bluuebunny/crossref_metadata_2025 huggingface dataset:
Evolutionary Relationships among Strains of
Mycobacterium tuberculosis
with Few Copies of IS
6110
Authors: Dale Jeremy W. , Al-Ghusein Hasan , Al-Hashmi Salim , Butcher Philip , Dickens Anne L. , Drobniewski Francis , Forbes Ken J. , Gillespie Stephen H. , Lamprecht Dianie , McHugh Timothy D. , Pitman Richard , Rastogi Nalin , Smith Andrew T. , Sola Christophe , Yesilkaya Hasan
Published: April 2003

Molecular typing of
Mycobacterium tuberculosis
by using IS
6110
shows low discrimination when there are fewer than five copies of the insertion sequence. Using a collection of such isolates from a study of the epidemiology of tuberculosis in London, we have shown a substantial degree of congruence between IS
6110
patterns and both spoligotype and PGRS type. This indicates that the IS
6110
types mainly represent distinct families of strains rather than arising through the convergent insertion of IS
6110
into favored positions. This is supported by identification of the genomic sites of the insertion of IS
6110
in these strains. The combined data enable identification of the putative evolutionary relationships of these strains, comprising three lineages broadly associated with patients born in South Asia (India and Pakistan), Africa, and Europe, respectively. These lineages appear to be quite distinct from
M. tuberculosis
isolates with multiple copies of IS
6110
.
Repression of
Escherichia coli
PhoP-PhoQ Signaling by Acetate Reveals a Regulatory Role for Acetyl Coenzyme A
Authors: Lesley Joseph A. , Waldburger Carey D.
Published: April 2003

The PhoP-PhoQ two-component system regulates the transcription of numerous genes in response to changes in extracellular divalent cation concentration and pH. Here we demonstrate that the
Escherichia coli
PhoP-PhoQ two-component system also responds to acetate. Signaling by the
E
.
coli
PhoP-PhoQ system was repressed during growth in acetate (≥25 mM) in a PhoQ-dependent manner. The periplasmic sensor domain of PhoQ was not required for acetate to repress signaling. Acetate-mediated repression of the PhoP-PhoQ system was not related to changes in the intracellular concentration of acetate metabolites such as acetyl-phosphate or acetyladenylate. Genetic analysis of acetate metabolism pathways suggested that a perturbation of acetyl coenzyme A turnover was the cause of decreased PhoP-PhoQ signaling during growth in acetate. Consistent with this hypothesis, intracellular acetyl coenzyme A levels rose during growth in the presence of exogenous acetate. Acetyl coenzyme A inhibited the autokinase activity of PhoQ in vitro, suggesting that the in vivo repressing effect may be due to a direct inhibition mechanism.
Genomic Diversity and Relatedness of Bifidobacteria Isolated from a Porcine Cecum
Authors: Simpson P. J. , Stanton C. , Fitzgerald G. F. , Ross R. P.
Published: April 2003

This study initially involved the isolation of a number of bifidobacteria from either the lumen or the epithelium of a porcine cecum. A total of 160 isolates were selected at random on MRS plates containing cysteine hydrochloride (0.5 g/liter) and mupirocin (50 mg/liter). All were identified as bifidobacteria based on fructose-6-phosphate phosphoketolase activity. Following genomic digestion with the restriction enzymeXbaI and pulsed-field gel electrophoresis (PFGE), the isolates produced 15 distinct macro-restriction patterns. Several of the PFGE patterns differed by only 1, 2, or 3 DNA fragments and were grouped as related patterns into seven PFGE types, termed A through G. The related patterns appeared to show genomic plasticity within the isolates arising from chromosomal mutations or possibly horizontal transfer of plasmids. The relative frequency of each PFGE type was maintained within each cecal sample, with PFGE type E representing approximately 50% of the isolates. Randomly amplified polymorphic DNA PCR, cell morphology, whole-cell protein profiling, 16S ribosomal DNA sequencing, and DNA-DNA hybridization were used to determine if the seven apparently unrelated PFGE types represented genetically distinct isolates. Four groups were identified: PFGE types A, C/D/G, B/E, and F, and these appeared to representBifidobacterium minimum, Bifidobacterium pseudolongumsubsp. pseudolongum,andBifidobacterium pseudolongumsubsp.globosumand two new species, respectively. The data demonstrate the presence of considerable genomic diversity within a relatively simple bifidobacteria population, consisting of 15 distinct strains representing four groups, which was maintained throughout the porcine cecal contents and epithelial layer.
Signal Transduction Protein P
II
Is Required for NtcA-Regulated Gene Expression during Nitrogen Deprivation in the Cyanobacterium
Synechococcus elongatus
Strain PCC 7942
Authors: Fadi Aldehni M. , Sauer Jörg , Spielhaupter Christian , Schmid Roland , Forchhammer Karl
Published: April 2003

The transcription factor of the cyclic AMP receptor protein/FNR family, NtcA, and the P
II
signaling protein play central roles in global nitrogen control in cyanobacteria. A dependence on P
II
for NtcA-regulated transcription, however, has not been observed. In the present investigation, we examined alterations in gene expression following nitrogen deprivation in
Synechococcus elongatus
strain PCC 7942 and specifically the roles of NtcA and P
II
. Global changes in de novo protein synthesis following combined-nitrogen deprivation were visualized by in vivo [
35
S]methionine labeling and two-dimensional polyacrylamide gel electrophoresis analysis. Nearly all proteins whose synthesis responded specifically to combined-nitrogen deprivation in wild-type cells of
S. elongatus
failed to respond in P
II
- and NtcA-deficient mutants. One of the proteins whose synthesis was down-regulated in a P
II
- and NtcA-dependent manner was RbcS, the small subunit of RubisCO. Quantification of its mRNA revealed that the abundance of the
rbcLS
transcript following combined-nitrogen deprivation rapidly declined in wild-type cells but not in P
II
and NtcA mutant cells. To investigate further the relationship between P
II
and NtcA, fusions of the promotorless
luxAB
reporter genes to the NtcA-regulated
glnB
gene were constructed and these constructs were used to transform wild-type cells and P
II
−
and NtcA
−
mutants. Determination of bioluminescence under different growth conditions showed that NtcA represses gene expression in the presence of ammonium in a P
II
-independent manner. By contrast, NtcA-dependent activation of
glnB
expression following combined-nitrogen deprivation was impaired in the absence of P
II
. Together, these results suggest that under conditions of combined-nitrogen deprivation, the regulation of NtcA-dependent gene expression requires the P
II
signal transduction protein.
Pseudomonas syringae
Exchangeable Effector Loci: Sequence Diversity in Representative Pathovars and Virulence Function in
P. syringae
pv. syringae B728a
Authors: Deng Wen-Ling , Rehm Amos H. , Charkowski Amy O. , Rojas Clemencia M. , Collmer Alan
Published: April 2003

Pseudomonas syringae
is a plant pathogen whose pathogenicity and host specificity are thought to be determined by Hop/Avr effector proteins injected into plant cells by a type III secretion system.
P. syringae
pv. syringae B728a, which causes brown spot of bean, is a particularly well-studied strain. The type III secretion system in
P. syringae
is encoded by
hrp
(hypersensitive response and pathogenicity) and
hrc
(
hrp
conserved) genes, which are clustered in a pathogenicity island with a tripartite structure such that the
hrp
/
hrc
genes are flanked by a conserved effector locus and an exchangeable effector locus (EEL). The EELs of
P. syringae
pv. syringae B728a,
P. syringae
strain 61, and
P. syringae
pv. tomato DC3000 differ in size and effector gene composition; the EEL of
P. syringae
pv. syringae B728a is the largest and most complex. The three putative effector proteins encoded by the
P. syringae
pv. syringae B728a EEL—HopPsyC, HopPsyE, and HopPsyV—were demonstrated to be secreted in an Hrp-dependent manner in culture. Heterologous expression of
hopPsyC
,
hopPsyE
, and
hopPsyV
in
P. syringae
pv. tabaci induced the hypersensitive response in tobacco leaves, demonstrating avirulence activity in a nonhost plant. Deletion of the
P. syringae
pv. syringae B728a EEL strongly reduced virulence in host bean leaves. EELs from nine additional strains representing nine
P. syringae
pathovars were isolated and sequenced. Homologs of
avrPphE
(e.g.,
hopPsyE
) and
hopPsyA
were particularly common. Comparative analyses of these effector genes and
hrpK
(which flanks the EEL) suggest that the EEL effector genes were acquired by horizontal transfer after the acquisition of the
hrp
/
hrc
gene cluster but before the divergence of modern pathovars and that some EELs underwent transpositions yielding effector exchanges or point mutations producing effector pseudogenes after their acquisition.
Peptide-Peptide Interactions between Human Transferrin and Transferrin-Binding Protein B from
Moraxella
catarrhalis
Authors: Sims Kurtis L. , Schryvers Anthony B.
Published: April 2003

Transferrin-binding protein B (TbpB) is one component of a bipartite receptor in several gram-negative bacterial species that binds host transferrin and mediates the uptake of iron for growth. Transferrin and TbpB are both bilobed proteins, and the interaction between these proteins seems to involve similar lobe-lobe interactions. Synthetic overlapping peptide libraries representing the N lobe of TbpB from
Moraxella
catarrhalis
were prepared and probed with labeled human transferrin. Transferrin-binding peptides were localized to six different regions of the TbpB N lobe, and reciprocal experiments identified six different regions of the C lobe of transferrin that bound TbpB. Truncations of the N lobe of TbpB that sequentially removed each transferrin-binding determinant were used to probe an overlapping peptide library of the C lobe of human transferrin. The removal of each TbpB N-lobe transferrin-binding determinant resulted in a loss of reactivity with peptides from the synthetic peptide library representing the C lobe of transferrin. Thus, individual peptide-peptide interactions between ligand and receptor were identified. A structural model of human transferrin was used to map surface regions capable of binding to TbpB.
Three-Dimensional Structure of the
Neisseria meningitidis
Secretin PilQ Determined from Negative-Stain Transmission Electron Microscopy
Authors: Collins Richard F. , Ford Robert C. , Kitmitto Ashraf , Olsen Ranveig O. , Tønjum Tone , Derrick Jeremy P.
Published: April 2003

The PilQ secretin from the pathogenic bacterium
Neisseria meningitidis
is an integral outer membrane protein complex which plays a crucial role in the biogenesis of type IV pili. We present here the first three-dimensional structure of this type of secretin at 2.5-nm resolution, obtained by single-particle averaging methods applied to the purified protein complex visualized in a negative stain. In projection, the PilQ complex is circular, with a donut-like appearance. When viewed from the side it has a rounded, conical profile. The complex was demonstrated to have 12-fold rotational symmetry, and this property was used to improve the quality of the density map by symmetry averaging. The dominant feature of the structure is a cavity, 10 nm deep, within the center of the molecule. The cavity is funnel-shaped in cross section, measures 6.5 nm in diameter at the top of the complex, and tapers to a closed point, effectively blocking formation of a continuous pore through the PilQ complex. These results suggest that the complex would have to undergo a conformational change in order to accommodate an assembled pilus fiber of diameter 6.5 nm running through the outer membrane.
The REP2 Repeats of the Genome ofNeisseria meningitidisAre Associated with Genes Coordinately Regulated during Bacterial Cell Interaction
Authors: Morelle Sandrine , Carbonnelle Etienne , Nassif Xavier
Published: April 2003

Interaction with host cells is essential in meningococcal pathogenesis especially at the blood-brain barrier. This step is likely to involve a common regulatory pathway allowing coordinate regulation of genes necessary for the interaction with endothelial cells. The analysis of the genomic sequence ofNeisseria meningitidisZ2491 revealed the presence of many repeats. One of these, designated REP2, contains a −24/−12 type promoter and a ribosome binding site 5 to 13 bp before an ATG. In addition most of these REP2 sequences are located immediately upstream of an ORF. Among these REP2-associated genes arepilC1andcrgA, described as being involved in steps essential for the interaction ofN. meningitidiswith host cells. Furthermore, the REP2 sequences located upstream ofpilC1andcrgAcorrespond to the previously identified promoters known to be induced during the initial localized adhesion ofN. meningitidiswith human cells. This characteristic led us to hypothesize that at least some of the REP2-associated genes were upregulated under the same circumstances aspilC1andcrgA. Quantitative PCR in real time demonstrated that the expression of 14 out of 16 REP2-associated genes were upregulated during the initial localized adhesion ofN. meningitidis. Taken together, these data suggest that these repeats control a set of genes necessary for the efficient interaction of this pathogen with host cells. Subsequent mutational analysis was performed to address the role of these genes during meningococcus-cell interaction.
The Mechanism of Bacterial Infection by Filamentous Phages Involves Molecular Interactions between TolA and Phage Protein 3 Domains
Authors: Karlsson Fredrik , Borrebaeck Carl A. K. , Nilsson Nina , Malmborg-Hager Ann-Christin
Published: April 2003

The early events in filamentous bacteriophage infection of gram-negative bacteria are mediated by the gene 3 protein (g3p) of the virus. This protein has a sophisticated domain organization consisting of two N-terminal domains and one C-terminal domain, separated by flexible linkers. The molecular interactions between these domains and the known bacterial coreceptor protein (TolA) were studied using a biosensor technique, and we report here on interactions of the viral coat protein with TolA, as well as on interactions between the TolA molecules. We detected an interaction between the pilus binding second domain (N2) of protein 3 and the bacterial TolA. This novel interaction was found to depend on the periplasmatic domain of TolA (TolAII). Furthermore, extensive interaction was detected between TolA molecules, demonstrating that bacterial TolA has the ability to interact functionally with itself during phage infection. The kinetics of g3p binding to TolA is also different from that of bacteriocins, since both N-terminal domains of g3p were found to interact with TolA. The multiple roles for each of the separate g3p and TolA domains imply a delicate interaction network during the phage infection process and a model for the infection mechanism is hypothesized.
The
Staphylococcus aureus cidAB
Operon: Evaluation of Its Role in Regulation of Murein Hydrolase Activity and Penicillin Tolerance
Authors: Rice Kelly C. , Firek Brian A. , Nelson Jeremy B. , Yang Soo-Jin , Patton Toni G. , Bayles Kenneth W.
Published: April 2003

Recent studies have shown that expression of the
Staphylococcus aureus lrgAB
operon inhibits murein hydrolase activity and decreases sensitivity to penicillin-induced killing. It was proposed that the
lrgAB
gene products function in a manner analogous to an antiholin, inhibiting a putative holin from transporting murein hydrolases out of the cell. In the present study the
cidAB
operon was identified and characterized based on the similarity of the
cidA
and
cidB
gene products to the products of the
lrgAB
operon. Zymographic and quantitative analyses of murein hydrolase activity revealed that mutation of the
cidA
gene results in decreased extracellular murein hydrolase activity compared to that of
S
.
aureus
RN6390, the parental strain. Complementation of
cidA
expression restored the wild-type phenotype, indicating that expression of the
cidAB
operon has a positive influence on extracellular murein hydrolase activity. The
cidA
mutant also displayed a significant decrease in sensitivity to the killing effects of penicillin. However, complementation of the
cidA
defect did not restore penicillin sensitivity to wild-type levels. Reverse transcriptase PCR also revealed that
cidAB
is maximally expressed during early exponential growth, opposite of what was previously observed for
lrgAB
expression. Based on these results, we propose that the
cidAB
operon encodes the holin-like counterpart of the
lrgAB
operon and acts in a manner opposite from that of
lrgAB
by increasing extracellular murein hydrolase activity and increasing sensitivity to penicillin-induced killing.
Phenotypic Mutants of the Intracellular Actinomycete
Rhodococcus
equi
Created by In Vivo
Himar1
Transposon Mutagenesis
Authors: Ashour Joseph , Hondalus Mary K.
Published: April 2003

Rhodococcus
equi
is a facultative intracellular opportunistic pathogen of immunocompromised people and a major cause of pneumonia in young horses. An effective live attenuated vaccine would be extremely useful in the prevention of
R
.
equi
disease in horses. Toward that end, we have developed an efficient transposon mutagenesis system that makes use of a
Himar1
minitransposon delivered by a conditionally replicating plasmid for construction of
R
.
equi
mutants. We show that
Himar1
transposition in
R
.
equi
is random and needs no apparent consensus sequence beyond the required TA dinucleotide. The diversity of the transposon library was demonstrated by the ease with which we were able to screen for auxotrophs and mutants with pigmentation and capsular phenotypes. One of the pigmentation mutants contained an insertion in a gene encoding phytoene desaturase, an enzyme of carotenoid biosynthesis, the pathway necessary for production of the characteristic salmon color of
R
.
equi
. We identified an auxotrophic mutant with a transposon insertion in the gene encoding a putative dual-functioning GTP cyclohydrolase II-3,4-dihydroxy-2-butanone-4-phosphate synthase, an enzyme essential for riboflavin biosynthesis. This mutant cannot grow in minimal medium in the absence of riboflavin supplementation. Experimental murine infection studies showed that, in contrast to wild-type
R
.
equi
, the riboflavin-requiring mutant is attenuated because it is unable to replicate in vivo. The mutagenesis methodology we have developed will allow the characterization of
R
.
equi
virulence mechanisms and the creation of other attenuated strains with vaccine potential.
Mutations at Residues 282, 286, and 293 of Phage λ Integrase Exert Pathway-Specific Effects on Synapsis and Catalysis in Recombination
Authors: Bankhead Troy M. , Etzel Bernard J. , Wolven Felise , Bordenave Sylvain , Boldt Jeffrey L. , Larsen Teresa A. , Segall Anca M.
Published: April 2003

Bacteriophage λ integrase (Int) catalyzes site-specific recombination between pairs of attachment (att) sites. Theattsites contain weak Int-binding sites called core-type sites that are separated by a 7-bp overlap region, where cleavage and strand exchange occur. We have characterized a number of mutant Int proteins with substitutions at positions S282 (S282A, S282F, and S282T), S286 (S286A, S286L, and S286T), and R293 (R293E, R293K, and R293Q). We investigated the core- and arm-binding properties and cooperativity of the mutant proteins, their ability to catalyze cleavage, and their ability to form and resolve Holliday junctions. Our kinetic analyses have identified synapsis as the rate-limiting step in excisive recombination. The IntS282 and IntS286 mutants show defects in synapsis in the bent-L and excisive pathways, respectively, while the IntR293 mutants exhibit synapsis defects in both the excision and bent-L pathways. The results of our study support earlier findings that the catalytic domain also serves a role in binding to core-type sites, that the core contacts made by this domain are important for both synapsis and catalysis, and that Int contacts core-type sites differently among the four recombination pathways. We speculate that these residues are important for the proper positioning of the catalytic residues involved in the recombination reaction and that their positions differ in the distinct nucleoprotein architectures formed during each pathway. Finally, we found that not all catalytic events in excision follow synapsis: theattLsite probably undergoes several rounds of cleavage and ligation before it synapses and exchanges DNA withattR.
Global Analysis of Genes Regulated by EvgA of the Two-Component Regulatory System in
Escherichia coli
Authors: Nishino Kunihiko , Inazumi Yoshihiko , Yamaguchi Akihito
Published: April 2003

The response regulator EvgA controls expression of multiple genes conferring antibiotic resistance in
Escherichia coli
(K. Nishino and A. Yamaguchi, J. Bacteriol. 184:2319-2323, 2002). To understand the whole picture of EvgA regulation, DNA macroarray analysis of the effect of EvgA overproduction was performed. EvgA activated genes related to acid resistance, osmotic adaptation, and drug resistance.
In Vitro Properties of RpoS (σ
S
) Mutants of
Escherichia coli
with Postulated N-Terminal Subregion 1.1 or C-Terminal Region 4 Deleted
Authors: Gowrishankar J. , Yamamoto Kaneyoshi , Subbarayan P. R. , Ishihama Akira
Published: April 2003

Derivatives of the stationary-phase sigma factor σ
S
of
Escherichia coli
lacking either of two conserved domains, the postulated N-terminal subregion 1.1 or the C-terminal region 4, were shown to be competent in vitro for transcription initiation from several σ
S
-dependent promoters on supercoiled DNA templates. Unlike wild-type σ
S
, however, the deletion derivatives were inactive on relaxed templates. The anomalous slow electrophoretic mobility of σ
S
on denaturing gels was corrected by deletion of subregion 1.1, suggesting that this domain in σ
S
may be structurally and functionally analogous to subregion 1.1 of σ
70
, substitutions in which have previously been shown to rectify the anomalous electrophoretic migration of σ
70
(V. Gopal and D. Chatterji, Eur. J. Biochem. 244:614-618, 1997).
Hydrogen-Oxidizing Capabilities ofHelicobacter hepaticusand In Vivo Availability of the Substrate
Authors: Maier Robert J. , Olson Jonathan , Olczak Adriana
Published: April 2003

Hydrogen-oxidizing hydrogenase activity was detected inHelicobacter hepaticusand compared to the activity inHelicobacter pylorifor characteristics associated with hydrogen uptake respiratory hydrogenases. Intact whole cells could couple H2oxidation to oxygen uptake, and no H2uptake was observed without oxygen available to complete the respiratory pathway. TheH. hepaticusenzyme coupled H2oxidation to reduction of many positive potential acceptors, and it underwent anaerobic or reductive activation.H. hepaticushad a strong affinity for molecular H2(apparentKmof 2.5 μM), and microelectrode measurements on the livers of live mice demonstrated that H2is available in the host tissue at levels 20-fold greater than the apparent whole-cellKmvalue.
Heat Shock Protein 90-Independent Activation of Truncated Hepadnavirus Reverse Transcriptase
Authors: Wang Xingtai , Qian Xiaofeng , Guo Hwai-Chen , Hu Jianming
Published: March 2003

The reverse transcriptase (RT) encoded by hepadnaviruses (hepatitis B viruses) is a multifunctional protein critical for several aspects of viral assembly and replication. Reverse transcription is triggered by the specific interaction between the RT and an RNA signal located on the viral pregenomic RNA, termed ε, and is initiated through a novel protein priming mechanism whereby the RT itself serves as a protein primer and ε serves as the obligatory template. Using the RT from duck hepatitis B virus as a model, we previously demonstrated that RT-ε interaction and protein priming require the assistance of a host cell chaperone complex, heat shock protein 90 (Hsp90) and its cochaperones, which associates with the RT and facilitates the folding of the RT into an active conformation. We now report that extensive truncation removing the entire C-terminal RNase H domain and part of the central RT domain could relieve this dependence on Hsp90 for RT folding such that the truncated RT variants could function in ε interaction and protein priming independently of Hsp90. The presence of certain nonionic or zwitterionic detergent was sufficient to establish and maintain the truncated RT proteins in an active, albeit labile, state. Furthermore, we were able to refold an RT truncation variant de novo after complete denaturation. In contrast, the full-length RT and also RT variants with less-extensive C-terminal truncations required Hsp90 for activation. Surprisingly, the presence of detergent plus some yet-to-be-identified cytoplasmic factor(s) led to a dramatic suppression of the RT activities. These results have important implications for RT folding and conformational maturation, Hsp90 chaperone function, and potential inhibition of RT functions by host cell factors.
Silencing of the Baculovirus Op-
iap3
Gene by RNA Interference Reveals that It Is Required for Prevention of Apoptosis during
Orgyia pseudotsugata M
Nucleopolyhedrovirus Infection of Ld652Y Cells
Authors: Means John C. , Muro Israel , Clem Rollie J.
Published: March 2003

The Op-
iap3
gene from the baculovirus
Orgyia pseudotsugata
M
nucleopolyhedrovirus (Op
M
NPV) inhibits apoptosis induced by a mutant of
Autographa californica
M
NPV (Ac
M
NPV) that lacks the antiapoptotic gene
p35
, as well as apoptosis induced by a wide range of other stimuli in both mammalian and insect cells. However, the role of Op-
iap3
during Op
M
NPV infection has not been previously examined. To determine the function of the Op-IAP3 protein during Op
M
NPV infection, we used RNA interference (RNAi) to silence Op-
iap3
expression during Op
M
NPV infection of Ld652Y cells. Infected cells treated with Op-
iap3
double-stranded RNA (dsRNA) did not accumulate detectable Op-
iap3
mRNA, confirming that the Op-
iap3
gene was effectively silenced. Op-IAP3 protein was found to be a component of the budded virion; however, in Op
M
NPV-infected cells treated with Op-
iap3
dsRNA, the Op-IAP3 protein that was introduced by the inoculum virus decreased to almost undetectable levels by 12 h after dsRNA addition. Apoptosis was observed in infected cells treated with Op-
iap3
dsRNA beginning at 12 h, and by 48 h, almost all of the cells had undergone apoptosis. These results show for the first time that Op-IAP3 is necessary to prevent apoptosis during Op
M
NPV infection. In addition, our results demonstrate that the RNAi technique can be an effective tool for studying baculovirus gene function.
Constitutive Inositol Phosphate Formation in Cytomegalovirus-Infected Human Fibroblasts Is due to Expression of the Chemokine Receptor Homologue pUS28
Authors: Minisini Rosalba , Tulone Calogero , Lüske Anke , Michel Detlef , Mertens Thomas , Gierschik Peter , Moepps Barbara
Published: March 2003

An open reading frame (ORF), US28, with homology to mammalian chemokine receptors has been identified in the genome of human cytomegalovirus (HCMV). Its protein product, pUS28, has been shown to bind several human CC chemokines, including RANTES, MCP-1, and MIP-1α, and the CX
3
C chemokine fractalkine with high affinity. Addition of CC chemokines to cells expressing pUS28 was reported to cause a pertussis toxin-sensitive increase in the concentration of cytosolic free Ca
2+
. Recently, pUS28 was shown to mediate constitutive, ligand-independent, and pertussis toxin-insensitive activation of phospholipase C via G
q/11
-dependent signaling pathways in transiently transfected COS-7 cells. Since these findings are not easily reconciled with the former observations, we analyzed the role of pUS28 in mediating CC chemokine activation of pertussis toxin-sensitive G proteins in cell membranes and phospholipase C in intact cells. The transmembrane signaling functions of pUS28 were studied in HCMV-infected cells rather than in cDNA-transfected cells. Since DNA sequence analysis of ORF US28 of different laboratory and clinical strains had revealed amino acid sequence differences in the amino-terminal portion of pUS28, we compared two laboratory HCMV strains, AD169 and Toledo, and one clinical strain, TB40/E. The results showed that infection of human fibroblasts with all three HCMV strains led to a vigorous, constitutively enhanced formation of inositol phosphates which was insensitive to pertussis toxin. This effect was critically dependent on the presence of the US28 ORF in the HCMV genome but was independent of the amino acid sequence divergence of the three HCMV strains investigated. The constitutive activity of pUS28 is not explained by expression of pUS28 at high density in HCMV-infected cells. The pUS28 ligands RANTES and MCP-1 failed to stimulate binding of guanosine 5′-O-(3-[
35
S]thiotriphosphate to membranes of HCMV-infected cells and did not enhance constitutive activation of phospholipase C in intact HCMV-infected cells. These findings raise the possibility that the effects of CC chemokines and pertussis toxin on G protein-mediated transmembrane signaling previously observed in HCMV-infected cells are either independent of or not directly mediated by the protein product of ORF US28.
CXCL10 Production from Cytomegalovirus-Stimulated Microglia Is Regulated by both Human and Viral Interleukin-10
Authors: Cheeran Maxim C.-J. , Hu Shuxian , Sheng Wen S. , Peterson Phillip K. , Lokensgard James R.
Published: March 2003

Glial cells orchestrate immunocyte recruitment to focal areas of viral infection within the brain and synchronize immune cell functions through a regulated network of cytokines and chemokines. Since recruitment of T lymphocytes plays a critical role in resolving cytomegalovirus (CMV) infection, we investigated the production of a T-cell chemoattractant, CXCL10 (gamma interferon-inducible protein 10) in response to viral infection of human glial cells. Infection with CMV was found to elicit the production of CXCL10 from primary microglial cells but not from astrocytes. This CXCL10 expression was not dependent on secondary protein synthesis but did require the phosphorylation of p38 mitogen-activated protein (MAP) kinase. In addition, migration of activated lymphocytes toward supernatants from CMV-stimulated microglial cells was partially suppressed by anti-CXCL10 antibodies. Since regulation of central nervous system inflammation is essential to allow viral clearance without immunopathology, microglial cells were then treated with anti-inflammatory cytokines. CMV-induced CXCL10 production from microglial cells was suppressed following treatment with interleukin-10 (IL-10) and IL-4 but not following treatment with transforming growth factor β. The IL-10-mediated inhibition of CXCL10 production was associated with decreased CMV-induced NF-κB activation but not decreased p38 MAP kinase phosphorylation. Finally, CMV infection of fully permissive astrocytes resulted in mRNA expression for the viral homologue to human IL-10 (i.e., cmvIL-10 [UL111a]) in its spliced form and conditioned medium from CMV-infected astrocytes inhibited virus-induced CXCL10 production from microglial cells through the IL-10 receptor. These findings present yet another mechanism through which CMV may subvert host immune responses.
Subcellular Localization of Feline Immunodeficiency Virus Integrase and Mapping of Its Karyophilic Determinant
Authors: Woodward Cora L. , Wang Yao , Dixon Wendy J. , Htun Han , Chow Samson A.
Published: March 2003

Feline immunodeficiency virus (FIV), like other members of the lentivirus subfamily, such as human immunodeficiency virus type 1 (HIV-1), can infect nondividing and terminally differentiated cells. The transport of the preintegration complex into the nucleus is cell cycle-independent, but the mechanism is not well understood. Integrase is a key component of the complex and has been suggested to play a role in nuclear import during HIV-1 replication. To determine its karyophilic property, FIV integrase fused with glutathione
S
-transferase and enhanced green fluorescent protein was expressed in various feline and human cells and the subcellular localization was visualized by fluorescence microscopy. Wild-type FIV integrase was karyophilic in all cell lines tested and capable of targeting the fusion protein to the nuclei of transfected cells. Analysis of deletion and point mutation variants of FIV integrase failed to reveal any canonical nuclear localization signal, and the karyophilic determinant was mapped to the highly conserved N-terminal zinc-binding HHCC motif. A region near the C-terminal domain enriched with basic amino acid residues also affected the nuclear import of integrase. However, the role of this region is only modulatory in comparison to that of the zinc-binding domain. The N-terminal zinc-binding domain does not bind DNA and instead is essential in integrase multimerization. We therefore postulate that the karyophilic property of FIV integrase requires subunit multimerization promoted by the HHCC motif. Alternatively, the HHCC motif may directly promote interaction between FIV integrase and cellular proteins involved in nuclear import.
Switching Species Tropism: an Effective Way To Manipulate the Feline Coronavirus Genome
Authors: Haijema Bert Jan , Volders Haukeliene , Rottier Peter J. M.
Published: March 2003

Feline infectious peritonitis virus (FIPV), a coronavirus, is the causative agent of an invariably lethal infection in cats. Like other coronaviruses, FIPV contains an extremely large positive-strand RNA genome of ca. 30 kb. We describe here the development and use of a reverse genetics strategy for FIPV based on targeted RNA recombination that is analogous to what has been described for the mouse hepatitis virus (MHV) (L. Kuo et al., J. Virol. 74:1393-1406, 2000). In this two-step process, we first constructed by targeted recombination a mutant of FIPV, designated mFIPV, in which the ectodomain of the spike glycoprotein was replaced by that of MHV. This switch allowed for the selection of the recombinant virus in murine cells: mFIPV grows to high titers in these cells but has lost the ability to grow in feline cells. In a second, reverse process, mFIPV was used as the recipient, and the reintroduction of the FIPV spike now allowed for selection of candidate recombinants by their regained ability to grow in feline cells. In this fashion, we reconstructed a wild-type recombinant virus (r-wtFIPV) and generated a directed mutant FIPV in which the initiation codon of the nonstructural gene 7b had been disrupted (FIPVΔ7b). The r-wtFIPV was indistinguishable from its parental virus FIPV 79-1146 not only for its growth characteristics in tissue culture but also in cats, exhibiting a highly lethal phenotype. FIPVΔ7b had lost the expression of its 7b gene but grew unimpaired in cell culture, confirming that the 7b glycoprotein is not required in vitro. We establish the second targeted RNA recombination system for coronaviruses and provide a powerful tool for the genetic engineering of the FIPV genome.
The Human Cytomegalovirus Protein UL16 Mediates Increased Resistance to Natural Killer Cell Cytotoxicity through Resistance to Cytolytic Proteins
Authors: Odeberg Jenny , Browne Helena , Metkar Sunil , Froelich Christopher J. , Brandén Lars , Cosman David , Söderberg-Nauclér Cecilia
Published: March 2003

Several reports have shown that human cytomegalovirus (HCMV)-infected cells are resistant to NK lysis. These studies have focused on receptor-ligand interactions, and different HCMV proteins have been indicated to mediate inhibitory NK signals. Here, we report that the HCMV protein UL16 is of major importance for the ability of HCMV-infected cells to resist NK cell-mediated cytotoxicity. Fibroblasts infected with the UL16 deletion mutant HCMV strain exhibited a 70% increased sensitivity to NK killing at 7 days postinfection compared to AD169-infected cells. Interestingly, HCMV-infected cells did not appear to engage inhibitory molecules on NK cells, since the levels of granzyme B were not reduced in supernatants obtained from NK cell cocultures with infected target cells compared to uninfected target cells. Furthermore, HCMV-infected cells, but not cells infected with the UL16 deletion mutant HCMV strain, exhibited a significantly increased resistance to the action of cytolytic proteins, including perforin, granzyme B, streptolysin O, and porcine NK lysin. In addition, fluorescence-activated cell sorting for UL16-positive transfected cells resulted in protection levels of 90% compared to control cells carrying the green fluorescent protein vector. Thus, the UL16 protein mediates an increased protection against the action of cytolytic proteins released by activated NK cells, possibly by a membrane-stabilizing mechanisms, rather than by delivering negative signals to NK cells.
Homo-Oligomerization of the Porcine Reproductive and Respiratory Syndrome Virus Nucleocapsid Protein and the Role of Disulfide Linkages
Authors: Wootton Sarah K. , Yoo Dongwan
Published: March 2003

As a step toward understanding the assembly pathway of the porcine reproductive and respiratory syndrome virus (PRRSV), the oligomeric properties of the nucleocapsid (N) protein were investigated. In this study, we have demonstrated that under nonreducing conditions the N protein forms disulfide-linked homodimers. However, inclusion of an alkylating agent (
N
-ethylmaleimide [NEM]) prevented disulfide bond formation, suggesting that these intermolecular disulfide linkages were formed as a result of spurious oxidation during cell lysis. In contrast, N protein homodimers isolated from extracellular virions were shown to have formed NEM-resistant intermolecular disulfide linkages, the function of which is probably to impart stability to the virion. Pulse-chase analysis revealed that N protein homodimers become specifically disulfide linked within the virus-infected cell, albeit at the later stages of infection, conceivably when the virus particle buds into the oxidizing environment of the endoplasmic reticulum. Moreover, NEM-resistant disulfide linkages were shown to occur only during productive PRRSV infection, since expression of recombinant N protein did not result in the formation of NEM-resistant disulfide-linked homodimers. Mutational analysis indicated that of the three conserved cysteine residues in the N protein, only the cysteine at position 23 was involved in the formation of disulfide linkages. The N protein dimer was shown to be stable both in the presence and absence of intermolecular disulfide linkages, indicating that noncovalent interactions also play a role in dimerization. Non-disulfide-mediated N protein interactions were subsequently demonstrated both in vitro by the glutathione
S
-transferase (GST) pull-down assay and in vivo by the mammalian two-hybrid assay. Using a series of N protein deletion mutants fused to GST, amino acids 30 to 37 were shown to be essential for N-N interactions. Furthermore, since RNase A treatment markedly decreased N protein-binding affinity, it appears that at least in vitro, RNA may be involved in bridging N-N interactions. In cross-linking experiments, the N protein was shown to assemble into higher-order structures, including dimers, trimers, tetramers, and pentamers. Together, these findings demonstrate that the N protein possesses self-associative properties, and these likely provide the basis for PRRSV nucleocapsid assembly.
Progesterone Increases Susceptibility and Decreases Immune Responses to Genital Herpes Infection
Authors: Kaushic Charu , Ashkar Ali A. , Reid Lesley A. , Rosenthal Kenneth L.
Published: March 2003

Depo-provera, a long-acting progestational formulation, is widely used to facilitate infection of sexually transmitted diseases in animal models. We have previously reported that hormone treatments change susceptibility and immune responses to genital tract infections. In this study we compared the changes in susceptibility of mice to genital herpes simplex virus type 2 (HSV-2) after Depo-provera or a saline suspension of progesterone (P-sal). We found that following Depo-provera-treatment, mice had prolonged diestrus that lasted more than 4 weeks. This coincided with a 100-fold increase in susceptibility to genital HSV-2 compared to that of untreated mice. Mice given P-sal were in diestrous stage for 4 to 6 days before returning to irregular reproductive cycles. When these mice were infected at diestrus they showed a 10-fold increase in susceptibility compared to that of normal, untreated mice. P-sal-treated mice infected at estrus were susceptible to HSV-2, depending on the infectious dose. Normal, untreated mice in estrus were not susceptible to HSV-2, even at a high infectious dose of 107PFU. In addition to alterations in susceptibility, Depo-provera treatment had inhibitory effects on immune responses to HSV-2. Mice immunized with HSV-2 protein (gB) and treated with Depo-provera showed significant lowering of local HSV-2-specific immunoglobulin G (IgG) and IgA in their vaginal washes. Mice immunized with an attenuated strain of HSV-2 2 weeks after Depo-provera treatment failed to develop protection when challenged intravaginally with wild-type HSV-2. In contrast, mice given progesterone and immunized at diestrus or estrus were completely protected from intravaginal challenge. These studies show that Depo-provera treatment changes susceptibility and local immune responses to genital HSV-2 infection. Animal models and vaccine strategies for sexually transmitted diseases need to consider the effect of hormone treatments on susceptibility and immune responses.
Human Immunodeficiency Virus Type 1 Genetic Recombination Is More Frequent Than That of Moloney Murine Leukemia Virus despite Similar Template Switching Rates
Authors: Onafuwa Adewunmi , An Wenfeng , Robson Nicole D. , Telesnitsky Alice
Published: March 2003

Retroviral recombinants result from template switching between copackaged viral genomes. Here, marker reassortment between coexpressed vectors was measured during single replication cycles, and human immunodeficiency virus type 1 (HIV-1) recombination was observed six- to sevenfold more frequently than murine leukemia virus (MLV) recombination. Template switching was also assayed by using transduction-type vectors in which donor and acceptor template regions were joined covalently. In this situation, where RNA copackaging could not vary, MLV and HIV-1 template switching rates were indistinguishable. These findings argue that MLV's lower intermolecular recombination frequency does not reflect enzymological differences. Instead, these data suggest that recombination rates differ because coexpressed MLV RNAs are less accessible to the recombination machinery than are coexpressed HIV RNAs. This hypothesis provides a plausible explanation for why most gammaretrovirus recombinants, although relatively rare, display evidence of multiple nonselected crossovers. By implying that recombinogenic template switching occurs roughly four times on average during the synthesis of every MLV or HIV-1 DNA, these results suggest that virtually all products of retroviral replication are biochemical recombinants.
The Small Envelope Protein E Is Not Essential for Murine Coronavirus Replication
Authors: Kuo Lili , Masters Paul S.
Published: March 2003

The importance of the small envelope (E) protein in the assembly of coronaviruses has been demonstrated in several studies. While its precise function is not clearly defined, E is a pivotal player in the morphogenesis of the virion envelope. Expression of the E protein alone results in its incorporation into vesicles that are released from cells, and the coexpression of the E protein with the membrane protein M leads to the assembly of coronavirus-like particles. We have previously generated E gene mutants of mouse hepatitis virus (MHV) that had marked defects in viral growth and produced virions that were aberrantly assembled in comparison to wild-type virions. We have now been able to obtain a viable MHV mutant in which the entire E gene, as well as the nonessential upstream genes 4 and 5a, has been deleted. This mutant (ΔE) was obtained by a targeted RNA recombination method that makes use of a powerful host range-based selection system. The ΔE mutant produces tiny plaques with an unusual morphology compared to plaques formed by wild-type MHV. Despite its low growth rate and low infectious titer, the ΔE mutant is genetically stable, showing no detectable phenotypic changes after several passages. The properties of this mutant provide further support for the importance of E protein in MHV replication, but surprisingly, they also show that E protein is not essential.
Respiratory Syncytial Virus (RSV) Fusion Protein Subunit F2, Not Attachment Protein G, Determines the Specificity of RSV Infection
Authors: Schlender Jörg , Zimmer Gert , Herrler Georg , Conzelmann Karl-Klaus
Published: March 2003

Human respiratory syncytial virus (HRSV) and bovine RSV (BRSV) infect human beings and cattle in a species-specific manner. We have here analyzed the contribution of RSV envelope proteins to species-specific entry into cells. In contrast to permanent cell lines, primary cells of human or bovine origin, including differentiated respiratory epithelia, peripheral blood lymphocytes, and macrophages, showed a pronounced species-specific permissivity for HRSV and BRSV infection, respectively. Recombinant BRSV deletion mutants lacking either the small hydrophobic (SH) protein gene or both SH and the attachment glycoprotein (G) gene retained their specificity for bovine cells, whereas corresponding mutants carrying the HRSV F gene specifically infected human cells. To further narrow the responsible region of F, two reciprocal chimeric F constructs were assembled from BRSV and HRSV F1 and F2 subunits. The specificity of recombinant RSV carrying only the chimeric F proteins strictly correlated with the origin of the membrane-distal F2 domain. A contribution of G to the specificity of entry could be excluded after reintroduction of BRSV or HRSV G. Virus with F1 and G from BRSV and with only F2 from HRSV specifically infected human cells, whereas virus expressing F1 and G from HRSV and F2 from BRSV specifically infected bovine cells. The introduction of G enhanced the infectivities of both chimeric viruses to equal degrees. Thus, the role of the nominal attachment protein G is confined to facilitating infection in a non-species-specific manner, most probably by binding to cell surface glycosaminoglycans. The identification of the F2 subunit as the determinant of RSV host cell specificity facilitates identification of virus receptors and should allow for development of reagents specifically interfering with RSV entry.
The Extreme Carboxyl Terminus of v-Abl Is Required for Lymphoid Cell Transformation by Abelson Virus
Authors: Warren David , Griffin Deborah S. , Mainville Celine , Rosenberg Naomi
Published: March 2003

The v-Abl protein tyrosine kinase encoded by Abelson murine leukemia virus (Ab-MLV) induces transformation of pre-B cells in vivo and in vitro and can transform immortalized fibroblast cell lines in vitro. Although the kinase activity of the protein is required for these events, most previously studied mutants encoding truncated v-Abl proteins that lack the extreme carboxyl terminus retain high transforming capacity in NIH 3T3 cells but transform lymphocytes poorly. To understand the mechanisms responsible for poor lymphoid transformation, mutants expressing a v-Abl protein lacking portions of the COOH terminus were compared for their ability to transform pre-B cells. Although all mutants lacking sequences within the COOH terminus were compromised for lymphoid transformation, loss of amino acids in the central region of the COOH terminus, including those implicated in JAK interaction and DNA binding, decreased transformation twofold or less. In contrast, loss of the extreme COOH terminus rendered the protein unstable and led to rapid proteosome-mediated degradation, a feature that was more prominent when the protein was expressed in Ab-MLV-transformed lymphoid cells. These data indicate that the central portion of the COOH terminus is not essential for lymphoid transformation and reveal that one important function of the COOH terminus is to stabilize the v-Abl protein in lymphoid cells.