutants. Taken together, these results indicate that nitrate reduction inR. sphaeroidesis mainly regulated at the level of enzyme activity by both nitrate and electron supply and confirm that the Nap system is involved in redox balancing using nitrate as an ancillary oxidant to dissipate excess reductant.
Detection of Sporadic Cases of Hepatitis E Virus (HEV) Infection in China Using Immunoassays Based on Recombinant Open Reading Frame 2 and 3 Polypeptides from HEV Genotype 4
Authors: Wang Youchun , Zhang Huayuan , Li Zhuo , Gu Wenjie , Lan Haiyuan , Hao Wa , Ling Roger , Li Hemin , Harrison Tim J.
Published: July 2002

We reported previously on the complete sequence of hepatitis E virus (HEV) genotype 4, isolated from patients with sporadic cases of acute HEV infection in China. At least eight HEV genotypes have now been described worldwide, and further isolates await classification. Current immunoassays for the detection of anti-HEV antibodies are based on polypeptides from genotypes 1 and 2 only and may be inadequate for the reliable detection of other genotypes. Because genotypes 1 and 4 predominate in China, we wished to investigate the antigenic reactivities of HEV genotype 4 proteins. Four overlapping regions of open reading frame 2 (ORF2) (FB5, amino acids [aa] 1 to 130; E4, aa 67 to 308; F2-2, aa 288 to 461; E5, aa 414 to 672) and the entire ORF3 product were expressed in
Escherichia coli
as fusion proteins. Enzyme immunoassays based on each of the five purified polypeptides were evaluated with sera from patients with sporadic cases of acute HEV infection. Individual immunoassays derived from HEV genotype 4 detected more cases of acute hepatitis E than a commercial assay. Some serum samples, which were positive for anti-HEV immunoglobulin G only by assays based on HEV genotype 4, were positive for HEV RNA by reverse transcription-PCR. Polypeptide FB5, from the N terminus of ORF2, had the greatest immunoreactivity with sera from patients with acute hepatitis E. These data indicate that the N terminus of ORF2 may provide epitopes which are highly reactive with acute-phase sera and that assays based on genotypes 1 and 2 alone may be inadequate for the detection of HEV infection in China, where sporadic cases of HEV infection are caused predominantly by HEV genotypes 4 and 1.
Outbreak of Subclinical Mastitis in a Flock of Dairy Sheep Associated with
Burkholderia cepacia
Complex Infection
Authors: Berriatua E. , Ziluaga I. , Miguel-Virto C. , Uribarren P. , Juste R. , Laevens S. , Vandamme P. , Govan J. R. W.
Published: July 2002

An outbreak of subclinical mastitis in a flock of 620 milking sheep was investigated. Microbiological and epidemiological analyses identified the causative agent as belonging to the
Burkholderia cepacia
complex (formerly
Pseudomonas cepacia
). Every ewe in the milking flock was individually tested for subclinical mastitis on two separate occasions, 6 weeks apart, by the California (rapid) mastitis test (CMT). The proportion of CMT-positive ewes was 69 of 393 (17.6%) on the first sampling and 27 of 490 (5.5%) on the second sampling. Pure
B. cepacia
cultures identified with the API 20 NE system were grown from 64 of 96 (66.7%) CMT-positive ewes and from 1 of 33 (3.0%) CMT-negative ewes. Statistical analysis confirmed the significant association between a positive CMT result and a positive culture result for
B. cepacia
complex. Additional polyphasic taxonomic analyses of eight isolates showed that seven belonged to
B. cepacia
genomovar III; the remaining isolate was identified as
Burkholderia vietnamiensis
(formerly
B. cepacia
genomovar V). Bacteriological investigation of samples from milking equipment and other environmental sites failed to identify “
B. cepacia
” in any of the samples taken. To our knowledge, this is the first report of an outbreak of natural infection in animals caused by
B. cepacia
complex and the first description of
B. cepacia
complex infection in sheep.
Electrophoretic Karyotype Analysis of Sequential
Candida parapsilosis
Isolates from Patients with Persistent or Recurrent Fungemia
Authors: Shin Jong Hee , Shin Dong Hyeon , Song Jeong Won , Kee Seung Jung , Suh Soon Pal , Ryang Dong Wook
Published: July 2002

We assessed the genetic relatedness of sequential isolates of
Candida parapsilosis
during persistent or recurrent fungemia and the effect of central venous catheter (CVC) removal. Serial isolates of
C. parapsilosis
were obtained from 17 patients with persistent or recurrent fungemia over periods of up to 5 months. Forty-eight
C. parapsilosis
isolates from the blood of 17 patients were analyzed by electrophoretic karyotyping (EK) with pulsed-field gel electrophoresis (PFGE), revealing 25 different karyotypes. The strains sequentially isolated from each of seven patients whose fungemia resolved following CVC removal had the same karyotype. Two patients with fungemia that cleared without CVC removal each had two sequential isolates with different karyotypes. In six (75%) of the eight patients whose fungemia was recurrent even after CVC removal, the karyotypes of the pre- and post-CVC removal isolates were different, implying the emergence of a new strain. Overall, the sequential strains from each patient had identical karyotypes in 53% (9 of 17) of the patients and two different karyotypes in 47% (8 of 17). This study shows that EK with PFGE is useful for investigating persistent or recurrent fungemia due to
C. parapsilosis
and that recurrent fungemia due to
C. parapsilosis
is more likely caused by reinfection with a second strain.
Evaluation of Mycobacteria Growth Indicator Tube for Direct and Indirect Drug Susceptibility Testing of
Mycobacterium tuberculosis
from Respiratory Specimens in a Siberian Prison Hospital
Authors: Goloubeva Vera , Lecocq Maryvonne , Lassowsky Piotr , Matthys Francine , Portaels Françoise , Bastian Ivan
Published: July 2002

The manual Mycobacteria Growth Indicator Tube (MGIT) method was evaluated for performing direct and indirect drug susceptibility testing (DST) of
Mycobacterium tuberculosis
for isoniazid and rifampin on 101 strongly smear-positive sputum specimens in a Siberian prison hospital. Using the indirect method of proportion (MOP) as the “gold standard,” the accuracies of isoniazid and rifampin susceptibility testing by the direct MGIT system were 97.0 and 94.1%, respectively. The accuracy of the indirect MGIT system was 98.0% for both drugs. The turnaround times from specimen processing to reporting of the DST results ranged between 4 and 23 (mean, 9.2) days by the direct MGIT method, 9 and 30 (mean, 15.3) days by the indirect MGIT method, and 26 and 101 (mean, 59.6) days by the indirect MOP. MGIT appears to be a reliable, rapid, and convenient method for performing direct and indirect DSTs in low-resource settings, but further studies are required to refine the direct DST protocol. Cost is the only factor prohibiting widespread implementation of MGIT.
Discrimination between
Candida albicans
and Other Pathogenic Species of the Genus
Candida
by Their Differential Sensitivities to Toxins of a Panel of Killer Yeasts
Authors: Buzzini P. , Martini A.
Published: July 2002

The differential sensitivities to toxins produced by a short panel of four killer yeasts allowed discrimination between 91 strains of the yeast
Candida albicans
and 223 non-
C. albicans Candida
strains. One hundred percent of
C. albicans
isolates exhibited negative results to the toxin panel, while 100% of non-
C. albicans
cultures gave well-defined and reproducible positive results to at least one of the four killer toxins. Among
C. albicans
strains only 96 and 87% gave germ tube (GT)- and chlamydospore-positive results, respectively. In addition a few GT-false-positive strains were detected among non-
C. albicans
isolates. Susceptibility to the toxin panel is apparently expressed more consistently than either GT or chlamydospore production and may constitute a promising basis for a new simple and easy-to-use procedure for routine discrimination between the species
C. albicans
and other species of the genus
Candida
.
Development and Evaluation of a DNA Enzyme Immunoassay Method for
env
Genotyping of Subtypes A through G of Human Immunodeficiency Virus Type 1 Group M, with Discrimination of the Circulating Recombinant Forms CRF01_AE and CRF02_AG
Authors: Plantier Jean-Christophe , Vergne Laurence , Damond Florence , MBoup Souleymane , MPoudi-NGole Eitel , Buzelay Laurence , Farfara Isabelle , Brand Denys , Peeters Martine , Brun-Vézinet Françoise , Delaporte Eric , Barin Francis
Published: July 2002

The tools currently available for genetic subtyping of human immunodeficiency virus type 1 are laborious or can be used only for the analysis of a limited number of samples and/or subtypes. We developed and evaluated a molecular biology-based method using subtype-specific oligonucleotide probes for
env
genotyping of subtypes A through G, CRF01_AE, and CRF02_AG. DNA enzyme immunoassay (DEIA) genotyping is based on nested PCR amplification of the 5′ end of the
env
gene (proviral DNA), followed by subtype-specific hybridization and immunoenzymatic detection on microplates. DEIA genotyping was validated with a large number of samples (
n
= 128) collected in Europe (France;
n
= 47), West-Central Africa (Cameroon;
n
= 36), and West Africa (Senegal;
n
= 45). Three different formats, depending on the distribution of subtypes in the three countries, were developed. The results were compared with those obtained by sequencing of the V3-V5 region and phylogenetic analysis or an
env
heteroduplex mobility assay. Additional sequencing and phylogenetic analyses of the DEIA region (the first codon of the env coding sequence to the middle of conserved region C1 of gp120) were performed to investigate the reasons for discrepancies. Intense and highly specific reactions between the oligonucleotide probes and the corresponding samples were observed. Overall, correct identification was achieved for 107 of 128 samples (83.6%). One sample was not amplified, 10 (8%) were nontypeable (NT), and 10 (8%) were misidentified. Six of the 10 discordant samples were further investigated by phylogenetic analysis, which indicated that these samples corresponded to recombinants involving the env 5′ end and the V3 and V5 regions of the two parental clades. Sequencing of NT samples showed numerous differences between sample and probe sequences, resulting in a lack of hybridization, and revealed the limitations of the selected probes in terms of specificity and sensitivity. We demonstrated the feasibility of DEIA genotyping: six subtypes plus the two most prevalent circulating recombinant forms were discriminated by using the 5′ end of the
env
gene. This method can be adapted to the local situation by including only probes that correspond to the prevalent strains.
In Vivo and In Vitro Identification of Structural and Sequence Elements of the Human Parechovirus 5′ Untranslated Region Required for Internal Initiation
Authors: Nateri Abdolrahman S. , Hughes Pamela J. , Stanway Glyn
Published: July 2002

Sequence analysis of the picornavirus echovirus 22 led to its classification as the first member of a new genus,
Parechovirus
, and renaming as human parechovirus type 1 (HPeV1). Although distinct from other genera in most of the genome, the 5′ untranslated region (5′UTR) shows similarities to that of cardio/aphthoviruses in some of its structural domains (A to L). The 5′UTR plays an important role in picornavirus translation initiation and in RNA synthesis. To investigate translation in HPeV1, we engineered an extensive range of mutations (including precise deletions and point mutations) into the 5′UTR. Their effects were studied both by in vitro transcription-translation using a bicistronic construct and by in vivo studies using an infectious, full-length HPeV1 cDNA. These approaches allowed the HPeV1 internal ribosome entry site (IRES) to be mapped. Deletions within the first 298 nucleotides had little impact in the in vitro system, while deletions of nucleotides 298 to 538 had a significant effect. Precise removal of domains H and L (nucleotides 287 to 316 and 664 to 682, respectively) did not significantly reduce translation efficiency in vitro, while domains I, J, and K (nucleotides 327 to 545, 551 to 661, and 614 to 645, respectively) appeared to have much more important roles. Mutation of a phylogenetically conserved GNRA motif (positions 421 to 424) within domain I severely reduced translation. We also confirmed the identity of the AUG (positions 710 to 712) which initiates the open reading frame, the positive identification of which has not been possible previously, as the N terminus of the polyprotein is blocked and not amenable to sequence analysis. This is therefore important in understanding parechovirus genome organization. Mutation of the AUG or an upstream polypyrimidine tract leads to aberrant translation, suggesting they both form part of the parechovirus Yn-Xm-AUG motif. In vivo experiments confirmed the importance of domains I, J, and K, the conserved GNRA motif, polypyrimidine sequences, and AUG, as mutations here were lethal. These features are also important in the IRES elements of cardio/aphthoviruses, but other features reported to be part of the IRES of some members of these genera, notably domains H and L, do not appear to be critical in HPeV1. This adds weight to the idea that there may be functional differences between the IRES elements of different picornaviruses, even when they share significant structural similarity.
Pathogenic Simian/Human Immunodeficiency Virus SHIV
KU
Inoculated into Immunized Macaques Caused Infection, but Virus Burdens Progressively Declined with Time
Authors: Silverstein Peter S. , Mackay Glenn A. , Mukherjee Sampa , Li Zhuang , Piatak Michael , Lifson Jeffrey D. , Narayan Opendra , Kumar Anil
Published: July 2002

Using the simian immunodeficiency virus/human immunodeficiency virus (SHIV)-macaque model of AIDS, we had shown in a previous report that a live, nonpathogenic strain of SHIV, further attenuated by deletion of the
vpu
gene and inoculated orally into adult macaques, had effectively prevented AIDS following vaginal inoculation with pathogenic SHIV
KU
. Examination of lymph nodes from the animals at 18 weeks postchallenge had shown that all six animals were persistently infected with challenge virus. We report here on a 2-year follow-up study on the nature of the persistent infections in these animals. DNA of the vaccine virus was present in the lymph nodes at all time points tested, as far as 135 weeks postchallenge. In contrast, the DNA of SHIV
KU
became undetectable in one animal by week 55 and in three others by week 63. These four macaques have remained negative for SHIV
KU
DNA as far as the last time point examined at week 135. Quantification of the total viral DNA concentration in lymph nodes during the observation period showed a steady decline. All animals developed neutralizing antibody and cytotoxic-T-lymphocyte responses to SHIV
KU
that persisted throughout the observation period. Vaccine-like viruses were isolated from two animals, and a SHIV
KU
-like virus was isolated from one of the two macaques that remained positive for SHIV
KU
DNA. There was no evidence of recombination between the vaccine and the challenge viruses. Thus, immunization with the live vaccine not only prevented disease but also contributed to the steady decline in the virus burdens in the animals.
Localization of a Binding Site for Herpes Simplex Virus Glycoprotein D on Herpesvirus Entry Mediator C by Using Antireceptor Monoclonal Antibodies
Authors: Krummenacher Claude , Baribaud Isabelle , Ponce de Leon Manuel , Whitbeck J. Charles , Lou Huan , Cohen Gary H. , Eisenberg Roselyn J.
Published: July 2002

The human herpesvirus entry mediator C (HveC), also known as the poliovirus receptor-related protein 1 (PRR1) and as nectin-1, allows the entry of herpes simplex virus type 1 (HSV-1) and HSV-2 into mammalian cells. The interaction of virus envelope glycoprotein D (gD) with such a receptor is an essential step in the process leading to membrane fusion. HveC is a member of the immunoglobulin (Ig) superfamily and contains three Ig-like domains in its extracellular portion. The gD binding site is located within the first Ig-like domain (V domain) of HveC. We generated a panel of monoclonal antibodies (MAbs) against the ectodomain of HveC. Eleven of these, which detect linear or conformational epitopes within the V domain, were used to map a gD binding site. They allowed the detection of HveC by enzyme-linked immunosorbent assay, Western blotting, and biosensor analysis or directly on the surface of HeLa cells and human neuroblastoma cell lines, as well as simian Vero cells. The anti-HveC V-domain MAbs CK6, CK8, and CK41, as well as the previously described MAb R1.302, blocked HSV entry. Their binding to soluble HveC was blocked by the association of gD with the receptor, indicating that their epitopes overlap a gD binding site. Competition assays on an optical biosensor showed that CK6 and CK8 (linear epitopes) inhibited the binding of CK41 and R1.302 (conformational epitopes) to HveC and vice versa. Epitope mapping showed that CK6 and CK8 bound between residues 80 and 104 of HveC, suggesting that part of the gD binding site colocalizes in the same region.
Recovery of NV Knockout Infectious Hematopoietic Necrosis Virus Expressing Foreign Genes
Authors: Biacchesi Stéphane , Thoulouze Maria-Isabel , Béarzotti Monique , Yu Yan-Xing , Brémont Michel
Published: July 2002

Infectious hematopoietic necrosis virus (IHNV) is a
Novirhabdovirus
and is the causative agent of a devastating acute, lethal disease in wild and farmed rainbow trout. The virus is enzootic throughout western North America and has spread to Asia and Europe. A full-length cDNA of the IHNV antigenome (pIHNV-Pst) was assembled from subgenomic overlapping cDNA fragments and cloned in a transcription plasmid between the T7 RNA polymerase promoter and the autocatalytic hepatitis delta virus ribozyme. Recombinant IHNV (rIHNV) was recovered from fish cells at 14°C, following infection with a recombinant vaccinia virus expressing the T7 RNA polymerase (vTF7-3) and cotransfection of pIHNV-Pst together with plasmids encoding the nucleoprotein N (pT7-N), the phosphoprotein P (pT7-P), the RNA polymerase L (pT7-L), and the nonvirion protein NV (pT7-NV). When pT7-N and pT7-NV were omitted, rIHNV was also recovered, although less efficiently. Incidental mutations introduced in pIHNV-Pst were all present in the rIHNV genome; however, a targeted mutation located in the L gene was eliminated from the recombinant genome by homologous recombination with the added pT7-L expression plasmid. To investigate the role of NV protein in virus replication, the pIHNV-Pst construct was engineered such that the entire NV open reading frame was deleted and replaced by the genes encoding green fluorescent protein or chloramphenicol acetyltransferase. The successful recovery of recombinant virus expressing foreign genes instead of the NV gene demonstrated that the NV protein was not absolutely required for viral replication in cell cultures, although its presence greatly improves virus growth. The ability to generate rIHNV from cDNA provides the basis to manipulate the genome in order to engineer new live viral vaccine strains.
Varicella-Zoster Virus Retains Major Histocompatibility Complex Class I Proteins in the Golgi Compartment of Infected Cells
Authors: Abendroth Allison , Lin Ines , Slobedman Barry , Ploegh Hidde , Arvin Ann M.
Published: July 2002

We sought to examine the effects of varicella-zoster virus (VZV) infection on the expression of major histocompatibility complex class I (MHC I) molecules by human fibroblasts and T lymphocytes. By flow cytometry, VZV infection reduced the cell surface expression of MHC I molecules on fibroblasts significantly, yet the expression of transferrin receptor was not affected. Importantly, when human fetal thymus/liver implants in SCID-hu mice were inoculated with VZV, cell surface MHC I expression was downregulated specifically on VZV-infected human CD3
+
T lymphocytes, a prominent target that sustains VZV viremia. The stage in the MHC I assembly process that was disrupted by VZV in fibroblasts was examined in pulse-chase and immunoprecipitation experiments in the presence of endoglycosidase H. MHC I complexes continued to be assembled in VZV-infected cells and were not retained in the endoplasmic reticulum. In contrast, immunofluorescence and confocal microscopy showed that VZV infection resulted in an accumulation of MHC I molecules which colocalized to the Golgi compartment. Inhibition of late viral gene expression by treatment of infected fibroblasts with phosphonoacetic acid did not influence the modulation of MHC I expression, nor did transfection of cells with plasmids expressing immediate early viral proteins. However, cells transfected with a plasmid carrying the early gene
ORF66
did result in a significant downregulation of MHC I expression, suggesting that this gene encodes a protein with an immunomodulatory function. Thus, VZV downregulates MHC I expression by impairing the transport of MHC I molecules from the Golgi compartment to the cell surface; this effect may enable the virus to evade CD8
+
T-cell immune recognition during VZV pathogenesis, including the critical phase of T-lymphocyte-associated viremia.
Augmentation of Human Immunodeficiency Virus Type 1 Subtype E (CRF01_AE) Multiple-Drug Resistance by Insertion of a Foreign 11-Amino-Acid Fragment into the Reverse Transcriptase
Authors: Sato Hironori , Tomita Yasuhiro , Ebisawa Kazuyoshi , Hachiya Atsuko , Shibamura Kayo , Shiino Teiichiro , Yang Rongge , Tatsumi Masashi , Gushi Kazuo , Umeyama Hideaki , Oka Shinichi , Takebe Yutaka , Nagai Yoshiyuki
Published: July 2002

A human immunodeficiency virus type 1 (HIV-1) subtype E (CRF01_AE) variant (99JP-NH3-II) possessing an in-frame 33-nucleotide insertion mutation in the β3-β4 loop coding region of the reverse transcriptase (RT) gene was isolated from a patient who had not responded to nucleoside analogue RT inhibitors. This virus exhibited an extremely high level of multiple nucleoside analog resistance (MNR). Neighbor-joining tree analysis of thepolsequences indicated that the 99JP-NH3-II variant had originated from the swarm of drug-sensitive predecessors in the patient. Population-based sequence analyses of 82 independently cloned RT segments from the patient suggested that the variants with the insertion, three or four 3′-azido-3′-deoxythymidine resistance mutations, and a T69I mutation in combination had strong selective advantages during chemotherapy. Consistently, in vitro mutagenesis of a drug-sensitive predecessor virus clone demonstrated that this mutation set functions cooperatively to confer a high level of MNR without deleterious effects on viral replication capability. Homology modeling of the parental RT and its MNR mutant showed that extension of the β3-β4 loop by an insertion caused reductions in the distances between the loop and the other subdomains, narrowing the template-primer binding cleft and deoxynucleoside triphosphate-binding pocket in a highly flexible manner. The origin of the insert is elusive, as every effort to find a homologue has been unsuccessful. Taken together, these data suggest that (i) HIV-1 tolerates in vivo insertions as long as 33 nucleotides into the highly conserved enzyme gene to survive multiple anti-HIV-1 inhibitors and (ii) the insertion mutation augments multiple-drug resistance, possibly by reducing the biochemical inaccuracy of substrate-enzyme interactions in the active center.
Adenovirus Vectors with the 100K Gene Deleted and Their Potential for Multiple Gene Therapy Applications
Authors: Hodges B. L. , Evans H. K. , Everett R. S. , Ding E. Y. , Serra D. , Amalfitano A.
Published: July 2002

The 100K protein has a number of critical roles vital for successful completion of the late phases of the adenovirus (Ad) life cycle. We hypothesized that the introduction of deletions within the 100K gene would allow for the production of a series of new classes of Ad vector, including one that is replication competent but blocked in the ability to carry out many late-phase Ad functions. Such a vector would have potential for several gene therapy applications, based upon its ability to increase the copy number of the transgene encoded by the vector (via genome replication) while decreasing the side effects associated with Ad late gene expression. To efficiently produce 100K-deleted Ad ([100K−]Ad) vectors, an E1- and 100K-complementing cell line (K-16) was successfully isolated. Transfection of an [E1−,100K−]Ad vector genome into the K-16 cells readily yielded high titers of the vector. After infection of noncomplementing cells, we demonstrated that [100K−]Ad vectors have a significantly decreased ability to express several Ad late genes. Additionally, if the E1 gene was present in the infected noncomplementing cells, [100K−]Ad vectors were capable of replicating their genomes to high copy number, but were significantly blocked in their ability to efficiently encapsidate the replicated genomes. Injection of an [E1−,100K−]Ad vector in vivo also correlated with significantly decreased hepatotoxicity, as well as prolonged vector persistence. In summary, the unique properties of [100K−]Ad vectors suggest that they may have utility in a variety of gene therapy applications.
Abundant Early Expression of gpUL4 from a Human Cytomegalovirus Mutant Lacking a Repressive Upstream Open Reading Frame
Authors: Alderete John P. , Child Stephanie J. , Geballe Adam P.
Published: July 2002

The human cytomegalovirus UL4 gene encodes a 48-kDa glycoprotein, expression of which is repressed at the translational level by a short upstream open reading frame (uORF2) within the UL4 transcript leader. Mutation of the uORF2 initiation codon in the viral genome eliminates ribosomal stalling at the uORF2 termination site, resulting in early and abundant gpUL4 protein synthesis. This mutation does not appear to affect viral replication kinetics in human fibroblasts. These results reveal that the unusual uORF2 inhibitory mechanism is a principal determinant of the abundance and timing of gpUL4 expression but is nonessential for replication in cell culture.
Sialic Acid Binding Activity of Transmissible Gastroenteritis Coronavirus Affects Sedimentation Behavior of Virions and Solubilized Glycoproteins
Authors: Krempl Christine , Herrler Georg
Published: July 2002

The sedimentation behavior of transmissible gastroenteritis coronavirus (TGEV) was analyzed. Upon sucrose gradient centrifugation, the major virus band was found at a density of 1.20 to 1.22 g/cm
3
. This high density was observed only when TGEV with a functional sialic acid binding activity was analyzed. Mutants of TGEV that lacked sialic acid binding activity due to a point mutation in the sialic acid binding site of the S protein were mainly recovered at a lower-density position on the sucrose gradient (1.18 to 1.19 g/cm
3
). Neuraminidase treatment of purified virions resulted in a shift of the sedimentation value from the higher to the lower density. These results suggest that binding of sialoglycoproteins to the virion surface is responsible for the sedimentation behavior of TGEV. When purified virions were treated with octylglucoside to solubilize viral glycoproteins, ultracentrifugation resulted in sedimentation of the S protein of TGEV. However, when neuraminidase-treated virions or mutants with a defective sialic acid binding activity were analyzed, the S protein remained in the supernatant rather than in the pellet fraction. These results indicate that the interaction of the surface protein S with sialoglycoconjugates is maintained after solubilization of this viral glycoprotein by detergent treatment.
Evidence for Common Structural Determinants of Human Immunodeficiency Virus Type 1 Coreceptor Activity Provided through Functional Analysis of CCR5/CXCR4 Chimeric Coreceptors
Authors: Pontow Suzanne , Ratner Lee
Published: July 2002

Human immunodeficiency virus type 1 (HIV-1) infection in vivo is dependent upon the interaction of the viral envelope glycoprotein gp120 with CC chemokine receptor 5 (CCR5) or CXC chemokine receptor 4 (CXCR4). To study the determinants of the gp120-coreceptor association, we generated a set of chimeric HIV-1 coreceptors which express all possible combinations of the four extracellular domains of CCR5 and CXCR4. Stable U87 astroglioma cell lines expressing CD4 and individual chimeric coreceptor proteins were tested against a variety of R5, X4, and R5X4 envelope glycoproteins and virus strains for their ability to support HIV-1-mediated cell fusion and infection, respectively. Each of the cell lines promoted fusion with cells expressing an HIV envelope glycoprotein, except for U87.CD4.5455, which presents the first extracellular loop (ECL1) and flanking sequences of CXCR4 in the context of CCR5. However, all of the chimeric coreceptors allowed productive infection by one or more of the viral strains tested. Viral phenotype was a predictive factor for the observed activity of the chimeric molecules; X4 and R5X4 HIV strains utilized a majority of the chimeras, while R5 strains were limited in their ability to infect cells expressing these chimeric molecules. The expression of CCR5 ECL2 within the CXCR4 backbone supported infection by an R5 primary isolate, but no chimeras bearing the N terminus of CCR5 exhibited activity with R5 strains. Remarkably, the introduction of any CXCR4 domain into the CCR5 backbone was sufficient to allow utilization by multiple X4 strains. However, critical determinants within ECL2 and/or ECL3 of CXCR4 were apparent for all X4 viruses upon replacement of these domains in CXCR4 with CCR5 sequences. Unexpectedly, chimeric coreceptor-facilitated entry was blocked in all cases by the presence of the CXCR4-specific inhibitor AMD3100. Our data provide proof that CCR5 contains elements that support usage by X4 viral strains and demonstrate that the gp120 interaction sites of CCR5 and CXCR4 are structurally related.
Identification of Novel Porcine Endogenous Betaretrovirus Sequences in Miniature Swine
Authors: Ericsson Thomas , Oldmixon Beth , Blomberg Jonas , Rosa Margaret , Patience Clive , Andersson Göran
Published: July 2002

PCR amplification of genomic DNA from miniature swine peripheral blood lymphocytes, using primers corresponding to highly conserved regions of the polymerase (
pol)
gene, allowed the identification of two novel porcine endogenous retrovirus (PERV) sequences, PMSN-1 and PMSN-4. Phylogenetic analyses of the nucleotide sequences of PMSN-1 and PMSN-4 revealed them to be most closely related to betaretroviruses. The identification of PERVs belonging to the
Betaretrovirus
genus shows that endogenous retroviruses of this family are more broadly represented in mammalian species than previously appreciated. Both sequences contained inactivating mutations, implying that these particular loci are defective. However, Southern blot analysis showed additional copies of closely related proviruses in the miniature swine genome. Analyses of fetal and adult miniature swine tissues revealed a broad mRNA expression pattern of both PMSN-1 and PMSN-4. The most abundant expression was detected in whole bone marrow c-
kit
+
(CD117
+
) progenitor bone marrow cells, fetal liver, salivary gland, and thymus. It appears unlikely that functional loci encoding these novel PERV sequences exist, but this remains to be established. The betaretrovirus sequences described in this report will allow such investigations to be actively pursued.
A Single Amino Acid Change in the Nuclear Localization Sequence of the nsP2 Protein Affects the Neurovirulence of Semliki Forest Virus
Authors: Fazakerley John K. , Boyd Amanda , Mikkola Marja L. , Kääriäinen Leevi
Published: July 2002

The replicase protein nsP2 of Semliki Forest virus (SFV) has a
648
RRR nuclear localization signal and is transported to the nucleus. SFV-RDR has a single amino acid change which disrupts this sequence and nsP2 nuclear transport. In BHK cells, SFV4 and SFV-RDR replicate to high titers, but SFV-RDR is less virulent in mice. We compared the replication of SFV4 and SFV-RDR in adult mouse brain. Both SFV4 and SFV-RDR were neuroinvasive following intraperitoneal inoculation. SFV4 spread rapidly throughout the brain, whereas SFV-RDR infection was confined to small foci of cells. Both viruses infected neurons and oligodendrocytes. Both viruses induced apoptosis in cultured BHK cells but not in the cells of the adult mouse brain. SFV-RDR infection of mice lacking alpha/beta interferon receptors resulted in widespread virus distribution in the brain. Thus, a component of the viral replicase plays an important role in the neuropathogenesis of SFV.
Modifications of the Human Immunodeficiency Virus Envelope Glycoprotein Enhance Immunogenicity for Genetic Immunization
Authors: Chakrabarti Bimal K. , Kong Wing-pui , Wu Bei-yue , Yang Zhi-Yong , Friborg Jacques , Ling Xu , King Steven R. , Montefiori David C. , Nabel Gary J.
Published: July 2002

In this study, we have investigated the effect of specific mutations in human immunodeficiency virus type 1 (HIV-1) envelope (Env) on antibody production in an effort to improve humoral immune responses to this glycoprotein by DNA vaccination. Mice were injected with plasmid expression vectors encoding HIV Env with modifications in regions that might affect this response. Elimination of conserved glycosylation sites did not substantially enhance humoral or cytotoxic-T-lymphocyte (CTL) immunity. In contrast, a modified gp140 with different COOH-terminal mutations intended to mimic a fusion intermediate and stabilize trimer formation enhanced humoral immunity without reducing the efficacy of the CTL response. This mutant, with deletions in the cleavage site, fusogenic domain, and spacing of heptad repeats 1 and 2, retained native antigenic conformational determinants as defined by binding to known monoclonal antibodies or CD4, oligomer formation, and virus neutralization in vitro. Importantly, this modified Env, gp140ΔCFI, stimulated the antibody response to native gp160 while it retained its ability to induce a CTL response, a desirable feature for an AIDS vaccine.