bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular Characterization and Regulation of the
aguBA
Operon, Responsible for Agmatine Utilization in
Pseudomonas aeruginosa
PAO1
Authors: Nakada Yuji , Jiang Ying , Nishijyo Takayuki , Itoh Yoshifumi , Lu Chung-Dar
Published: July 2002

Pseudomonas aeruginosa
PAO1 utilizes agmatine as the sole carbon and nitrogen source via two reactions catalyzed successively by agmatine deiminase (encoded by
aguA
; also called agmatine iminohydrolase) and
N
-carbamoylputrescine amidohydrolase (encoded by
aguB
). The
aguBA
and adjacent
aguR
genes were cloned and characterized. The predicted AguB protein (
M
r
32,759; 292 amino acids) displayed sequence similarity (≤60% identity) to enzymes of the β-alanine synthase/nitrilase family. While the deduced AguA protein (
M
r
41,190; 368 amino acids) showed no significant similarity to any protein of known function, assignment of agmatine deiminase to AguA in this report discovered a new family of carbon-nitrogen hydrolases widely distributed in organisms ranging from bacteria to
Arabidopsis
. The
aguR
gene encoded a putative regulatory protein (
M
r
24,424; 221 amino acids) of the TetR protein family. Measurements of agmatine deiminase and
N
-carbamoylputrescine amidohydrolase activities indicated the induction effect of agmatine and
N
-carbamoylputrescine on expression of the
aguBA
operon. The presence of an inducible promoter for the
aguBA
operon in the
aguR
-
aguB
intergenic region was demonstrated by
lacZ
fusion experiments, and the transcription start of this promoter was localized 99 bp upstream from the initiation codon of
aguB
by S1 nuclease mapping. Experiments with knockout mutants of
aguR
established that expression of the
aguBA
operon became constitutive in the
aguR
background. Interaction of AguR overproduced in
Escherichia coli
with the
aguBA
regulatory region was demonstrated by gel retardation assays, supporting the hypothesis that AguR serves as the negative regulator of the
aguBA
operon, and binding of agmatine and
N
-carbamoylputrescine to AguR would antagonize its repressor function.
Stability and Subcellular Localization of Cytadherence-Associated Protein P65 in
Mycoplasma pneumoniae
Authors: Jordan Jarrat L. , Berry Karen M. , Balish Mitchell F. , Krause Duncan C.
Published: July 2002

The surface protein P65 is a constituent of the
Mycoplasma pneumoniae
cytoskeleton and is present at reduced levels in mutants lacking the cytadherence accessory protein HMW2. Pulse-chase studies demonstrated that P65 is subject to accelerated turnover in the absence of HMW2. P65 was also less abundant in noncytadhering mutants lacking HMW1 or P30 but was present at wild-type levels in mutants lacking proteins A, B, C, and P1. P65 exhibited a polar localization like that in wild-type
M. pneumoniae
in all mutants having normal levels of HMW1 and HMW2. Partial or complete loss of these proteins, however, correlated with severe reduction in the P65 level and the inability to localize P65 properly.
A Putatively Phase Variable Gene (
dca
) Required for Natural Competence in
Neisseria gonorrhoeae
but Not
Neisseria meningitidis
Is Located within the Division Cell Wall (
dcw
) Gene Cluster
Authors: Snyder Lori A. S. , Saunders Nigel J. , Shafer William M.
Published: July 2002

A cluster of 18 open reading frames (ORFs), 15 of which are homologous to genes involved in division and cell wall synthesis, has been identified in
Neisseria gonorrhoeae
and
Neisseria meningitidis
. The three additional ORFs, internal to the
dcw
cluster, are not homologous to
dcw
-related genes present in other bacterial species. Analysis of the
N. meningitidis
strain MC58 genome for foreign DNA suggests that these additional ORFs have not been acquired by recent horizontal exchange, indicating that they are a long-standing, integral part of the neisserial
dcw
gene cluster. Reverse transcription-PCR analysis of RNA extracted from
N. gonorrhoeae
strain FA19 confirmed that all three ORFs are transcribed in gonococci. One of these ORFs (
dca
, for division cluster competence associated), located between
murE
and
murF
, was studied in detail and found to be essential for competence in the gonococcal but not in the meningococcal strains tested. Computer analysis predicts that
dca
encodes an inner membrane protein similar to hypothetical proteins produced by other gram-negative bacteria. In some meningococcal strains
dca
is prematurely terminated following a homopolymeric tract of G's, the length of which differs between isolates of
N. meningitidis
, suggesting that
dca
is phase variable in this species. A deletion and insertional mutation was made in the
dca
gene of
N. gonorrhoeae
strain FA19 and
N. meningitidis
strain NMB. This mutation abrogated the ability of the gonococci to be transformed with chromosomal DNA. Thus, we conclude that the
dca
-encoded gene product is an essential competence factor for gonococci.
Interaction ofProteus mirabilisUrease Apoenzyme and Accessory Proteins Identified with Yeast Two-Hybrid Technology
Authors: Heimer Susan R. , Mobley Harry L. T.
Published: July 2002

Proteus mirabilis, a gram-negative bacterium associated with complicated urinary tract infections, produces a metalloenzyme urease which hydrolyzes urea to ammonia and carbon dioxide. The apourease is comprised of three structural subunits, UreA, UreB, and UreC, assembled as a homotrimer of individual UreABC heterotrimers (UreABC)3. To become catalytically active, apourease acquires divalent nickel ions through a poorly understood process involving four accessory proteins, UreD, UreE, UreF, and UreG. While homologues of UreD, UreF, and UreG have been copurified with apourease, it remains unclear specifically how these polypeptides associate with the apourease or each other. To identify interactions amongP. mirabilisaccessory proteins, in vitro immunoprecipitation and in vivo yeast two-hybrid assays were employed. A complex containing accessory protein UreD and structural protein UreC was isolated by immunoprecipitation and characterized with immunoblots. This association occurs independently of coaccessory proteins UreE, UreF, and UreG and structural protein UreA. In a yeast two-hybrid screen, UreD was found to directly interact in vivo with coaccessory protein UreF. Unique homomultimeric interactions of UreD and UreF were also detected in vivo. To substantiate the study of urease proteins with a yeast two-hybrid assay, previously described UreE dimers and homomultimeric UreA interactions among apourease trimers were confirmed in vivo. Similarly, a known structural interaction involving UreA and UreC was also verified. This report suggests that in vivo,P. mirabilisUreD may be important for recruitment of UreF to the apourease and that crucial homomultimeric associations occur among these accessory proteins.
Rapid Dephosphorylation of the TorR Response Regulator by the TorS Unorthodox Sensor in
Escherichia coli
Authors: Ansaldi Mireille , Jourlin-Castelli Cécile , Lepelletier Michèle , Théraulaz Laurence , Méjean Vincent
Published: July 2002

Induction of the
torCAD
operon, encoding the trimethylamine
N
-oxide (TMAO) respiratory system, is tightly controlled by the TorS-TorR phosphorelay system in response to TMAO availability. TorS is an unorthodox sensor that contains three phosphorylation sites and transphosphorylates TorR via a four-step phosphorelay, His443→Asp723→His850→Asp(TorR). In this study, we provide genetic evidence that TorS can dephosphorylate phospho-TorR when TMAO is removed. Dephosphorylation probably occurs by a reverse phosphorelay, Asp(TorR)→His850→Asp723, since His850 and Asp723 are both essential in this process. By using reverse transcriptase PCR, we also show that TMAO removal results in shutoff of
tor
operon transcription in less than 2 min. Based on our results and on analogy to other phosphorelay signal transduction systems, we propose that reverse phosphotransfer could be a rapid and efficient mechanism to inactivate response regulators.
IntI2 Integron Integrase in Tn7
Authors: Hansson Karin , Sundström Lars , Pelletier Alex , Roy Paul H.
Published: July 2002

Integrons can insert and excise antibiotic resistance genes on plasmids in bacteria by site-specific recombination. Class 1 integrons code for an integrase, IntI1 (337 amino acids in length), and are generally borne on elements derived from Tn5090, such as that found in the central part of Tn21. A second class of integron is found on transposon Tn7and its relatives. We have completed the sequence of the Tn7integrase gene,intI2, which contains an internal stop codon. This codon was found to be conserved amongintI2genes on three other Tn7-like transposons harboring different cassettes. The predicted peptide sequence (IntI2*) is 325 amino acids long and is 46% identical to IntI1. In order to detect recombination activity, the internal stop codon at position 179 in the parental allele was changed to a triplet coding for glutamic acid. The sequences flanking the cassette arrays in the class 1 and 2 integrons are not closely related, but a common pool of mobile cassettes is used by the different integron classes; two of the three antibiotic resistance cassettes on Tn7and its close relatives are also found in various class 1 integrons. We also observed a fourth excisable cassette downstream of those described previously in Tn7. The fourth cassette encodes a 165-amino-acid protein of unknown function with 6.5 contiguous repeats of a sequence coding for 7 amino acids. IntI2*179E promoted site-specific excision of each of the cassettes in Tn7at different frequencies. The integrases from Tn21and Tn7showed limited cross-specificity in that IntI1 could excise all cassettes from both Tn21and Tn7. However, we did not observe a corresponding excision of theaadA1cassette from Tn21by IntI2*179E.
Transcriptional Interference by a Complex Formed at the Centromere-Like Partition Site of Plasmid P1
Authors: Sawitzke James A. , Li Yongfang , Sergueev Kirill , Youngren Brenda , Brendler Therese , Jones Kristine , Austin Stuart
Published: July 2002

The partition site,
parS
, promotes accurate segregation of the replicated P1 plasmid to daughter cells when the P1-encoded ParA and ParB proteins are supplied. The
parS
site was inserted into the
Escherichia coli
chromosome between the promoter and the structural gene for β-galactosidase,
lacZ
. There was little interference with
lacZ
expression when ParA and ParB were supplied in
trans
. However, when a mutant ParA protein, ParAM314I, was supplied along with ParB, expression of
lacZ
was shut down. ParAM314I, ParB, and
parS
appear to form a nucleoprotein complex that blocks transcription. Mutations in
parA
and
parB
that relieved the
parA
M314I-dependent block were found. In addition, new mutations which impose the block were selected. Five of the latter mapped to
parA
and one to
parB
; all had a propagation-defective phenotype (Par
PD
) similar to that of
parA
M314I. Thus, whereas a null
par
mutant P1 plasmid segregates its DNA randomly, these mutants prevent even random distribution of the plasmid. We propose that ParA protein normally interacts transiently with the ParB-
parS
complex for partition to proceed but that the mutations block ParA dissociation. This “permanent” ParA-ParB-
parS
complex acts as a transcription block. Consistent with this hypothesis, we found that three of the seven blocking mutations lie within regions of ParA and ParB that are known to interact with each other. When the transcription block is imposed, regional silencing of nearby genes occurs. However, the requirement for ParA and a mutant
parA
or
parB
allele distinguishes the transcription block from the regional ParB-dependent gene silencing previously described.
Application of a Simple Multiplex PCR To Aid in Routine Work of the Mycobacterium Reference Laboratory
Authors: Yeboah-Manu Dorothy , Yates Malcolm D. , Wilson Stuart Mark
Published: July 2002

A PCR specific for spacer regions 33 and 34 of the direct repeat region of the
Mycobacterium tuberculosis
complex was developed to complement the biochemical differentiation of
M
.
tuberculosis
,
Mycobacterium bovis
,
M
.
bovis
BCG, and
Mycobacterium africanum
subtypes I and II. In addition, this approach was incorporated into a multiplex PCR that included primers specific for IS
6110
and the 65-kDa antigen gene in order to differentiate members of the
M
.
tuberculosis
complex from atypical mycobacteria.
Clonal Relationships among
Shigella
Serotypes Suggested by Cryptic Flagellin Gene Polymorphism
Authors: Coimbra Roney S. , Lefevre Martine , Grimont Francine , Grimont Patrick A. D.
Published: July 2002

The presence of cryptic
fliC
alleles in the genomes of 120 strains representative of the four
Shigella
species was investigated. One fragment was obtained by PCR amplification of
fliC
, with a size varying from 1.2 to 3.2 kbp, depending on the species or serotype. After digestion with endonuclease
Hha
I, the number of fragments in patterns varied from three to nine, with sizes of between 115 and 1,020 bp. Patterns sharing most of their bands were grouped to constitute an F type. A total of 17 different F types were obtained from all strains included in this study. A unique pattern was observed for each the following serotypes:
Shigella dysenteriae
1, 2, 8, and 10 and
S. boydii
7, 13, 15, 16, and 17. On the contrary,
S. dysenteriae
serotype 13 and
S. sonnei
biotype e were each subdivided into two different F types.
S. flexneri
serotypes 3a and X could be distinguished from the cluster containing
S. flexneri
serotypes 1 to 5 and Y.
S. flexneri
serotype 6 clustered with
S. boydii
serotypes 1, 2, 3, 4, 6, 8, 10, 11, 14, and 18 and
S. dysenteriae
serotypes 4, 5, 6, 7, 9, 11, and 12. Two other clusters were outlined: one comprising
S. dysenteriae
serotypes 3, 12, 13 (strain CDC598-77), 14, and 15 and the other one joining
S. boydii
serotypes 5 and 9. None of the 17
fliC
patterns was found in the
fliC Hha
I pattern database previously described for
Escherichia coli
. Overall, this work supports the hypothesis that
Shigella
evolved from different ancestral strains of
E. coli
. Moreover, the method outlined here is a promising tool for the identification of some clinically important
Shigella
strains as well as for confirmation of atypical isolates as
Shigella
spp.
Multicenter Evaluation of the BDProbeTec ET System for Detection of
Chlamydia trachomatis
and
Neisseria gonorrhoeae
in Urine Specimens, Female Endocervical Swabs, and Male Urethral Swabs
Authors: Van Der Pol Barbara , Ferrero Dennis V. , Buck-Barrington Linda , Hook Edward , Lenderman Connie , Quinn Thomas , Gaydos Charlotte A. , Lovchik Judith , Schachter Julius , Moncada Jeanne , Hall Geraldine , Tuohy Marion J. , Jones Robert B.
Published: July 2002

The performance of the Becton Dickinson BDProbe Tec ET System
Chlamydia trachomatis
and
Neisseria gonorrhoeae
Amplified DNA Assays (BD Biosciences, Sparks, Md.) was evaluated in a multicenter study. Specimens were collected from 2,109 men and women, with or without symptoms, attending sexually transmitted disease, family planning, and obstetrics and gynecology clinics. Both swab and urine samples were collected, and the results obtained from 4,131 specimens were compared to those from culture and the LCx nucleic acid amplification test (Abbott Industries, Abbott Park, Ill.). PCR and cytospin of the culture transport medium with chlamydia direct fluorescent antibody staining were used to adjudicate chlamydia culture-negative results. Sensitivity and specificity were calculated both with and without use of the amplification control (AC), with little apparent difference in the results. Without the AC result, sensitivity for
C. trachomatis
and
N. gonorrhoeae
were 92.8 and 96.6%, respectively, for cervical swabs and 80.5 and 84.9% for urine from women.
C. trachomatis
and
N. gonorrhoeae
sensitivities were 92.5 and 98.5%, respectively, for male urethral swabs and 93.1 and 97.9% for urine from men. This amplified DNA system for simultaneous detection of chlamydial and gonococcal infections demonstrated superior sensitivity compared to chlamydia culture and has performance characteristics comparable to those of other commercially available nucleic acid-based assays for these organisms.
Pneumocystis carinii
Carriage among Cystic Fibrosis Patients, as Detected by Nested PCR
Authors: Sing Andreas , Geiger Anna Maria , Hogardt Michael , Heesemann Jürgen
Published: July 2002

A total of 137 sputa from 95 consecutive cystic fibrosis (CF) patients undergoing routine bacteriological surveillance were analyzed for
Pneumocystis carinii
colonization using nested PCR. Seven of 95 patients (7.4%) were PCR positive, suggesting that
P. carinii
carriage may exist among CF patients due to their underlying pulmonary disease.
Group G Beta-Hemolytic Streptococcal Bacteremia Characterized by 16S Ribosomal RNA Gene Sequencing
Authors: Woo Patrick C. Y. , Fung Ami M. Y. , Lau Susanna K. P. , Wong Samson S. Y. , Yuen Kwok-Yung
Published: July 2002

Little is known about the relative importance of the four species of Lancefield group G beta-hemolytic streptococci in causing bacteremia and the factors that determine the outcome for patients with group G beta-hemolytic streptococcal bacteremia. From 1997 to 2000, 75 group G beta-hemolytic streptococcal strains were isolated from the blood cultures of 66 patients. Sequencing of the 16S rRNA genes of the group G beta-hemolytic streptococci showed that all 75 isolates were
Streptococcus dysgalactiae
subspecies
equisimilis
. The API system (20 STREP) and Vitek system (GPI) successfully identified 65 (98.5%) and 62 (93.9%) isolates, respectively, as
S. dysgalactiae
subspecies
equisimilis
with >95% confidence, whereas the ATB Expression system (ID32 STREP) only successfully identified 49 isolates (74.2%) as
S. dysgalactiae
subspecies
equisimilis
with >95% confidence. The median age of the patients was 76 years (range, 33 to 99 years). Fifty-six patients (85%) were over 60 years old. All patients had underlying diseases. No source of the bacteremia was identified (primary bacteremia) in 34 patients (52%), whereas 17 (26%) had cellulitis and 8 (12%) had bed sore or wound infections. Fifty-eight patients (88%) had community-acquired group G streptococcal bacteremia. Sixty-two patients (94%) had group G
Streptococcus
recovered in one blood culture, whereas 4 patients (6%) had it recovered in multiple blood cultures. Fifty-nine patients (89%) had group G
Streptococcus
as the only bacterium recovered in their blood cultures, whereas in 7 patients other bacteria were recovered concomitantly with the group G
Streptococcus
in the blood cultures (
Staphylococcus aureus
in 3,
Clostridium perfringens
in 2,
Citrobacter freundii
in 1, and
Bacteroides fragilis
in 1). Overall, 10 patients (15%) died. Male sex, diagnosis other than cellulitis, hospital-acquired bacteremia, and multiple positive blood cultures were associated with mortality {
P
< 0.005 (relative risk [RR] = 7.6),
P
< 0.05 (RR = 3.7),
P
< 0.005 (RR = 5.6), and
P
< 0.05 (RR = 5.6), respectively}. Unlike group C beta-hemolytic streptococcal bacteremia, group G beta-hemolytic streptococcal bacteremia is not a zoonotic infection in Hong Kong.
Application of Restriction Fragment Length Polymorphism Analysis of VP7-Encoding Genes: Fine Comparison of Irish and Global Rotavirus Isolates
Authors: O'Halloran F. , Lynch M. , Cryan B. , Fanning S.
Published: July 2002

A restriction fragment length polymorphism (RFLP) detection assay was developed to examine the genetic relationship(s) among VP7-encoding genes from 100 Irish rotavirus isolates and 30 randomly selected global rotavirus isolates (from the current databases). RFLP analysis of the VP7 gene segments was performed independently with three enzymes (
Rsa
I,
Alu
I, and
Eco
RV) in separate reactions by direct digestion of the DNA product amplified by reverse transcriptase (RT)-mediated PCR (RT-PCR) or by using computational methods. Thirty-six RFLP patterns were identified for all 130 strains, and of these, only nine patterns were associated with the Irish isolates. A correlation between the G type of the Irish isolates and certain single or combined enzyme profiles was apparent. These data suggested that the Irish wild-type rotavirus population was homogeneous and could be distinguished by RFLP analysis from global isolates of the same serotype(s). The deduced amino acid sequences of the VP7 RT-PCR products from six Irish isolates known to be of the G serotype revealed significant amino acid substitutions within major antigenic regions. In addition, these data identified the existence of at least two genetic lineages within serotype G1 strains which were distinguishable by RFLP analysis.
Genetic Profiling of Enterohemorrhagic
Escherichia coli
Strains in Relation to Clonality and Clinical Signs of Infection
Authors: Welinder-Olsson Christina , Badenfors Maria , Cheasty Tom , Kjellin Eva , Kaijser Bertil
Published: July 2002

Sixty-seven human strains of enterohemorrhagic
Escherichia coli
(EHEC) (from patients with more or less severe symptoms) were serogrouped and arranged according to pulsed-field gel electrophoresis (PFGE) patterns. We used PCR to investigate the strains according to known or putative virulence factors, and associations with disease were studied. All EHEC strains with the same PFGE pattern belonged to the same serogroup. On the contrary, two serogroups (O157 and O8) included strains with different PFGE patterns. We found several different combinations of chromosomal and plasmid-borne determinants, encoding the putative virulence factors, among the strains. As judged from clinical symptoms, there was no marked difference in pathogenicity among the strains and their combinations of virulence traits. All strains of O157 had the genes coding for verocytotoxin (VT) 2, intimin (eaeA),
E. coli
hemolysin (E-hly), and secreted serine protease (espP). Among EHEC non-O157 strains, the genes coding for VT1 and VT2 were equally dispersed. EaeA positivity was just as common among VT1- as VT2-positive strains. Among the plasmid-borne determinants, E-hly and espP were the most common and E-hly might be a pathogenicity marker among EHEC non-O157 strains. The conclusion is that PFGE is a very useful tool in epidemiological studies. The EHEC plasmids are heterogeneous in their gene composition, with the four plasmid-borne determinants found in many combinations. There was no reliable correlation between chromosomal and plasmid-borne virulence factors and human disease.
Six-Year Molecular Analysis of
Burkholderia cepacia
Complex Isolates among Cystic Fibrosis Patients at a Referral Center for Lung Transplantation
Authors: Heath David G. , Hohneker Kathy , Carriker Charlene , Smith Kelly , Routh Jonathan , LiPuma John J. , Aris Robert M. , Weber David , Gilligan Peter H.
Published: July 2002

Over a 6-year period,
Burkholderia cepacia
complex species were isolated from cystic fibrosis (CF) patients receiving care at The University of North Carolina Hospitals (clinic CF patients) and from those referred from other treatment centers. Fifty-six isolates collected from 30 referred patients and 26 clinic CF patients were characterized by pulsed-field gel electrophoresis (PFGE) and were assayed by PCR to detect the cable pilin gene,
cblA
. PFGE results indicated that six separate clusters (clusters A to F) were present among the 56 isolates and that three clusters (clusters A, B, and E) consisted only of isolates from referred patients infected with
B. cepacia
complex isolates prior to referral. However, one cluster (cluster C) consisted of isolates from four CF patients, and hospital records indicate that this cluster began with an isolate that came from a referred patient and that spread to three clinic CF patients. Cluster D consisted of two isolates from clinic CF patients, and hospitalization records are consistent with nosocomial, patient-to-patient spread.
cblA
was present in only 4 of the 56 isolates and included isolates in cluster E from the referred patients. Our results indicate a lack of spread of a previously characterized, transmissible clone from referred patients to our clinic CF population. Only two instances of nosocomial, patient-to-patient spread could be documented over the 6-year period. An additional spread of an isolate (cluster F) from a referred patient to a clinic patient could not be documented as nosocomial and may have been the result of spread in a nonhospitalized setting. The majority (36 of 56) of our
B. cepacia
complex-infected CF patients harbor isolates with unique genotypes, indicating that a diversity of sources account for infection. These data suggest that CF patients infected with
B. cepacia
complex and referred for lung transplantation evaluation were not a major source of
B. cepacia
complex strains that infected our resident CF clinic population.
The Surface Conformation of Sindbis Virus Glycoproteins E1 and E2 at Neutral and Low pH, as Determined by Mass Spectrometry-Based Mapping
Authors: Phinney Brett S. , Blackburn Kevin , Brown Dennis T.
Published: July 2002

Sindbis virus contains two membrane glycoproteins, E1 and E2, which are organized into 80 trimers of heterodimers (spikes). These trimers form a precise T=4 icosahedral protein lattice on the surface of the virus. Very little is known about the organization of the E1 and E2 glycoproteins within the spike trimer. To gain a better understanding of how the proteins E1 and E2 are arranged in the virus membrane, we have used the techniques of limited proteolysis and amino acid chemical modification in combination with mass spectrometry. We have determined that at neutral pH the E1 protein regions that are accessible to proteases include domains 1–21 (region encompassing amino acids 1 to 21), 161–176, and 212–220, while the E2 regions that are accessible include domains 31–84, 134–148, 158–186, 231–260, 299–314, and 324–337. When Sindbis virus is exposed to low pH, E2 amino acid domains 99–102 and 262–309 became exposed while other domains became inaccessible. Many new E1 regions became accessible after exposure to low pH, including region 86–91, which is in the putative fusion domain of E1 of Semliki Forest virus (SFV) (M. C. Kielian et al., J. Cell Biol. 134:863–872, 1996). E1 273–287 and region 145–158 were also exposed at low pH. These data support a model for the structure of the alphavirus spike in which the E1 glycoproteins are centrally located as trimers which are surrounded and protected by the E2 glycoprotein. These data improve our understanding of the structure of the virus membrane and have implications for understanding the protein conformational changes which accompany the process of virus-cell membrane fusion.
In Vivo Induction of a High-Avidity, High-Frequency Cytotoxic T-Lymphocyte Response Is Associated with Antiviral Protective Immunity
Authors: Sedlik C. , Dadaglio G. , Saron M. F. , Deriaud E. , Rojas M. , Casal S. I. , Leclerc C.
Published: July 2002

Many approaches are currently being developed to deliver exogenous antigen into the major histocompatibility complex class I-restricted antigen pathway, leading to in vivo priming of CD8+cytotoxic T cells. One attractive possibility consists of targeting the antigen to phagocytic or macropinocytic antigen-presenting cells. In this study, we demonstrate that strong CD8+class I-restricted cytotoxic responses are induced upon intraperitoneal immunization of mice with different peptides, characterized as CD8+T-cell epitopes, bound to 1-μm synthetic latex microspheres and injected in the absence of adjuvant. The cytotoxic response induced against a lymphocytic choriomeningitis virus (LCMV) peptide linked to these microspheres was compared to the cytotoxic T-lymphocyte (CTL) response obtained upon immunization with the nonreplicative porcine parvovirus-like particles (PPV:VLP) carrying the same peptide (PPV:VLP-LCMV) previously described (C. Sedlik, M. F. Saron, J. Sarraseca, I. Casal, and C. Leclerc, Proc. Natl. Acad. Sci. USA 94:7503–7508, 1997). We show that the induction of specific CTL activity by peptides bound to microspheres requires CD4+T-cell help in contrast to the CTL response obtained with the peptide delivered by viral pseudoparticles. Furthermore, PPV:VLP are 100-fold more efficient than microspheres in generating a strong CTL response characterized by a high frequency of specific T cells of high avidity. Moreover, PPV:VLP-LCMV are able to protect mice against a lethal LCMV challenge whereas microspheres carrying the LCMV epitope fail to confer such protection. This study demonstrates the crucial involvement of the frequency and avidity of CTLs in conferring antiviral protective immunity and highlights the importance of considering these parameters when developing new vaccine strategies.
Functional Heterogeneity and High Frequencies of Cytomegalovirus-Specific CD8+T Lymphocytes in Healthy Seropositive Donors
Authors: Gillespie Geraldine M. A. , Wills Mark R. , Appay Victor , O'Callaghan Chris , Murphy Mike , Smith Neil , Sissons Patrick , Rowland-Jones Sarah , Bell John I. , Moss Paul A. H.
Published: July 2002

Human cytomegalovirus (HCMV) infection is largely asymptomatic in the immunocompetent host, but remains a major cause of morbidity in immunosuppressed individuals. Using the recently described technique of staining antigen-specific CD8+T cells with peptide-HLA tetrameric complexes, we have demonstrated high levels of antigen-specific cells specific for HCMV peptides and show that this may exceed 4% of CD8+T cells in immunocompetent donors. Moreover, by staining with tetramers in combination with antibodies to cell surface markers and intracellular cytokines, we demonstrate functional heterogeneity of HCMV-specific populations. A substantial proportion of these are effector cytotoxic T lymphocytes, as demonstrated by their ability to lyse peptide-pulsed targets in “fresh” killing assays. These data suggest that the immune response to HCMV is periodically boosted by a low level of HCMV replication and that sustained immunological surveillance contributes to the maintenance of host-pathogen homeostasis. These observations should improve our understanding of the immunobiology of persistent viral infection.
In Vitro and In Vivo Characterization of a Murine Cytomegalovirus with a Transposon Insertional Mutation at Open Reading Frame M43
Authors: Xiao Jianqiao , Tong Tuong , Zhan Xiaoyan , Haghjoo Erik , Liu Fenyong
Published: July 2002

We have recently generated a pool of murine cytomegalovirus (MCMV) mutants by using a Tn
3
-based transposon mutagenesis approach. In this study, one of the MCMV mutants, RvM43, which contained the transposon inserted in open reading frame M43, was characterized. Our results provide the first direct evidence to suggest that M43 is not essential for viral replication in vitro in NIH 3T3 cells. Moreover, RvM43 exhibited a titer similar to that of the wild-type virus in the lungs, livers, spleens, and kidneys of both BALB/c and SCID mice and was as virulent as the wild-type virus in killing SCID mice that had been intraperitoneally infected with the viruses. In contrast, titers of the mutant virus in the salivary glands of the infected animals at 21 days postinfection were significantly (100 to 1,000-fold) lower than those of the wild-type virus and a rescued virus that restored the M43 region and its expression. Thus, M43 appears to be not essential for viral growth in vivo in the lungs, livers, spleens, and kidneys of infected animals and is also dispensable for virulence in killing SCID mice. Moreover, our results suggest that M43 is an MCMV determinant for growth in the salivary glands. Studies of viral genes required for replication in the salivary glands are important in understanding the mechanism of viral tropism for the salivary glands and shedding in saliva, which is believed to be one of the major routes of CMV transmission among healthy human populations.
Multi-PDZ Domain Protein MUPP1 Is a Cellular Target for both Adenovirus E4-ORF1 and High-Risk Papillomavirus Type 18 E6 Oncoproteins
Authors: Lee Siu Sylvia , Glaunsinger Britt , Mantovani Fiamma , Banks Lawrence , Javier Ronald T.
Published: July 2002

A general theme that has emerged from studies of DNA tumor viruses is that otherwise unrelated oncoproteins encoded by these viruses often target the same important cellular factors. Major oncogenic determinants for human adenovirus type 9 (Ad9) and high-risk human papillomaviruses (HPV) are the E4-ORF1 and E6 oncoproteins, respectively, and although otherwise unrelated, both of these viral proteins possess a functional PDZ domain-binding motif that is essential for their transforming activity and for binding to the PDZ domain-containing and putative tumor suppressor protein DLG. We report here that the PDZ domain-binding motifs of Ad9 E4-ORF1 and high-risk HPV-18 E6 also mediate binding to the widely expressed cellular factor MUPP1, a large multi-PDZ domain protein predicted to function as an adapter in signal transduction. With regard to the consequences of these interactions in cells, we showed that Ad9 E4-ORF1 aberrantly sequesters MUPP1 within the cytoplasm of cells whereas HPV-18 E6 targets this cellular protein for degradation. These effects were specific because mutant viral proteins unable to bind MUPP1 lack these activities. From these results, we propose that the multi-PDZ domain protein MUPP1 is involved in negatively regulating cellular proliferation and that the transforming activities of two different viral oncoproteins depend, in part, on their ability to inactivate this cellular factor.
A Dominant-Negative Herpesvirus Protein Inhibits Intranuclear Targeting of Viral Proteins: Effects on DNA Replication and Late Gene Expression
Authors: McNamee Elizabeth E. , Taylor Travis J , Knipe David M.
Published: July 2002

The
d
105 dominant-negative mutant form of the herpes simplex virus 1 (HSV-1) single-stranded DNA-binding protein, ICP8 (
d
105 ICP8), inhibits wild-type viral replication, and it blocks both viral DNA replication and late gene transcription, although to different degrees (M. Gao and D. M. Knipe, J. Virol. 65:2666–2675, 1991; Y. M. Chen and D. M. Knipe, Virology 221:281–290, 1996). We demonstrate here that this protein is also capable of preventing the formation of intranuclear prereplicative sites and replication compartments during HSV infection. We defined three patterns of ICP8 localization using indirect immunofluorescence staining of HSV-1-infected cells: large replication compartments, small compartments, and no specific intranuclear localization of ICP8. Cells that form large replication compartments replicate viral DNA and express late genes. Cells that form small replication compartments replicate viral DNA but do not express late genes, while cells without viral replication compartments are incapable of both DNA replication and late gene expression. The
d
105 ICP8 protein blocks formation of prereplicative sites and large replication compartments in 80% of infected cells and formation of large replication compartments in the remaining 20% of infected cells. The phenotype of
d
105 suggests a correlation between formation of large replication compartments and late gene expression and a role for intranuclear rearrangement of viral DNA and bound proteins in activation of late gene transcription. Thus, these results provide evidence for specialized machinery for late gene expression within replication compartments.
Adeno-Associated Virus (AAV) Rep Protein Enhances the Generation of a Recombinant Mini-Adenovirus (Ad) Utilizing an Ad/AAV Hybrid Virus
Authors: Sandalon Ziv , Gnatenko Dimitri V. , Bahou Wadie F. , Hearing Patrick
Published: July 2002

Mini-adenoviruses (mAd) deleted of all viral coding regions represent an emerging approach for transgene expression. We have exploited the unique features of the adeno-associated virus (AAV) terminal repeats within the context of an adenovirus–adeno-associated hybrid virus (Ad/AAV) as a strategy for rapid and efficient generation of mAd. Excision and generation of mAd from the parental Ad/AAV hybrid vector was achieved in 293 cells through recombination but without selection for mAd production. Analysis of mAd isolated from 293 cells indicated that mAd DNA exists as monomer and dimer forms within the recombinant viral capsid. Formation of recombinant mAd was significantly increased using an AAV Rep78- or Rep68-expressing cell line through Rep-mediated excision utilizing the AAV terminal repeat sequences present in the Ad/AAV hybrid virus genome. The mAd viruses were infectious and able to transfer functional gene to A549 and HeLa cells. This approach is rapid and efficient, thereby providing a simplified methodology for generating mAd with functional transducing capabilities.
Mechanism of Interference Mediated by Human Parainfluenza Virus Type 3 Infection
Authors: Horga Maria-Arantxa , Gusella G. Luca , Greengard Olga , Poltoratskaia Natalia , Porotto Matteo , Moscona Anne
Published: July 2002

Viral interference is characterized by the resistance of infected cells to infection by a challenge virus. Mechanisms of viral interference have not been characterized for human parainfluenza virus type 3 (HPF3), and the possible role of the neuraminidase (receptor-destroying) enzyme of the hemagglutinin-neuraminidase (HN) glycoprotein has not been assessed. To determine whether continual HN expression results in depletion of the viral receptors and thus prevents entry and cell fusion, we tested whether cells expressing wild-type HPF3 HN are resistant to viral infection. Stable expression of wild-type HN-green fluorescent protein (GFP) on cell membranes in different amounts allowed us to establish a correlation between the level of HN expression, the level of neuraminidase activity, and the level of protection from HPF3 infection. Cells with the highest levels of HN expression and neuraminidase activity on the cell surface were most resistant to infection by HPF3. To determine whether this resistance is attributable to the viral neuraminidase, we used a cloned variant HPF3 HN that has two amino acid alterations in HN leading to the loss of detectable neuraminidase activity. Cells expressing the neuraminidase-deficient variant HN-GFP were not protected from infection, despite expressing HN on their surface at levels even higher than the wild-type cell clones. Our results demonstrate that the HPF3 HN-mediated interference effect can be attributed to the presence of an active neuraminidase enzyme activity and provide the first definitive evidence that the mechanism for attachment interference by a paramyxovirus is attributable to the viral neuraminidase.
Clustered Charge-to-Alanine Mutagenesis of the Vaccinia Virus H5 Gene: Isolation of a Dominant, Temperature-Sensitive Mutant with a Profound Defect in Morphogenesis
Authors: DeMasi Joseph , Traktman Paula
Published: July 2002

The vaccinia virus H5 gene encodes a 22.3-kDa phosphoprotein that is expressed during both the early and late phases of viral gene expression. It is a major component of virosomes and has been implicated in viral transcription and, as a substrate of the B1 kinase, may participate in genome replication. To enable a genetic analysis of the role of H5 during the viral life cycle, we used clustered charge-to-alanine mutagenesis in an attempt to create a temperature-sensitive (
ts
) virus with a lesion in the H5 gene. Five mutant viruses were isolated, with one of them,
ts
H5-4, having a strong
ts
phenotype as assayed by plaque formation and measurements of 24-h viral yield. Surprisingly, no defects in genome replication or viral gene expression were detected at the nonpermissive temperature. By electron microscopy, we observed a profound defect in the early stages of virion morphogenesis, with arrest occurring prior to the formation of crescent membranes or immature particles. Nonfunctional, “curdled” virosomes were detected in
ts
H5-4 infections at the nonpermissive temperature. These structures appeared to revert to functional virosomes after a temperature shift to permissive conditions. We suggest an essential role for H5 in normal virosome formation and the initiation of virion morphogenesis. By constructing recombinant genomes containing two H5 alleles, wild type and H5-4, we determined that H5-4 exerted a dominant phenotype.
ts
H5-4 is the first example of a dominant
ts
mutant isolated and characterized in vaccinia virus.
Pathogenesis of Primary R5 Human Immunodeficiency Virus Type 1 Clones in SCID-hu Mice
Authors: Scoggins Robert M. , Taylor James R. , Patrie James , van't Wout Angélique B. , Schuitemaker Hanneke , Camerini David
Published: July 2002

We studied the replication and cytopathicity in SCID-hu mice of R5 human immunodeficiency virus type 1 (HIV-1) biological clones from early and late stages of infection of three patients who never developed MT-2 cell syncytium-inducing (SI; R5X4 or X4) viruses. Several of the late-stage non-MT-2 cell syncytium-inducing (NSI; R5) viruses from these patients depleted human CD4+thymocytes from SCID-hu mice. Earlier clones from the same patients did not deplete CD4+thymocytes from SCID-hu mice as well as later clones. We studied three R5 HIV-1 clones from patient ACH142 in greater detail. Two of these clones were obtained prior to the onset of AIDS; the third was obtained following the AIDS diagnosis. In GHOST cell infection assays, all three ACH142 R5 HIV-1 clones could infect GHOST cells expressing CCR5 but not GHOST cells expressing any of nine other HIV coreceptors tested. Furthermore, these patient clones efficiently infected stimulated peripheral blood mononuclear cells from a normal donor but not those from a homozygous CCR5Δ32 individual. Statistical analyses of data obtained from infection of SCID-hu mice with patient ACH142 R5 clones revealed that only the AIDS-associated clone significantly depleted CD4+thymocytes from SCID-hu mice. This clone also replicated to higher levels in SCID-hu mice than the two earlier clones, and a significant correlation between viral replication and CD4+thymocyte depletion was observed. Our results indicate that an intrinsic property of AIDS-associated R5 patient clones causes their increased replication and cytopathic effects in SCID-hu mice and likely contributes to the development of AIDS in patients who harbor only R5 quasispecies of HIV-1.
Expression of Functional Influenza Virus RNA Polymerase in the Methylotrophic Yeast
Pichia pastoris
Authors: Hwang Jung-Shan , Yamada Kazunori , Honda Ayae , Nakade Kohji , Ishihama Akira
Published: July 2002

Influenza virus RNA polymerase with the subunit composition PB1-PB2-PA is a multifunctional enzyme with the activities of both synthesis and cleavage of RNA and is involved in both transcription and replication of the viral genome. In order to produce large amounts of the functional viral RNA polymerase sufficient for analysis of its structure-function relationships, the cDNAs for RNA segments 1, 2, and 3 of influenza virus A/PR/8, each under independent control of the alcohol oxidase gene promoter, were integrated into the chromosome of the methylotrophic yeast
Pichia pastoris
. Simultaneous expression of all three P proteins in the yeast
P. pastoris
was achieved by the addition of methanol. To purify the P protein complexes, a sequence coding for a histidine tag was added to the PB2 protein gene at its N terminus. Starting from the induced
P. pastoris
cell lysate, we partially purified a 3P complex by Ni
2+
-agarose affinity column chromatography. The 3P complex showed influenza virus model RNA-directed and ApG-primed RNA synthesis in vitro but was virtually inactive without addition of template or primer. The kinetic properties of model template-directed RNA synthesis and the requirements for template sequence were analyzed using the 3P complex. Furthermore, the 3P complex showed capped RNA-primed RNA synthesis. Thus, we conclude that functional influenza virus RNA polymerase with the catalytic properties of a transcriptase is formed in the methylotrophic yeast
P. pastoris.
Dysregulation of Cyclin E Gene Expression in Human Cytomegalovirus-Infected Cells Requires Viral Early Gene Expression and Is Associated with Changes in the Rb-Related Protein p130
Authors: McElroy Anita K. , Dwarakanath Roopashree S. , Spector Deborah H.
Published: July 2002

We have previously shown that many cell cycle regulatory gene products are markedly affected by infection of primary fibroblasts with human cytomegalovirus (HCMV) (F. M. Jault, J. M. Jault, F. Ruchti, E. A. Fortunato, C. Clark, J. Corbeil, D. D. Richman, and D. H. Spector, J. Virol. 69:6697–6704, 1995). One of these proteins, cyclin E, is a key determinant of cell cycle progression during G
1
, and its mRNA levels are significantly increased in HCMV-infected fibroblasts (B. S. Salvant, E. A. Fortunato, and D. H. Spector, J. Virol. 72:3729–3741, 1998). To determine the molecular basis of this effect, we have examined the events that occur at the endogenous cyclin E promoter during the course of infection. In vivo dimethyl sulfate footprinting of the cyclin E promoter revealed several regions of protection and hypersensitivity that were unique to infected cells. In accord with this observation, we find that the virus-induced cyclin E transcripts initiate downstream of the start site identified in mock-infected cells, in regions where these newly appearing protected and hypersensitive sites occur. Viral gene expression is required for this induction. However, the viral immediate-early proteins IE1-72 and IE2-86, either alone or in combination, cannot induce expression of the endogenous cyclin E. The virus must progress past the immediate-early phase and express an early gene product(s) for activation of cyclin E expression. Moreover, IE1-72 does not appear to be required, as infection of cells with an HCMV mutant containing a deletion in the IE1-72 gene leads to full upregulation of cyclin E expression. Using electrophoretic mobility shift assays with infected cell extracts and a region of the cyclin E promoter that includes two previously defined E2F sites as the probe, we detected the appearance of an infection-specific banding pattern. One of the infection-specific bands contained the proteins E2F-4, DP-1, and p130, which were maintained in the infected cells as uniquely phosphorylated species. These results suggest that an altered E2F-4–DP-1–p130 complex along with viral early gene expression may play a role in the transcriptional regulation of cyclin E mRNA during HCMV infection.
Identification of a Novel Posttranscriptional Regulatory Element by Using a
rev-
and RRE-Mutated Human Immunodeficiency Virus Type 1 DNA Proviral Clone as a Molecular Trap
Authors: Nappi Filomena , Schneider Ralf , Zolotukhin Andrei , Smulevitch Sergey , Michalowski Daniel , Bear Jenifer , Felber Barbara K. , Pavlakis George N.
Published: July 2002

Human immunodeficiency virus (HIV) and all other lentiviruses utilize the essential viral protein Rev, which binds to RRE RNA, to export their unspliced and partially spliced mRNAs from the nucleus. We used a
rev-
and RRE-defective HIV type 1 (HIV-1) molecular clone in complementation experiments to establish a method for the rapid isolation of posttranscriptional regulatory elements from the mammalian genome by selecting for rescue of virus replication. Viruses rescued by this method contained a novel element with homology to rodent intracisternal A-particle (IAP) retroelements. A functional element was contained within a 247-nucleotide fragment named RNA transport element (RTE), which was able to promote replication of the Rev- and RRE-defective HIV-1 in both human lymphoid cell lines and primary lymphocytes, demonstrating its potent posttranscriptional function. RTE was functional in many cell types, indicating that the cellular factors that recognize RTE are widely expressed and evolutionarily conserved. RTE also promoted RNA export from
Xenopus
oocyte nuclei. RTE-mediated RNA transport was CRM1 independent, and RTE did not show high affinity for binding to mRNA export factor TAP/NXF1. Since CRM1 and TAP/NXF1 are critical export receptors associated with the two recognized mRNA export pathways, these results suggest that RTE functions via a distinct export mechanism. Taken together, our results identify a novel posttranscriptional control element that uses a conserved cellular export mechanism.