bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of Hsp90 function delays and impairs recovery from heat shock
Authors: Duncan Roger F
Published: September 2005

The induction of the heat shock response as well as its termination is autoregulated by heat shock protein activities. In this study we have investigated whether Hsp90 functional protein levels influence the characteristics and duration of the heat shock response. Treatment of cells with several benzoquinone ansamycin inhibitors of Hsp90 (geldanamycin, herbimycin A) activated a heat shock response in the absence of heat shock, as reported previously. Pretreatment of cells with the Hsp90 inhibitors significantly delayed the rate of restoration of normal protein synthesis following a brief heat shock. Concurrently, the rate of Hsp synthesis and accumulation was substantially increased and prolonged. The cessation of heat shock protein synthesis did not occur until the levels of Hsp70 were substantially elevated relative to its standard threshold for autoregulation. The elevated levels of HSPS 22–28 (the small HSPS) and Hsp70 are not able to promote thermotolerance when Hsp90 activity is repressed by ansamycins; rather a suppression of thermotolerance is observed. These results suggest that a multicomponent protein chaperone complex involving both Hsp90 and Hsp70 signals the cessation of heat shock protein synthesis, the restoration of normal translation, and likely the establishment of thermotolerance. Impaired function of either component is sufficient to alter the heat shock response.
The redshift distribution of gamma-ray bursts revisited
Authors: Natarajan P. , Albanna B. , Hjorth J. , Ramirez-Ruiz E. , Tanvir N. , Wijers R.
Published: September 2005

In this Letter, we calculate the redshift distribution of gamma-ray bursts assuming that they trace (i) the globally averaged star formation rate or (ii) the average metallicity in the Universe. While at redshifts 5 and below both the star formation rate and the metallicity are observationally determined modulo some uncertainties, at higher redshifts there are few constraints. We extrapolate the star formation rate and metallicity to higher redshifts and explore models that are broadly consistent with bounds on the optical depth from WMAP results. In addition, we also include parametric descriptions of the luminosity function, and the typical spectrum for gamma-ray bursts (GRBs). With these essential ingredients included in the modelling, we find that a substantial fraction (75 per cent) of GRBs are expected to originate at redshifts below 4, in variance with some previous estimates. Conversely, if we assume as expected for the collapsar model that gamma-ray bursts favour a low-metallicity environment, and therefore relate the GRB rate to a simple model of the average metallicity as a function of redshift, we find that a higher fraction of bursts, about 40 per cent, originate from z > 4. We conclude with the implications of Swift GRB detections.
Gap pattern and colonization opportunities in plant communities: effects of species richness, mortality, and spatial aggregation
Authors: Qing Li Zhen , Bogaert Jan , Nijs Ivan
Published: September 2005

We investigate how perturbations that induce mortality transform original spatial patterns in plant communities into binary spatial patterns of survivors and perished individuals. By means of computer simulation, we analyse effects of average mortality, interspecific variation of mortality around the mean, spatial distribution of the species (clumping degree), and species richness. Gap spatial pattern is quantified by four spatial indices or landscape metrics (gap area, density, shape and coherence). In single‐species communities, the emerging gap patterns are subject to critical phenomena: opportunities for colonizers to establish increase with mortality, but more rapidly at specific mortality thresholds. In multi‐species communities, neither species richness nor interspecific variation of mortality influences gap spatial pattern when community assembly is random. Colonization opportunities would therefore not be affected by local species extinction in such a system, nor by the presence of species with divergent sensitivities to perturbation. In a community that is highly spatially aggregated, increases in interspecific mortality variation shift the pattern towards fewer gaps that are larger and more isodiametric, which suggests increased establishment chances for colonizers. Similar changes are induced in communities characterized by large interspecific mortality differences if clumping degree is increased. Loss of species richness only modifies gap spatial pattern to a substantial extent if mortality variation is high: in this case, depauperate communities exhibit a wider variety of colonization opportunities (more gaps which are on average smaller, but the largest gap is larger) than species‐richer ones. These findings may explain the contrast between the negative diversity‐invasibility relationship often found in small‐scale experimental studies and the positive diversity‐invasibility relationship found in observational studies at larger scale. They also demonstrate that the pre‐disturbance spatial structure of a community significantly affects colonization opportunities for alien species, and is therefore a likely determinant of the trajectory of secondary succession following perturbation.
Long‐term population dynamics of the epiphytic bromeliad, Werauhia sanguinolenta
Authors: Zotz Gerhard , Laube Stefan , Schmidt Gerold
Published: September 2005

The population dynamics of the epiphytic bromeliad, Werauhia sanguinolenta, growing in the moist tropical forest of Barro Colorado Island, Panama, was studied for seven years from 1997 through 2004. In contrast to the generally held notion of the great importance of moisture availability for growth and survival of vascular epiphytes, no demographic process showed a significant correlation with the amount of annual precipitation or the varying number of rainy days per year. Most deaths, for example, were rather related to substrate instability (tree falls, branch breakage, or flaking bark) in all but the smallest size classes. We found evidence for both competition and facilitation. Elasticity analysis revealed that the finite rate of population increase, which invariably exceeded unity, was mostly influenced by survival (stasis) and to a lesser extent by growth, and very little by fecundity. In contrast to earlier reports on disastrous outbreaks of herbivores in this epiphyte species, the long‐term impact of herbivory on the population dynamics of W. sanguinolenta was negligible. Being at least facultatively autogamous, reproduction seems to be controlled by resource availability alone: this is suggested by long intervals between reproductive events, and a decrease in size and an increased mortality after reproduction. We conclude that the demography of this epiphytic bromeliad is probably influenced at least as much by biotic factors (i.e. the dynamics of the substratum) as by abiotic limitations.
Is the population turnover of patchy‐distributed annuals determined by dormancy dynamics or dispersal processes?
Authors: Dostál Petr
Published: September 2005

In species with fragmented distribution, regional turnover dynamics is given by the processes of local population extinction and patch (re)colonization by migrants spreading from neighboring occupied patches. In plants with dormant stages (e.g. seeds) and limited dispersal capacity, regional dynamics based on dispersal processes can be overridden by pseudo‐turnover determined by signals inducing or breaking dormancy (e.g. due to changes in habitat quality) resulting in a low importance of habitat configuration and size.  In this study, I investigated the turnover dynamics of 5 annual plant species growing on ant mounds ofLasius flavusover three years. I analyzed whether the grassland‐scale dynamics of these annuals is influenced by dispersal processes, or alternatively, by pseudo‐turnover of soil seed populations. For that purpose I 1) searched for populations formed from soil seeds only, 2) compared the relative contribution of the soil seed bank and seed rain for population restoration after disappearance from the vegetation and 3) investigated whether colonization and extinction events are affected by patch isolation. I assumed if population turnover was rather a result of the soil seed bank dynamics then spatial effects would be hard to detect.  In spite of the presence of populations formed from soil seed and the relatively more important soil seed bank for potential population reestablishment, turnover dynamics followed the predictions of metapopulation theory. Population appearance was more probable in larger and less isolated patches. Probability of disappearance increased with decrease of population size that was negatively influenced by the patch size and its isolation. These findings indicate dispersal processes to be important in the turnover dynamics and only limited contribution of soil seed populations. Their small effectiveness is probably related to the low chance of recurrent disturbance on the mound surface.
Ceftiofur-Resistant
Salmonella
Strains Isolated from Dairy Farms Represent Multiple Widely Distributed Subtypes That Evolved by Independent Horizontal Gene Transfer
Authors: Alcaine S. D. , Sukhnanand S. S. , Warnick L. D. , Su W.-L. , McGann P. , McDonough P. , Wiedmann M.
Published: September 2005

Salmonella
is the leading cause of known food-borne bacterial infections in the United States, with an incidence rate of approximately 15 cases per 100,000 people. The rise of antimicrobial-resistant
Salmonella
subtypes, including the appearance of subtypes resistant to ceftriaxone, represents a particular concern. Ceftriaxone is used to treat invasive cases of
Salmonella
in children and is closely related to ceftiofur, an antibiotic commonly used to treat diseases of cattle. In order to develop a better understanding of the evolution and transmission of ceftiofur resistance in
Salmonella
, we characterized ceftiofur-resistant and -sensitive
Salmonella
isolates from seven New York dairy farms. A total of 39 isolates from these seven farms were analyzed for evolutionary relatedness (by DNA sequencing of the
Salmonella
genes
fimA
,
manB
, and
mdh
), antibiotic resistance profiles, and the presence of
bla
CMY-2
, a beta-lactamase gene associated with resistance to cephalosporins. Our data indicate that (i) resistance to ceftriaxone and ceftiofur was highly correlated with the presence of
bla
CMY-2
; (ii) ceftiofur-resistant
Salmonella
strains were geographically widespread, as shown by their isolation from farms located throughout New York State; (iii) ceftiofur-resistant
Salmonella
strains isolated from farms represent multiple distinct subtypes and evolutionary lineages, as determined by serotyping, DNA sequence typing, and antimicrobial-resistance profiles; and (iv) ceftiofur-resistant
Salmonella
strains evolved by multiple independent acquisitions of an identical
bla
CMY-2
allele and by clonal spread of ceftiofur-resistant subtypes.
Transcriptional Inhibitor of Virulence Factors in EnteropathogenicEscherichia coli
Authors: Gauthier Annick , Robertson Marilyn L. , Lowden Michael , Ibarra J. Antonio , Puente José Luis , Finlay B. Brett
Published: September 2005

The type III secretion system (TTSS) is a key virulence mechanism of many important gram-negative bacterial pathogens. The TTSS is conserved among different bacterial pathogens, and mutations and deletions to the system significantly decrease virulence, making the TTSS an important potential therapeutic target. We have developed a high-throughput assay to search for inhibitors of the TTSS. We screened a commercial library of 20,000 small molecules for their ability to inhibit type III secretion by enteropathogenicEscherichia coli(EPEC). After discarding compounds that had no effect on secretion, inhibited bacterial growth, and/or caused degradation of EPEC-secreted proteins, the search was focused on a class of compounds that, while not direct inhibitors of type III secretion, inhibit expression of TTSS-related genes and other genes involved in virulence. This class of compounds does not affect bacterial viability or motility, indicating that it is not significantly affecting the expression of essential genes and is specific to virulence-associated genes. Transcriptional fusion assays confirmed that virulence-associated promoters were more sensitive to inhibition by this class of compounds. Overall, we have identified a class of compounds that can be used as a tool to probe the mechanism(s) that regulates virulence gene expression in EPEC.
Characterization of the Naturally Occurring Oxacillinase ofAcinetobacter baumannii
Authors: Héritier Claire , Poirel Laurent , Fournier Pierre-Edouard , Claverie Jean-Michel , Raoult Didier , Nordmann Patrice
Published: September 2005

A chromosomally encoded oxacillinase, OXA-69, was characterized fromAcinetobacter baumanniiAYE. β-Lactamase OXA-69 shared 97% amino acid identity with the recently described OXA-51 enzyme ofA. baumanniiand 62 and 56% amino acid identity with the carbapenem-hydrolyzing oxacillinases OXA-24 and OXA-23, respectively. Biochemical characterization of the purified OXA-69 revealed a narrow-spectrum hydrolysis profile but including, at a low level, imipenem and meropenem. By PCR and sequencingblaOXA-69-like genes were identified in allA. baumanniistrains tested (n= 12), suggesting that this oxacillinase is naturally occurring in that species.
Prevalence and Molecular Genetics of Macrolide Resistance among
Streptococcus pneumoniae
Isolates Collected in Finland in 2002
Authors: Rantala M. , Huikko S. , Huovinen P. , Jalava J.
Published: September 2005

The prevalence and mechanisms of macrolide resistance among 1,007 clinical pneumococcal isolates collected in Finland were investigated. Of these, 217 (21.5%) were resistant to erythromycin and 11% to clindamycin. Among the erythromycin-resistant isolates,
mef
(E) was present in 95 isolates (44%),
mef
(A) was present in 12 isolates (6%), and
erm
(B) was present in 90 isolates (41%). A double mechanism,
mef
(E) and
erm
(B), was detected in five isolates (2%). Ribosomal mutation was detected in 14 (6%) macrolide-resistant isolates in which no other determinant was found. Based on the telithromycin MICs, two groups of isolates were formed: 83.3% of the isolates belonged to a major group for which the telithromycin MIC range was ≤0.008 to 0.063 μg/ml, and 16.7% belonged to a minor group for which the telithromycin MIC range was 0.125 to 8 μg/ml. All except three isolates in the minor population carried a macrolide resistance gene.
Macrolide Efflux in
Streptococcus pneumoniae
Is Mediated by a Dual Efflux Pump (
mel
and
mef
) and Is Erythromycin Inducible
Authors: Ambrose Karita D. , Nisbet Rebecca , Stephens David S.
Published: September 2005

Macrolide resistance in
Streptococcus pneumoniae
due to efflux has emerged as an important worldwide clinical problem over the past decade. Efflux is mediated by the genes of the genetic element mega (
m
acrolide
e
fflux
g
enetic
a
ssembly) and related elements, such as Tn
1207.1
. These elements contain two adjacent genes,
mef
(
mefE
or
mefA
) and the closely related
mel
gene (
msrA
homolog), encoding a proton motive force pump and a putative ATP-binding cassette transporter homolog, and are transcribed as an operon (M. Del Grosso et al., J. Clin. Microbiol.
40:
774-778, 2004; K. Gay and D. S. Stephens, J. Infect. Dis.
184:
56-65, 2001; and M. Santagati et al., Antimicrob. Agents Chemother.
44:
2585-2587, 2000). Previous studies have shown that Mef is required for macrolide resistance in
S. pneumoniae
; however, the contribution of Mel has not been fully determined. Independent deletions were constructed in
mefE
and
mel
in the serotype 14 macrolide-resistant strains GA16638 (erythromycin [Em] MIC, 8 to 16 μg/ml) and GA17719 (Em MIC, 2 to 4 μg/ml), which contain allelic variations in the mega element. The MICs to erythromycin were significantly reduced for the independent deletion mutants of both
mefE
and
mel
compared to those of the parent strains and further reduced threefold to fourfold to Em MICs of <0.15 μg/ml with
mefE mel
double mutants. Using quantitative reverse transcription-PCR, the expression of
mefE
in the
mel
deletion mutants was increased more than 10-fold. However, in the
mefE
deletion mutants, the expression of
mel
did not differ significantly from the parent strains. The expression of both
mefE
and
mel
was inducible by erythromycin. These data indicate a requirement for both Mef and Mel in the novel efflux-mediated macrolide resistance system in
S. pneumoniae
and other gram-positive bacteria and that the system is inducible by macrolides.
Efficacy and Pharmacodynamics of Flucytosine Monotherapy in a Nonneutropenic Murine Model of Invasive Aspergillosis
Authors: te Dorsthorst D. T. A. , Verweij P. E. , Meis J. F. G. M. , Mouton J. W.
Published: September 2005

The therapeutic efficacy of flucytosine (5FC) monotherapy and the pharmacodynamic index predictive of efficacy were evaluated in a nonneutropenic mouse model of acute invasive aspergillosis. Mice were infected intravenously with an
Aspergillus fumigatus
isolate (the median MICs of 5FC were 128 μg/ml under the standard condition, 0.5 μg/ml at pH 6.0, and 0.031 μg/ml at pH 5.0) 2 h prior to the start of therapy and were treated for 7 days with different 5FC dosing regimens. The total doses ranged from 50 to 800 mg/kg of body weight/day and were administered at 6-, 12-, and 24-h intervals. The efficacy was assessed by means of survival. The survival rates of the treatment groups ranged from 40 to 90%, while the survival rate of the control group was 20%. The efficacy found depended primarily on the total daily dose. However, the power of our sample size may have been too low to exclude an effect of dose fractionation. The pharmacodynamic index that most strongly correlated with the efficacy was the area under the serum concentration-time curve and MIC ratio (
R
2
= 0.86). We conclude that 5FC monotherapy is efficacious in a murine
Aspergillus fumigatus
infection model.
Mode of Action and Biochemical Characterization of REP8839, a Novel Inhibitor of Methionyl-tRNA Synthetase
Authors: Ochsner Urs A. , Young Casey L. , Stone Kimberley C. , Dean Frank B. , Janjic Nebojsa , Critchley Ian A.
Published: September 2005

Aminoacyl-tRNA synthetases have attracted interest as essential and novel targets involved in bacterial protein synthesis. REP8839 is a potent inhibitor of MetS, the methionyl-tRNA synthetase in
Staphylococcus aureus
, including methicillin-resistant
S. aureus
(MRSA), and in
Streptococcus pyogenes
. The biochemical activity of REP8839 was shown by specific inhibition of purified
S. aureus
MetS (50% inhibitory concentration, <1.9 nM). Target specificity was confirmed by overexpression of the
metS
gene in
S. aureus
, resulting in an eightfold increase in the MIC for REP8839. Macromolecular synthesis assays in the presence of REP8839 demonstrated a dose-dependent inhibition of protein synthesis and RNA synthesis in
S. pneumoniae
R6, but only protein synthesis was affected in an isogenic
rel
mutant deficient in the stringent response. Strains with reduced susceptibility to REP8839 were generated by selection of strains with spontaneous mutations and through serial passages. Point mutations within the
metS
gene were mapped, leading to a total of 23 different amino acid substitutions within MetS that were located around the modeled active site. The most frequent MetS mutations were I57N, leading to a shift in the MIC from 0.06 μg/ml to 4 μg/ml, and G54S, resulting in a MIC of 32 μg/ml that was associated with a reduced growth rate. The mutation prevention concentration was 32 μg/ml in four
S. aureus
strains (methicillin-sensitive
S. aureus
and MRSA), which is well below the drug concentration of 2% (20,000 μg/ml) in a topical formulation. In conclusion, we demonstrate by biochemical, physiologic, and genetic mode-of-action studies that REP8839 exerts its antibacterial activity through specific inhibition of MetS, a novel target.
Different Clonal Complexes of Methicillin-Resistant
Staphylococcus aureus
Are Disseminated in the Euregio Meuse-Rhine Region
Authors: Deurenberg Ruud H. , Vink Cornelis , Oudhuis Guy J. , Mooij Jascha E. , Driessen Christel , Coppens Guy , Craeghs Jos , De Brauwer Els , Lemmen Sebastian , Wagenvoort Hans , Friedrich Alexander W. , Scheres Jacques , Stobberingh Ellen E.
Published: September 2005

The Euregio Meuse-Rhine (EMR) is formed by the border regions of Belgium, Germany, and The Netherlands. Cross-border health care requires infection control measures, in particular since the prevalence of methicillin-resistant
Staphylococcus aureus
(MRSA) differs among the three countries. To investigate the dissemination of MRSA in the EMR, 152 MRSA isolates were characterized by pulsed-field gel electrophoresis (PFGE), SCC
mec
typing, and multilocus sequence typing. PFGE revealed major clonal groups A, G, L, and Q, suggesting dissemination of MRSA in the EMR. Group A harbored mainly SCC
mec
type III and sequence types (STs) 239 and 241. The majority of the strains from group G harbored SCC
mec
type I and ST8 and ST247, whereas most strains from group L carried either SCC
mec
type IV or type I. Within group L, ST8 and ST228 were found, belonging to clonal complexes 8 and 5, respectively. Most strains from group Q included SCC
mec
type II and were sequence typed as ST225. Both ST225-MRSA-II and ST241-MRSA-III were novel findings in Germany. In addition, the SCC
mec
type of two isolates has not been described previously. One strain was classified as SCC
mec
type III but harbored the
pls
gene and the
dcs
region. Another strain was characterized as SCC
mec
type IV but lacked the
dcs
region. In addition, one isolate harbored both SCC
mec
type V and Panton-Valentine leukocidin. Finally, the SCC
mec
type of the strains was found to be correlated with the antibiotic susceptibility pattern.
Epigallocatechin Gallate Inhibits Biofilm Formation by Ocular Staphylococcal Isolates
Authors: Blanco Anna Rita , Sudano-Roccaro Andrea , Spoto Giovanna Carmela , Nostro Antonia , Rusciano Dario
Published: September 2005

Epigallocatechin gallate (EGCg), the main polyphenol component of green tea, has several antibacterial properties. Here we show that sub-MICs of EGCg appear to decrease slime production, therefore inhibiting biofilm formation by ocular staphylococcal isolates previously characterized for the presence of
ica
genes by the Congo red agar plate assay and for adhesion to microtiter plates.
In Vitro Susceptibility of
Mycobacterium tuberculosis
Clinical Isolates to Garenoxacin and DA-7867
Authors: Vera-Cabrera Lucio , Castro-Garza Jorge , Rendon Adrian , Ocampo-Candiani Jorge , Welsh Oliverio , Choi Sung Hak , Blackwood Kym , Molina-Torres Carmen
Published: September 2005

The in vitro activities of DA-7867, a novel oxazolidinone, and garenoxacin (BMS-284756) were compared to those of linezolid in 67 susceptible and drug-resistant clinical isolates of
Mycobacterium tuberculosis
. DA-7867 was the most active drug with an MIC
90
of 0.125 μg/ml, compared to the MIC
90
s of 4 μg/ml of garenoxacin and 2 μg/ml of linezolid.
Comparative In Vitro Activity of Ceftobiprole against Staphylococci Displaying Normal and Small-Colony Variant Phenotypes
Authors: von Eiff Christof , Friedrich Alexander W. , Becker Karsten , Peters Georg
Published: September 2005

The antistaphylococcal activity of ceftobiprole was compared with those of cefuroxime, linezolid, and moxifloxacin by using the agar dilution method. Apart from three strains with small-colony variant phenotypes, all
Staphylococcus aureus
isolates tested were inhibited by ≤2 μg/ml of ceftobiprole. This compound exhibited an excellent antistaphylococcal activity, comparable to that of linezolid.
Lack of Pharmacokinetic Drug Interaction between Tenofovir Disoproxil Fumarate and Nelfinavir Mesylate
Authors: Boffito Marta , Pozniak Anton , Kearney Brian P. , Higgs Christopher , Mathias Anita , Zhong Lijie , Shah Jaymin
Published: September 2005

A study explored the pharmacokinetics of tenofovir (300 mg administered once daily) and nelfinavir (1,250 mg twice daily) when coadministered in 29 healthy volunteers. Tenofovir, nelfinavir, and M8 pharmacokinetics was unaltered when tenofovir and nelfinavir were coadministered, and tenofovir administration did not affect the M8/nelfinavir area under the concentration-versus-time curve over the dosing interval (AUCtau) ratio. No interaction between tenofovir and nelfinavir was observed.
L-Domain Flanking Sequences Are Important for Host Interactions and Efficient Budding of Vesicular Stomatitis Virus Recombinants
Authors: Irie Takashi , Harty Ronald N.
Published: September 2005

Vesicular stomatitis virus (VSV) possesses a PPPY and a PSAP motif within the matrix (M) protein. The PPPY motif has significant L-domain activity in BHK-21 cells, whereas the PSAP motif does not. Since the core PSAP motif alone is insufficient to provide L-domain activity, we modified upstream or downstream amino acids flanking the PSAP core motif to determine their effect on L-domain activity. VSV recombinants were recovered that contained single or multiple amino acid mutations in upstream or downstream sequences flanking the PSAP core. Recombinant viruses were examined for growth kinetics, budding efficiency, and functional interactions with host proteins. We demonstrate that the composition of amino acids surrounding the L-domain core motifs are critical for efficient L-domain activity and for interactions with host proteins in the context of a VSV infection.
Comparative Sequence Analysis of the Hexon Gene in the Entire Spectrum of Human Adenovirus Serotypes: Phylogenetic, Taxonomic, and Clinical Implications
Authors: Ebner K. , Pinsker W. , Lion T.
Published: September 2005

The adenovirus (AdV) hexon constitutes the major virus capsid protein. The epitopes located on the hexon protein are targets of neutralizing antibodies in vivo, serve in the recognition by cytotoxic T cells, and provide the basis for the classification of adenoviruses into the 51 serotypes known to date. We have sequenced the entire hexon gene from human serotypes with incomplete or no sequence information available (
n
= 34) and performed a comparative analysis of all sequences. The overall sequence divergence between the 51 human serotypes ranged from 0.7 to 25.4% at the protein level. The sequence information has been exploited to assess the phylogeny of the adenovirus family, and protein distances between the six AdV species (A to F) and among individual serotypes within each species were calculated. The analysis revealed that the differences among serotypes within individual species range from 0.3 to 5.4% in the conserved regions (765 amino acids [aa]) and from 1.5 to 59.6% in the variable regions (154 to 221 aa). Serotypes of different species showed an expectedly greater divergence both in the conserved (5.9 to 12.3%) and variable (49.0 to 74.7%) regions. Construction of a phylogenetic tree revealed three major clades comprising the species B+D+E, A+F, and C, respectively. For serotypes 50 and 51, the original assignment to species B and D, respectively, is not in accordance with the hexon DNA and protein sequence data, which placed serotype 50 within species D and serotype 51 within species B. Moreover, the hexon gene of serotype 16, a member of species B, was identified as the product of recombination between sequences of species B and E. In addition to providing a basis for improved molecular diagnostics and classification, the elucidation of the complete hexon gene sequence in all AdV serotypes yields information on putative epitopes for virus recognition, which may have important implications for future treatment strategies permitting efficient targeting of any AdV serotype.
Rapid Recruitment of Virus-Specific CD8 T Cells Restructures Immunodominance during Protective Secondary Responses
Authors: Tebo Anne E. , Fuller Michael J. , Gaddis Dalia E. , Kojima Kyoko , Rehani Kunal , Zajac Allan J.
Published: September 2005

In this study we investigate the attributes of virus-specific memory CD8 T cells which most effectively control secondary infections. By rechallenging mice that had cleared primary lymphocytic choriomeningitis virus infections, we revealed that the secondary response is remarkably swift. Within 6 h following secondary infection, the production of gamma interferon becomes detectable directly ex vivo. During this protective phase of the secondary response, a very early elaboration of effector activities is preferentially exhibited by T cells specific for the viral NP396 epitope. This wave of activation contains the infection primarily before the initiation of the proliferative phase of the secondary response. Marked expansion is observed, but its magnitude differs depending on the epitope specificity of the responding cells; between 42 and 48 h following infection, ∼70% of NP396-specific memory cells are in the S phase of the cell cycle, as assessed by bromodeoxyuridine incorporation studies. Epitope-dependent differences during the proliferative phase of the secondary response were confirmed by adoptive transfer studies with CFSE-labeled T cells. Although NP396-specific T cells typically dominate secondary responses, the broader multiepitope-specific population of antiviral T cells is beneficial for controlling a variant virus with an escape mutation in this epitope. These findings indicate that the induction and maintenance of a focused response contribute to the clearance of secondary infections; however, a more diverse pool of antiviral T cells facilitates long-term immunity to mutable pathogens.
Cathepsin L Is Involved in Proteolytic Processing of the Hendra Virus Fusion Protein
Authors: Pager Cara Theresia , Dutch Rebecca Ellis
Published: September 2005

Proteolytic processing of paramyxovirus fusion (F) proteins is essential for the generation of a mature and fusogenic form of the F protein. Although many paramyxovirus F proteins are proteolytically processed by the cellular protease furin at a multibasic cleavage motif, cleavage of the newly emerged Hendra virus F protein occurs by a previously unidentified cellular protease following a single lysine at residue 109. We demonstrate here that the cellular protease cathepsin L is involved in converting the Hendra virus precursor F protein (F
0
) to the active F
1
+ F
2
disulfide-linked heterodimer. To initially identify the class of protease involved in Hendra virus F protein cleavage, Vero cells transfected with pCAGGS-Hendra F or pCAGGS-SV5 F (known to be proteolytically processed by furin) were metabolically labeled and chased in the absence or presence of serine, cysteine, aspartyl, and metalloprotease inhibitors. Nonspecific and specific protease inhibitors known to decrease cathepsin activity inhibited proteolytic processing of Hendra virus F but had no effect on simian virus 5 F processing. We next designed shRNA oligonucleotides to cathepsin L which dramatically reduced cathepsin L protein expression and enzyme activity. Cathepsin L shRNA-expressing Vero cells transfected with pCAGGS-Hendra F demonstrated a nondetectable amount of cleavage of the Hendra virus F protein and significantly decreased membrane fusion activity. Additionally, we found that purified human cathepsin L processed immunopurified Hendra virus F
0
into F
1
and F
2
fragments. These studies introduce a novel mechanism for primary proteolytic processing of viral glycoproteins and also suggest a previously unreported biological role for cathepsin L.
ADP-Ribose-1"-Monophosphatase: a Conserved Coronavirus Enzyme That Is Dispensable for Viral Replication in Tissue Culture
Authors: Putics Ákos , Filipowicz Witold , Hall Jonathan , Gorbalenya Alexander E. , Ziebuhr John
Published: September 2005

Replication of the ∼30-kb plus-strand RNA genome of coronaviruses and synthesis of an extensive set of subgenome-length RNAs are mediated by the replicase-transcriptase, a membrane-bound protein complex containing several cellular proteins and up to 16 viral nonstructural proteins (nsps) with multiple enzymatic activities, including protease, polymerase, helicase, methyltransferase, and RNase activities. To get further insight into the replicase gene-encoded functions, we characterized the coronavirus X domain, which is part of nsp3 and has been predicted to be an ADP-ribose-1"-monophosphate (Appr-1"-p) processing enzyme. Bacterially expressed forms of human coronavirus 229E (HCoV-229E) and severe acute respiratory syndrome-coronavirus X domains were shown to dephosphorylate Appr-1"-p, a side product of cellular tRNA splicing, to ADP-ribose in a highly specific manner. The enzyme had no detectable activity on several other nucleoside phosphates. Guided by the crystal structure of AF1521, an X domain homolog from
Archaeoglobus fulgidus
, potential active-site residues of the HCoV-229E X domain were targeted by site-directed mutagenesis. The data suggest that the HCoV-229E replicase polyprotein residues, Asn 1302, Asn 1305, His 1310, Gly 1312, and Gly 1313, are part of the enzyme's active site. Characterization of an Appr-1"-pase-deficient HCoV-229E mutant revealed no significant effects on viral RNA synthesis and virus titer, and no reversion to the wild-type sequence was observed when the mutant virus was passaged in cell culture. The apparent dispensability of the conserved X domain activity in vitro indicates that coronavirus replicase polyproteins have evolved to include nonessential functions. The biological significance of the novel enzymatic activity in vivo remains to be investigated.
Reconstitution and Molecular Analysis of an Active Human Immunodeficiency Virus Type 1 Nef/p21-Activated Kinase 2 Complex
Authors: Raney Alexa , Kuo Lillian S. , Baugh Laura L. , Foster John L. , Garcia J. Victor
Published: September 2005

Human immunodeficiency virus type 1 (HIV-1) Nef activation of p21-activated kinase 2 (PAK-2) was recapitulated in a cell-free system consisting of in vitro-transcribed RNA, rabbit reticulocyte lysate, and microsomal membranes on the basis of the following observations: (i) Nef associated with a kinase endogenous to the rabbit reticulocyte lysate that was identified as PAK-2, (ii) Nef-associated kinase activity was detected with Nefs from HIV-1
SF2
, HIV-1
YU2
, and SIV
mac239
, (iii) kinase activation was not detected with a myristoylation-defective Nef (HIV-1
SF2
NefG2A) or with a Nef defective in PAK-2 activation but fully competent in other Nef functions (HIV-1
SF2
NefF195I), and (iv) Nef-associated kinase activation required activated endogenous p21 GTPases (Rac1 or Cdc42). The cell-free system was used to analyze the mechanism of Nef activation of PAK-2. First, studies suggest that the p21 GTPases may act transiently to enhance Nef activation of PAK-2 in vitro. Second, addition of wortmannin to the cell-free system demonstrated that Nef activation of PAK-2 does not require PI 3-kinase activity. Third, ultracentrifugation analysis revealed that whereas the majority of Nef and PAK-2 partitioned to the supernatant, Nef-associated PAK-2 activity partitioned to the membrane-containing pellet as a low-abundance complex. Lastly, Nef activation of PAK-2 in vitro requires addition of microsomal membranes either during or after translation of the Nef RNA. These results are consistent with a model in which activation of PAK-2 by Nef occurs by recruiting PAK-2 to membranes. As demonstrated herein, the cell-free system is a new and important tool in the investigation of the mechanism of PAK-2 activation by Nef.
Coronaviruses as Vectors: Stability of Foreign Gene Expression
Authors: de Haan Cornelis A. M. , Haijema Bert Jan , Boss David , Heuts Frank W. H. , Rottier Peter J. M.
Published: September 2005

Coronaviruses are enveloped, positive-stranded RNA viruses considered to be promising vectors for vaccine development, as (i) genes can be deleted, resulting in attenuated viruses; (ii) their tropism can be modified by manipulation of their spike protein; and (iii) heterologous genes can be expressed by simply inserting them with appropriate coronaviral transcription signals into the genome. For any live vector, genetic stability is an essential requirement. However, little is known about the genetic stability of recombinant coronaviruses expressing foreign genes. In this study, the
Renilla
and the firefly luciferase genes were systematically analyzed for their stability after insertion at various genomic positions in the group 1 coronavirus feline infectious peritonitis virus and in the group 2 coronavirus mouse hepatitis virus. It appeared that the two genes exhibit intrinsic differences, the
Renilla
gene consistently being maintained more stably than the firefly gene. This difference was not caused by genome size restrictions, by different effects of the encoded proteins, or by different consequences of the synthesis of the additional subgenomic mRNAs. The loss of expression of the firefly luciferase was found to result from various, often large deletions of the gene, probably due to RNA recombination. The extent of this process appeared to depend strongly on the coronaviral genomic background, the luciferase gene being much more stable in the feline than in the mouse coronavirus genome. It also depended significantly on the particular genomic location at which the gene was inserted. The data indicate that foreign sequences are more stably maintained when replacing nonessential coronaviral genes.
TMC125 Displays a High Genetic Barrier to the Development of Resistance: Evidence from In Vitro Selection Experiments
Authors: Vingerhoets Johan , Azijn Hilde , Fransen Els , De Baere Inky , Smeulders Liesbet , Jochmans Dirk , Andries Koen , Pauwels Rudi , de Béthune Marie-Pierre
Published: September 2005

TMC125 is a potent new investigational nonnucleoside reverse transcriptase inhibitor (NNRTI) that is active against human immunodeficiency virus type 1 (HIV-1) with resistance to currently licensed NNRTIs. Sequential passage experiments with both wild-type virus and NNRTI-resistant virus were performed to identify mutations selected by TMC125 in vitro. In addition to “classic” selection experiments at a low multiplicity of infection (MOI) with increasing concentrations of inhibitors, experiments at a high MOI with fixed concentrations of inhibitors were performed to ensure a standardized comparison between TMC125 and current NNRTIs. Both low- and high-MOI experiments demonstrated that the development of resistance to TMC125 required multiple mutations which frequently conferred cross-resistance to efavirenz and nevirapine. In high-MOI experiments, 1 μM TMC125 completely inhibited the breakthrough of resistant virus from wild-type and NNRTI-resistant HIV-1, in contrast to efavirenz and nevirapine. Furthermore, breakthrough of virus from site-directed mutant (SDM) SDM-K103N/Y181C occurred at the same time or later with TMC125 as breakthrough from wild-type HIV-1 with efavirenz or nevirapine. The selection experiments identified mutations selected by TMC125 that included known NNRTI-associated mutations L100I, Y181C, G190E, M230L, and Y318F and the novel mutations V179I and V179F. Testing the antiviral activity of TMC125 against a panel of SDMs indicated that the impact of these individual mutations on resistance was highly dependent upon the presence and identity of coexisting mutations. These results demonstrate that TMC125 has a unique profile of activity against NNRTI-resistant virus and possesses a high genetic barrier to the development of resistance in vitro.
T-bet Is Required for Protection against Vaccinia Virus Infection
Authors: Matsui Masanori , Moriya Osamu , Yoshimoto Takayuki , Akatsuka Toshitaka
Published: September 2005

The transcription factor T-bet regulates the differentiation of CD4+T-helper type 1 (Th1) cells and represses Th2 lineage commitment. Since Th1 cells are crucial in the defense against pathogens, several studies addressed the role of T-bet in immunity to infection using T-bet knockout (T-bet−/−) mice. Nevertheless, it is still unclear whether T-bet is required for defense. Although vaccinia virus (VV) has extensively been used as an expression vector and the smallpox vaccine, there is only limited knowledge about immunity to VV infection. The urgency to understand the immune responses has been increased because of concerns about bioterrorism. Here, we show that T-bet is critical in the defense against VV infection as follows: (i) the survival rate of T-bet−/−mice was lower than that of control littermates postinfection; (ii) T-bet−/−mice lost more weight postinfection; and (iii) control mice cleared VV faster than T-bet−/−mice. As expected, a significant Th2 shift was observed in CD4+T cells of T-bet−/−mice. Furthermore, absence of T-bet impaired VV-specific CD8+cytotoxic T-lymphocyte (CTL) function, including cytolytic activity, antiviral cytokine production, and proliferation. Cytolytic capacity of natural killer (NK) cells was also diminished in T-bet−/−mice, whereas anti-VV antibody production was not impaired. These data reveal that the enhanced susceptibility to VV infection in T-bet−/−mice was at least partially due to the Th2 shift of CD4+T cells and the diminished function of VV-specific CTLs and NK cells but not due to downregulation of antibody production.
Priming-Boosting Vaccination with Recombinant
Mycobacterium bovis
Bacillus Calmette-Guérin and a Nonreplicating Vaccinia Virus Recombinant Leads to Long-Lasting and Effective Immunity
Authors: Ami Yasushi , Izumi Yasuyuki , Matsuo Kazuhiro , Someya Kenji , Kanekiyo Masaru , Horibata Shigeo , Yoshino Naoto , Sakai Koji , Shinohara Katsuaki , Matsumoto Sohkichi , Yamada Takeshi , Yamazaki Shudo , Yamamoto Naoki , Honda Mitsuo
Published: September 2005

Virus-specific T-cell responses can limit immunodeficiency virus type 1 (HIV-1) transmission and prevent disease progression and so could serve as the basis for an affordable, safe, and effective vaccine in humans. To assess their potential for a vaccine, we used
Mycobacterium bovis
bacillus Calmette-Guérin (BCG)-Tokyo and a replication-deficient vaccinia virus strain (DIs) as vectors to express full-length
gag
from simian immunodeficiency viruses (SIVs) (rBCG-SIVgag and rDIsSIVgag). Cynomolgus macaques were vaccinated with either rBCG-SIVgag dermally as a single modality or in combination with rDIsSIVgag intravenously. When cynomologus macaques were primed with rBCG-SIVgag and then boosted with rDIsSIVgag, high levels of gamma interferon (IFN-γ) spot-forming cells specific for SIV Gag were induced. This combination regimen elicited effective protective immunity against mucosal challenge with pathogenic simian-human immunodeficiency virus for the 1 year the macaques were under observation. Antigen-specific intracellular IFN-γ activity was similarly induced in each of the macaques with the priming-boosting regimen. Other groups receiving the opposite combination or the single-modality vaccines were not effectively protected. These results suggest that a recombinant
M. bovis
BCG-based vector may have potential as an HIV/AIDS vaccine when administered in combination with a replication-deficient vaccinia virus DIs vector in a priming-boosting strategy.
Virus Infection Switches TLR-3-Positive Human Neurons To Become Strong Producers of Beta Interferon
Authors: Préhaud Christophe , Mégret Françoise , Lafage Mireille , Lafon Monique
Published: September 2005

To study the capacity of human neurons to mount innate immunity responses to viral infections, we infected cells of a human postmitotic neuron-derivative cell line, NT2-N, with rabies virus (RABV) and herpes simplex type 1 (HSV-1). Changes in neuronal gene expression were analyzed by use of Affymetrix microarrays. Applying a twofold cutoff, RABV increased the transcription of 228 genes, and HSV-1 increased the transcription of 263 genes. The most striking difference between the two infections concerns genes involved in immunity. These genes represent 24% of the RABV-upregulated genes and only 4.9% of the HSV-1-upregulated genes. Following RABV infection, the most upregulated genes belong to the immunity cluster and included almost exclusively genes for beta interferon (
IFN
-β) primary and secondary responses as well as genes for chemokines (
CCL-5
,
CXCL-10
) and inflammatory cytokines (interleukin 6 [IL-6], tumor necrosis factor alpha, interleukin 1 alpha). In contrast, HSV-1 infection did not increase
IFN
-β gene transcripts and triggered the production of only
IL-6
and interferon regulatory factor 1 mRNAs. The microarray results were confirmed by real-time PCR, immunocytochemistry, and enzyme-linked immunosorbent assay. Human neurons were found to express Toll-like receptor 3. They produced
IFN
-β after treatment with poly(I:C) but not with lipopolysaccharide. Thus, human neurons can mount an innate immunity response to double-stranded RNA. These observations firmly establish that human neurons, in absence of glia, have the intrinsic machinery to sense virus infection.
Structural Genomics of the Severe Acute Respiratory Syndrome Coronavirus: Nuclear Magnetic Resonance Structure of the Protein nsP7
Authors: Peti Wolfgang , Johnson Margaret A. , Herrmann Torsten , Neuman Benjamin W. , Buchmeier Michael J. , Nelson Mike , Joseph Jeremiah , Page Rebecca , Stevens Raymond C. , Kuhn Peter , Wüthrich Kurt
Published: September 2005

Here, we report the three-dimensional structure of severe acute respiratory syndrome coronavirus (SARS-CoV) nsP7, a component of the SARS-CoV replicase polyprotein. The coronavirus replicase carries out regulatory tasks involved in the maintenance, transcription, and replication of the coronavirus genome. nsP7 was found to assume a compact architecture in solution, which is comprised primarily of helical secondary structures. Three helices (α2 to α4) form a flat up-down-up antiparallel α-helix sheet. The N-terminal segment of residues 1 to 22, containing two turns of α-helix and one turn of 310-helix, is packed across the surface of α2 and α3 in the helix sheet, with the α-helical region oriented at a 60° angle relative to α2 and α3. The surface charge distribution is pronouncedly asymmetrical, with the flat surface of the helical sheet showing a large negatively charged region adjacent to a large hydrophobic patch and the opposite side containing a positively charged groove that extends along the helix α1. Each of these three areas is thus implicated as a potential site for protein-protein interactions.
A Short Double-Stranded RNA Motif of
Peach Latent Mosaic Viroid
Contains the Initiation and the Self-Cleavage Sites of Both Polarity Strands
Authors: Delgado Sonia , Martínez de Alba Ángel E. , Hernández Carmen , Flores Ricardo
Published: September 2005

The transcription initiation sites of viroid RNAs, despite their relevance for replication and in vivo folding, are poorly characterized. Here we have examined this question for
Peach latent mosaic viroid
(PLMVd), which belongs to the family of chloroplastic viroids with hammerhead ribozymes (
Avsunviroidae
), by adapting an RNA ligase-mediated rapid amplification of cDNA ends methodology developed for mapping the genuine capped 5′ termini of eukaryotic messenger RNAs. To this aim, the characteristic free 5′-triphosphate group of chloroplastic primary transcripts from PLMVd-infected young fruits was previously capped in vitro with GTP and guanylyltransferase. PLMVd plus and minus initiation sites map at similar double-stranded motifs of 6 to 7 bp that also contain the conserved GUC triplet preceding the self-cleavage site in both polarity strands. Within the branched secondary structures predicted for the two PLMVd strands, this motif is located at the base of a similar long hairpin that presumably contains the promoters for a chloroplastic RNA polymerase. The transcription templates could be the circular viroid RNAs or their most abundant linear counterparts, assuming the involvement of an RNA polymerase able to jump over template discontinuities. Both PLMVd initiation sites were confirmed by applying the same methodology to two purified PLMVd subgenomic RNAs and by primer extension, and they therefore likely reflect the in vivo situation. The location of the PLMVd initiation sites provides a mechanistic view into how the nascent strands may fold and self-cleave during transcription. The approach described here may be extended to other chloroplastic RNA replicons and transcripts accumulating at low levels.