bluuebunny/crossref_metadata_2025 huggingface dataset:
Domain Interactions in the Yeast ATP Binding Cassette Transporter Ycf1p: Intragenic Suppressor Analysis of Mutations in the Nucleotide Binding Domains
Authors: Falcón-Pérez Juan M. , Martı́nez-Burgos Mónica , Molano Jesús , Mazón Marı́a J. , Eraso Pilar
Published: July 2002

The yeast cadmium factor (Ycf1p) is a vacuolar ATP binding cassette (ABC) transporter required for heavy metal and drug detoxification. Cluster analysis shows that Ycf1p is strongly related to the human multidrug-associated protein (MRP1) and cystic fibrosis transmembrane conductance regulator and therefore may serve as an excellent model for the study of eukaryotic ABC transporter structure and function. Identifying intramolecular interactions in these transporters may help to elucidate energy transfer mechanisms during transport. To identify regions in Ycf1p that may interact to couple ATPase activity to substrate binding and/or movement across the membrane, we sought intragenic suppressors of
ycf1
mutations that affect highly conserved residues presumably involved in ATP binding and/or hydrolysis. Thirteen intragenic second-site suppressors were identified for the D777N mutation which affects the invariant Asp residue in the Walker B motif of the first nucleotide binding domain (NBD1). Two of the suppressor mutations (V543I and F565L) are located in the first transmembrane domain (TMD1), nine (A1003V, A1021T, A1021V, N1027D, Q1107R, G1207D, G1207S, S1212L, and W1225C) are found within TMD2, one (S674L) is in NBD1, and another one (R1415G) is in NBD2, indicating either physical proximity or functional interactions between NBD1 and the other three domains. The original D777N mutant protein exhibits a strong defect in the apparent affinity for ATP and
V
max
of transport. The phenotypic characterization of the suppressor mutants shows that suppression does not result from restoring these alterations but rather from a change in substrate specificity. We discuss the possible involvement of Asp777 in coupling ATPase activity to substrate binding and/or transport across the membrane.
Evidence for the Contribution of Point Mutations tovlsEVariation and for Apparent Constraints on the Net Accumulation of Sequence Changes invlsEduring Infection with Lyme Disease Spirochetes
Authors: Sung Shian-Ying , McDowell John V. , Marconi Richard T.
Published: July 2002

In the Lyme disease spirochetes, both theospEandvlsEgene families have been demonstrated to undergo sequence variation during infection. To further investigate the mechanisms associated with the generation ofvlsvariation, single-nucleotide polymorphism and subsequent DNA sequence analyses were performed on thevlsEgene and its paralog, BBJ51, a related gene with a frameshift mutation. These analyses focused on a series of postinfection clonal populations obtained from mice infected withBorrelia burgdorferiB31MIpc or its clonal derivative, B31MIc53.vlsE, but not BBJ51, was found to undergo sequence changes during infection. Consistent with that reported previously (J.-R. Zhang et al., Cell 89:275–285, 1997) many of the sequence changes appear to have arisen through gene conversion events and to be localized to the variable regions ofvlsE. However, analysis of thevlsEnucleotide sequences revealed that some sequence changes were the result of point mutations, as these changes did not have potential contributing sources in thevlscassettes. To determine if sequence changes accumulate invlsEover long-term infection, thevlsEgenes of clonal populations recovered after 7 months of infection in mice were analyzed. While new sequence changes developed, a significant number of these changes resulted in the restoration of thevlsEsequence of the original infecting clone. In addition, we noted that some positions within the variable regions (VR) are stable even though the cassettes possess residues that could contribute to sequence variation through gene conversion. These analyses suggest that the total number of amino acid sequence changes that can be maintained by VlsE levels off during infection. In summary, in this report we demonstrate that the development of point mutations serves as a second mechanism by whichvlsEsequence variation can be generated and that the capacity forvlsEvariation, while still significant, is less than previously postulated.
Controlled Expression in
Klebsiella pneumoniae
and
Shigella flexneri
Using a Bacteriophage P1-Derived C1-Regulated Promoter System
Authors: Schofield David A. , Westwater Caroline , Dolan Joseph W. , Schmidt Michael G. , Norris James S.
Published: July 2002

The utility of promoters regulated by the bacteriophage P1 temperature-sensitive C1 repressor was examined in
Shigella flexneri
and
Klebsiella pneumoniae
. Promoters carrying C1 operator sites driving LacZ expression had induction/repression ratios of up to 240-fold in
S. flexneri
and up to 50-fold in
K. pneumoniae
. The promoters exhibited remarkably low basal expression, demonstrated modulation by temperature, and showed rapid induction. This system will provide a new opportunity for controlled gene expression in enteric gram-negative bacteria.
Systematic Identification of Selective Essential Genes in
Helicobacter pylori
by Genome Prioritization and Allelic Replacement Mutagenesis
Authors: Chalker Alison F. , Minehart Heather W. , Hughes Nicky J. , Koretke Kristin K. , Lonetto Michael A. , Brinkman Kerry K. , Warren Patrick V. , Lupas Andrei , Stanhope Michael J. , Brown James R. , Hoffman Paul S.
Published: July 2002

A comparative genomic approach was used to identify
Helicobacter pylori
26695 open reading frames (ORFs) which are conserved in
H. pylori
J99 but highly diverged in other eubacteria. A survey of selected pathways of central intermediary metabolism was also carried out, and genes with a potentially selective role in
H. pylori
were identified. Forty-five ORFs identified in these two analyses were screened using a rapid vector-free allelic replacement mutagenesis technique, and 33 were shown to be essential in vitro. Notably, 13 ORFs gave essentiality results which are unexpected in view of their known or proposed functions, and phylogenetic analysis was used to investigate the annotation of 7 such ORFs which are highly diverged. We propose that the products of a number of these
H. pylori
-specific essential genes may be suitable targets for novel anti-
H. pylori
therapies.
Hydrogen Peroxide-Forming NADH Oxidase Belonging to the Peroxiredoxin Oxidoreductase Family: Existence and Physiological Role in Bacteria
Authors: Nishiyama Yoshitaka , Massey Vincent , Takeda Kouji , Kawasaki Shinji , Sato Junichi , Watanabe Toshihiro , Niimura Youichi
Published: July 2002

Amphibacillus xylanus
and
Sporolactobacillus inulinus
NADH oxidases belonging to the peroxiredoxin oxidoreductase family show extremely high peroxide reductase activity for hydrogen peroxide and alkyl hydroperoxides in the presence of the small disulfide redox protein, AhpC (peroxiredoxin). In order to investigate the distribution of this enzyme system in bacteria, 15 bacterial strains were selected from typical aerobic, facultatively anaerobic, and anaerobic bacteria. AhpC-linked alkyl hydroperoxide reductase activities were detected in most of the tested strains, and especially high activities were shown in six bacterial species that grow well under aerobic conditions, including aerobic bacteria (
Alcaligenes faecalis
and
Bacillus licheniformis
) and facultatively anaerobic bacteria (
Amphibacillus xylanus
,
Sporolactobacillus inulinus
,
Escherichia coli
, and
Salmonella enterica
serovar Typhimurium). In the absence of AhpC, the purified enzymes from
A. xylanus
and
S. inulinus
catalyze the NADH-linked reduction of oxygen to hydrogen peroxide. Similar activities were observed in the cell extracts from each of these six strains. The cell extract of
B. licheniformis
revealed the highest AhpC-linked alkyl hydroperoxide reductase activity in the four strains, with
V
max
values for hydrogen peroxide and alkyl hydroperoxides being similar to those for the enzymes from
A. xylanus
and
S. inulinus
. Southern blot analysis of the three strains probed with the
A. xylanus
peroxiredoxin reductase gene revealed single strong bands, which are presumably derived from the individual peroxiredoxin reductase genes. Single bands were also revealed in other strains which show high AhpC-linked reductase activities, suggesting that the NADH oxidases belonging to the peroxiredoxin oxidoreductase family are widely distributed and possibly play an important role both in the peroxide-scavenging systems and in an effective regeneration system for NAD in aerobically growing bacteria.
Western Blot Analysis of Sera Reactive to Human Monocytic Ehrlichiosis and Human Granulocytic Ehrlichiosis Agents
Authors: Unver Ahmet , Felek Suleyman , Paddock Christopher D. , Zhi Ning , Horowitz Harold W. , Wormser Gary P. , Cullman Louis C. , Rikihisa Yasuko
Published: July 2002

Laboratory diagnosis of human ehrlichioses is routinely made by an indirect immunofluorescence assay (IFA) using cultured ehrlichia-infected whole cells as antigen. Concern has been raised that incorrect diagnoses of human monocytic ehrlichiosis (HME) or human granulocytic ehrlichiosis (HGE) may be made on the basis of serologic cross-reactivity between
Ehrlichia chaffeensis
and the agent of HGE. The present study examined whether two recombinant major outer membrane proteins, rP30 and rP44, that were previously shown to be sensitive and specific serodiagnostic antigens for HME and HGE, respectively, could be used to discriminate IFA dually reacting sera. Thirteen dually IFA-reactive sera, three sera that were IFA positive only with
E. chaffeensis
, and three sera that were IFA positive only with the HGE agent were examined by Western immunoblot analysis using purified whole organisms and recombinant proteins as antigens. All 16
E. chaffeensis
IFA-positive sera reacted with rP30. However, none of these sera reacted with rP44, regardless of IFA reactivity with the HGE agent. The three HGE-agent-only IFA-positive sera reacted only with rP44, not with rP30. Western immunoblotting using purified
E. chaffeensis
and the HGE agent as antigens suggested that heat shock and other proteins, but not major outer membrane proteins, cross-react between the two organisms. Therefore, Western immunoblot analysis using rP44 and rP30 may be useful in discriminating dually HME and HGE IFA-reactive sera.
Isolation of a
Nodulisporium
Species from a Case of Cerebral Phaeohyphomycosis
Authors: Umabala P. , Lakshmi V. , Murthy A. R. , Prasad V. S. S. V. , Sundaram C. , Beguin H.
Published: July 2002

A fungal infection of the brain of a 55-year-old male patient is reported. The lesion and involved fungus were located exclusively in the right medial temporo-parietal region. The patient was successfully treated with surgical resection of the lesion and antifungal chemotherapy. Few pathogenic dematiaceous fungi exhibit neurotropism and can cause primary infection in the central nervous system (CNS). The etiological agent is described as a
Nodulisporium
species. To date
Nodulisporium
has never been reported as an agent of CNS infection in humans.
Genetic Diversity of
Mycobacterium tuberculosis
in Sicily Based on Spoligotyping and Variable Number of Tandem DNA Repeats and Comparison with a Spoligotyping Database for Population-Based Analysis
Authors: Sola Christophe , Ferdinand Severine , Mammina Caterina , Nastasi Antonino , Rastogi Nalin
Published: July 2002

In a previous study, we proposed to associate spoligotyping and typing with the variable number of tandem DNA repeats (VNTR) as an alternative strategy to IS
6110
-restriction fragment length polymorphism (RFLP) for molecular epidemiological studies on tuberculosis. The aim of the present study was to further evaluate this PCR-based typing strategy and to describe the population structure of
Mycobacterium tuberculosis
in another insular setting, Sicily. A collection of 106 DNA samples from
M. tuberculosis
patient isolates was characterized by spoligotyping and VNTR typing. All isolates were independently genotyped by the standard IS
6110
-RFLP method, and clustering results between the three methods were compared. The totals for the clustered isolates were, respectively, 15, 60, and 82% by IS
6110
-RFLP, spoligotyping, and VNTR typing. The most frequent spoligotype included type 42 that missed spacers 21 to 24 and spacers 33 to 36 and derived types 33, 213, and 273 that, together represented as much as 26% of all isolates, whereas the Haarlem clade of strains (types 47 and 50, VNTR allele 32333) accounted for 9% of the total strains. The combination of spoligotyping and VNTR typing results reduced the number of clusters to 43% but remained superior to the level of IS
6110
-RFLP clustering (ca. 15%). All but one IS
6110
-defined cluster were identified by the combination of spoligotyping and VNTR clustering results, whereas 9 of 15 spoligotyping-defined clusters could be further subdivided by IS
6110
-RFLP. Reinterpretation of previous IS
6110
-RFLP results in the light of spoligotyping-VNTR typing results allowed us to detect an additional cluster that was previously missed. Although less discriminative than IS
6110
-RFLP, our results suggest that the use of the combination of spoligotyping and VNTR typing is a good screening strategy for detecting epidemiological links for the study of tuberculosis epidemiology at the molecular level.
Rotavirus G and P Genotypes in Rural Ghana
Authors: Asmah Richard H. , Green Jon , Armah George E. , Gallimore Chris I. , Gray Jim J. , Iturriza-Gómara Mirren , Anto Francis , Oduro Abraham , Binka Fred N. , Brown David W. G. , Cutts Felicity
Published: July 2002

An epidemiological study of rotavirus infection was conducted on specimens collected from patients with gastroenteritis and domiciled in the rural Upper Eastern Region of Ghana during 1998. Fifty isolates, randomly selected from 165 human group A rotavirus-positive samples, were G and P characterized by a reverse transcription (RT)-PCR assay using a seminested multiplex method. Rotaviruses of the G3 genotype were found to be the predominant strain (78%), followed by G2 (14%) and G1 (2%). Mixed infections, as shown by combinations of G3 and G2 (4%) and G3 and G1 (2%), were also observed. P typing showed P[4] (72.34%) to be the prevalent strain, followed by P[6] (21.3%), P[8] (2.13%), and a combination of P[4] and P[6] (4.3%).
Comparison of the BacT/Alert PF Pediatric FAN Blood Culture Bottle with the Standard Pediatric Blood Culture Bottle, the Pedi-BacT
Authors: Krisher Karen K. , Gibb Patrick , Corbett Sandra , Church Deidre
Published: July 2002

The performance of the BacT/Alert PF (Organon-Teknika Corp., Durham, N.C.), a new nonvented pediatric FAN blood culture bottle, was compared to that of the original pediatric bottle, the Pedi-BacT, with matched aerobic cultures obtained from two separate facilities. A total of 244 clinically significant isolates were recovered from 4,015 compliant pairs. Among the positive cultures, 170 (70%) isolates were detected in both the BacT/Alert PF and the Pedi-BacT bottles, while 47 (19%) isolates were recovered in the BacT/Alert PF bottle only and 27 (11%) isolates were recovered in the Pedi-BacT bottle only. Although isolation of specific microorganisms was comparable for the two bottles, the total number of organisms recovered by the BacT/Alert PF was greater than that by the Pedi-BacT (
P
= 0.0272). In addition, more organisms were recovered by the BacT/Alert PF bottle from the blood of patients receiving antimicrobial therapy (
P
= 0.0180). Overall time to detection was similar for the two bottles; however, a significantly decreased mean time to detection was recorded for yeast from the BacT/Alert PF bottle (22.9 h;
P
= 0.0001) and staphylococci from the Pedi-BacT bottle (22.5 h;
P
= 0.0056). One false-negative culture and five false-positive cultures occurred with the Pedi-BacT bottle, compared to one false-positive culture with the BacT/Alert PF bottle. The BacT/Alert PF bottle is a reliable blood culture bottle for pediatric blood culture specimens and may offer improved recovery of microbes from patients on antimicrobial therapy. The use of the nonvented bottle will both facilitate bottle processing and decrease expenditures for materials due to the elimination of the venting needles required for the original vented bottles.
Determination of
Enterococcus faecalis groESL
Full-Length Sequence and Application for Species Identification
Authors: Teng Lee-Jene , Hsueh Po-Ren , Wang Yi-Hui , Lin Hsiao-Mann , Luh Kwen-Tay , Ho Shen-Wu
Published: July 2002

Amplification of the partial Cpn60 (or GroEL) gene segment has been used for identification of many bacteria, including
Enterococcus
species. To obtain more sequence data from
groESL
genes of
Enterococcus faecalis
, the full-length sequence of the
E. faecalis groESL
genes containing
groES
(285 bp), spacer (57 bp), and
groEL
(1,626 bp) was determined. A database search of GenBank revealed that the deduced
E. faecalis
GroES and GroEL proteins show significant homology to the GroES and GroEL proteins of other bacteria. The GroEL (
groEL
) of
E. faecalis
had the highest identity with
Streptococcus pneumoniae
(81.8% amino acid sequence identity and 73.0% nucleotide sequence identity), followed by
Lactococcus zeae
, while GroES (
groES
) had 60.2% (64.6%) identity with
Lactobacillus zeae
and 58.5% (66.2%) identity with
Lactococcus lactis
, followed by 57.0% (65.5%) identity with
Bacillus subtilis.
Based on the
groES
sequence, an
E. faecalis
-specific PCR assay was developed, and this PCR assay was positive for all the
E. faecalis
strains tested. Dot blot hybridization using either
groES
or
groEL
as the probe distinguished
E. faecalis
clearly from other species, indicating that both genes can be used as suitable targets for
E. faecalis
identification. Moreover, broad-range PCR-restriction fragment length polymorphism of
groESL
was designed to differentiate eight commonly encountered
Enterococcus
species. The
Enterococcus
species of reference strains could be easily differentiated on the basis of restriction patterns produced by
Hae
III and
Rsa
I. The DNA-based assays developed in this study provide an alternative to currently used methods of identification for clinically important enterococcal species.
Isolation and Characterization of a Species-Specific DNA Fragment for Identification of
Candida
(
Torulopsis
)
glabrata
by PCR
Authors: Becker Karsten , Badehorn Dörte , Keller Birgit , Schulte Martina , Böhm Karl Heinz , Peters Georg , Fegeler Wolfgang
Published: July 2002

A PCR specific for
Candida glabrata
that amplifies a mitochondrial rRNA gene fragment was developed by analysis of
C. glabrata
-specific agarose gel bands, which were generated by arbitrarily primed PCR. The expected PCR product was successfully amplified with genomic DNA from 95
C. glabrata
isolates but not from a number of other fungal isolates.
Machinery To Support Genome Segment Inversion Exists in a Herpesvirus Which Does Not Naturally Contain Invertible Elements
Authors: McVoy Michael A. , Ramnarain D.
Published: July 2002

In many herpesviruses, genome segments flanked by inverted repeats invert during DNA replication. It is not known whether this inversion is a consequence of an inherently recombinagenic replicative mechanism common to all herpesviruses or whether the replication enzymes of viruses with invertible segments have specifically evolved additional enzymatic activities to drive inversion. By artificially inserting a fusion of terminal sequences into the genome of a virus which normally lacks invertible elements (murine cytomegalovirus), we created a genome composed of long and short segments flanked by 1,359- and 543-bp inverted repeats. Analysis of genomic DNA from this virus revealed that inversion of both segments generates equimolar amounts of four isomers during the viral propagation necessary to produce DNA for analysis from a single viral particle. We conclude that a herpesvirus which naturally lacks invertible elements is able to support efficient segment inversion. Thus, the potential to invert is probably inherent in the replication machinery of all herpesviruses, irrespective of genome structure, and therefore genomes with invertible elements could have evolved simply by acquisition of inverted repeats and without concomitant evolution of enzymatic activities to mediate inversion. Furthermore, the recombinagenicity of herpesvirus DNA replication must have some importance independent of genome segment inversion.
A Stem-Loop Motif Formed by the Immediate 5′ Terminus of the Bovine Viral Diarrhea Virus Genome Modulates Translation as well as Replication of the Viral RNA
Authors: Yu Haiying , Isken Olaf , Grassmann Claus W. , Behrens Sven-Erik
Published: July 2002

Bovine viral diarrhea virus
(BVDV), a
Pestivirus
member of the
Flaviviridae
family, has a positive-stranded RNA genome which consists of a single open reading frame (ORF) and untranslated regions (UTRs) at the 5′ and 3′ ends. The 5′ UTR harbors extensive RNA structure motifs; most of them were shown to contribute to an internal ribosomal entry site (IRES), which mediates cap-independent translation of the ORF. The extreme 5′-terminal region of the BVDV genome had so far been believed not to be required for IRES function. By structure probing techniques, we initially verified the existence of a computer-predicted stem-loop motif at the 5′ end of the viral genome (hairpin Ia) as well as at the 3′ end of the complementary negative-strand replication intermediate [termed hairpin Ia (−)]. While the stem of this structure is mainly constituted of nucleotides that are conserved among pestiviruses, the loop region is predominantly composed of variable residues. Taking a reverse genetics approach to a subgenomic BVDV replicon RNA (DI9c) which could be equally employed in a translation as well as replication assay system based on BHK-21 cells, we obtained the following results. (i) Proper folding of the Ia stem was found to be crucial for efficient translation. Thus, in the context of an authentic replication-competent viral RNA, the 5′-terminal motif operates apparently as an integral functional part of the ribosome entry. (ii) An intact loop structure and a stretch of nucleotide residues that constitute a portion of the stem of the Ia or the Ia (−) motif, respectively, were defined to represent important determinants of the RNA replication pathway. (iii) Formation of the stem structure of the Ia (−) motif was determined to be not critical for RNA replication. In summary, our findings affirmed that the 5′-terminal region of the BVDV genome encodes a bifunctional secondary structure motif which may enable the viral RNA to switch from the translation to the replicative cycle and vice versa.
Functional Implications of the Human T-Lymphotropic Virus Type 1 Transmembrane Glycoprotein Helical Hairpin Structure
Authors: Maerz Anne L. , Center Rob J. , Kemp Bruce E. , Kobe Bostjan , Poumbourios Pantelis
Published: July 2002

Retrovirus entry into cells follows receptor binding by the surface-exposed envelope glycoprotein (Env) subunit (SU), which triggers the membrane fusion activity of the transmembrane (TM) protein. TM protein fragments expressed in the absence of SU adopt helical hairpin structures comprising a central coiled coil, a region of chain reversal containing a disulfide-bonded loop, and a C-terminal segment that packs onto the exterior of the coiled coil in an antiparallel manner. Here we used in vitro mutagenesis to test the functional role of structural elements observed in a model helical hairpin, gp21 of human T-lymphotropic virus type 1. Membrane fusion activity requires the stabilization of the N and C termini of the central coiled coil by a hydrophobic N cap and a small hydrophobic core, respectively. A conserved Gly-Gly hinge motif preceding the disulfide-bonded loop, a salt bridge that stabilizes the chain reversal region, and interactions between the C-terminal segment and the coiled coil are also critical for fusion activity. Our data support a model whereby the chain reversal region transmits a conformational signal from receptor-bound SU to induce the fusion-activated helical hairpin conformation of the TM protein.
Cooperation of Multiple CCR5 Coreceptors Is Required for Infections by Human Immunodeficiency Virus Type 1
Authors: Kuhmann Shawn E. , Platt Emily J. , Kozak Susan L. , Kabat David
Published: July 2002

In addition to the primary cell surface receptor CD4, CCR5 or another coreceptor is necessary for infections by human immunodeficiency virus type 1 (HIV-1), yet the mechanisms of coreceptor function and their stoichiometries in the infection pathway remain substantially unknown. To address these issues, we studied the effects of CCR5 concentrations on HIV-1 infections using wild-type CCR5 and two attenuated mutant CCR5s, one with the mutation Y14N at a critical tyrosine sulfation site in the amino terminus and one with the mutation G163R in extracellular loop 2. The Y14N mutation converted a YYT sequence at positions 14 to 16 to an NYT consensus site for N-linked glycosylation, and the mutant protein was shown to be glycosylated at that position. The relationships between HIV-1 infectivity values and CCR5 concentrations took the form of sigmoidal (S-shaped) curves, which were dramatically altered in different ways by these mutations. Both mutations shifted the curves by factors of approximately 30- to 150-fold along the CCR5 concentration axis, consistent with evidence that they reduce affinities of virus for the coreceptor. In addition, the Y14N mutation specifically reduced the maximum efficiencies of infection that could be obtained at saturating CCR5 concentrations. The sigmoidal curves for all R5 HIV-1 isolates were quantitatively consistent with a simple mathematical model, implying that CCR5s reversibly associate with cell surface HIV-1 in a concentration-dependent manner, that approximately four to six CCR5s assemble around the virus to form a complex needed for infection, and that both mutations inhibit assembly of this complex but only the Y14N mutation also significantly reduces its ability to successfully mediate HIV-1 infections. Although several alternative models would be compatible with our data, a common feature of these alternatives is the cooperation of multiple CCR5s in the HIV-1 infection pathway. This cooperativity will need to be considered in future studies to address in detail the mechanism of CCR5-mediated HIV-1 membrane fusion.
Intracellular Cre-Mediated Deletion of the Unique Packaging Signal Carried by a Herpes Simplex Virus Type 1 Recombinant and Its Relationship to the Cleavage-Packaging Process
Authors: Logvinoff Carine , Epstein Alberto L.
Published: July 2002

To gain further insight on the function of the herpes simplex virus type 1 (HSV-1) packaging signal (
a
sequence), we constructed a recombinant virus containing a unique
a
sequence, which was flanked by two loxP sites in parallel orientation. The phenotype of this recombinant, named HSV-1 LaL, was studied in cell lines which either express or do not express Cre recombinase. Although LaL virus multiplication was only slightly reduced in standard cell lines, its growth was strongly inhibited in Cre-expressing cells. In these cells,
a
sequences were detected mostly in low-molecular-weight DNA circles, indicating that they had been excised from virus DNA by site-specific recombination. Deletion of the
a
sequences from the viral genome resulted in the accumulation of uncleaved replication intermediates, as observed by pulsed-field gel electrophoresis. B-type capsids also accumulated in these cells, as shown both by electron microscopy and by sucrose gradient sedimentation. Further examination of the status of
a
sequences in Cre-expressing cells indicated that high-level amplification of this sequence can occur in the absence of the cleavage-packaging process. Moreover, the amplified
a
signals in small circular DNA molecules remained uncleaved, indicating that these molecules were not able to efficiently interact with the cleavage-packaging machinery. The cleavage-packaging machinery and the structural proteins required to assemble virions were, however, functional in HSV-1 LaL-infected Cre-expressing cells, since this system could be used to package plasmid DNA harboring an origin of virus replication and one normal
a
signal. This is the first study in which accumulation both of uncleaved replication intermediates and of B capsids has been obtained in the presence of the full set of proteins required to package virus DNA.
Intracellular Localization of Vaccinia Virus Extracellular Enveloped Virus Envelope Proteins Individually Expressed Using a Semliki Forest Virus Replicon
Authors: Lorenzo María M. , Galindo Inmaculada , Griffiths Gareth , Blasco Rafael
Published: July 2002

The extracellular enveloped virus (EEV) form of vaccinia virus is bound by an envelope which is acquired by wrapping of intracellular virus particles with cytoplasmic vesicles containing trans-Golgi network markers. Six virus-encoded proteins have been reported as components of the EEV envelope. Of these, four proteins (A33R, A34R, A56R, and B5R) are glycoproteins, one (A36R) is a nonglycosylated transmembrane protein, and one (F13L) is a palmitylated peripheral membrane protein. During infection, these proteins localize to the Golgi complex, where they are incorporated into infectious virus that is then transported and released into the extracellular medium. We have investigated the fates of these proteins after expressing them individually in the absence of vaccinia infection, using a Semliki Forest virus expression system. Significant amounts of proteins A33R and A56R efficiently reached the cell surface, suggesting that they do not contain retention signals for intracellular compartments. In contrast, proteins A34R and F13L were retained intracellularly but showed distributions different from that of the normal infection. Protein A36R was partially retained intracellularly, decorating both the Golgi complex and structures associated with actin fibers. A36R was also transported to the plasma membrane, where it accumulated at the tips of cell projections. Protein B5R was efficiently targeted to the Golgi region. A green fluorescent protein fusion with the last 42 C-terminal amino acids of B5R was sufficient to target the chimeric protein to the Golgi region. However, B5R-deficient vaccinia virus showed a normal localization pattern for other EEV envelope proteins. These results point to the transmembrane or cytosolic domain of B5R protein as one, but not the only, determinant of the retention of EEV proteins in the wrapping compartment.
The Rice Tungro Bacilliform Virus Gene II Product Interacts with the Coat Protein Domain of the Viral Gene III Polyprotein
Authors: Herzog Etienne , Guerra-Peraza Orlene , Hohn Thomas
Published: July 2002

Rice tungro bacilliform virus
(RTBV) is a plant pararetrovirus whose DNA genome contains four genes encoding three proteins and a large polyprotein. The function of most of the viral proteins is still unknown. To investigate the role of the gene II product (P2), we searched for interactions between this protein and other RTBV proteins. P2 was shown to interact with the coat protein (CP) domain of the viral gene III polyprotein (P3) both in the yeast two-hybrid system and in vitro. Domains involved in the P2-CP association have been identified and mapped on both proteins. To determine the importance of this interaction for viral multiplication, the infectivity of RTBV gene II mutants was investigated by agroinoculation of rice plants. The results showed that virus viability correlates with the ability of P2 to interact with the CP domain of P3. This study suggests that P2 could participate in RTBV capsid assembly.
Regulation of Adenovirus Membrane Penetration by the Cytoplasmic Tail of Integrin β5
Authors: Wang Kena , Guan Tinglu , Cheresh David A. , Nemerow Glen R.
Published: July 2002

Adenovirus (Ad) cell entry involves sequential interactions with host cell receptors that mediate attachment (CAR), internalization (αvβ3 and αvβ5), and penetration (αvβ5) of the endosomal membrane. These events allow the virus to deliver its genome to the nucleus. While integrins αvβ3 and αvβ5 both promote Ad internalization into cells, integrin αvβ5 selectively facilitates Ad-mediated membrane permeabilization and endosome rupture. In the experiments reported herein, we demonstrate that the intracellular domain of the integrin β5 subunit specifically regulates Ad-mediated membrane permeabilization and gene delivery. CS-1 melanoma cells expressing a truncated integrin β5 or a chimeric (β5-β3) cytoplasmic tail (CT) supported normal levels of Ad endocytosis but had reduced Ad-mediated gene delivery and membrane permeabilization relative to cells expressing a wild-type integrin β5. Thin-section electron microscopy revealed that virion particles were capable of being endocytosed into cells expressing a truncated β5CT, but they failed to escape cytoplasmic vesicles and translocate to the nucleus. Site-specific mutagenesis studies suggest that a C-terminal TVD motif in the β5CT plays a major role in Ad membrane penetration.
cis
- and
trans
-Acting Elements in Flavivirus RNA Replication
Authors: Khromykh Alexander A. , Sedlak Petra L. , Westaway Edwin G.
Published: July 2002

Most of the seven flavivirus nonstructural proteins (NS1 to NS5) encoded in the distal two-thirds of the RNA positive-sense genome are believed to be essential components of RNA replication complexes. To explore the functional relationships of these components in RNA replication, we used
trans
-complementation analysis of full-length infectious RNAs of Kunjin (KUN) virus with a range of lethal in-frame deletions in the nonstructural coding region, using as helper a repBHK cell line stably producing functional replication complexes from KUN replicon RNA. Recently we showed that replication of KUN RNAs with large carboxy-terminal deletions including the entire RNA polymerase region in the NS5 gene, representing 34 to 75% of the NS5 coding content, could be complemented after transfection into repBHK cells. In this study we have demonstrated that KUN RNAs with deletions of 84 to 97% of the NS1 gene, or of 13 to 63% of the NS3 gene including the entire helicase region, were also complemented in repBHK cells with variable efficiencies. In contrast, KUN RNAs with deletions in any of the other four nonstructural genes NS2A, NS2B, NS4A, and NS4B were not complemented. We have also demonstrated successful
trans
complementation of KUN RNAs containing either combined double deletions in the NS1 and NS5 genes or triple deletions in the NS1, NS3, and NS5 genes comprising as much as 38% of the entire nonstructural coding content. Based on these and our previous complementation results, we have generated a map of
cis
- and
trans
-acting elements in RNA replication for the nonstructural coding region of the flavivirus genome. These results are discussed in the context of our model on formation and composition of the flavivirus replication complex, and we suggest molecular mechanisms by which functions of some defective components of the replication complex can be complemented by their wild-type counterparts expressed from another (helper) RNA molecule.
Synaptic Pathology in Borna Disease Virus Persistent Infection
Authors: Gonzalez-Dunia Daniel , Watanabe Michiko , Syan Sylvie , Mallory Margaret , Masliah Eliezer , Carlos de la Torre Juan
Published: July 2002

Borna disease virus (BDV) infection of newborn rats leads to a persistent infection of the brain, which is associated with behavioral and neuroanatonomical abnormalities. These disorders occur in the absence of lymphoid cell infiltrates, and BDV-induced cell damage is restricted to defined brain areas. To investigate if damage to synaptic structures anteceded neuronal loss in BDV neonatally infected rats, we analyzed at different times postinfection the expression levels of growth-associated protein 43 and synaptophysin, two molecules involved in neuroplasticity processes. We found that BDV induced a progressive and marked decrease in the expression of these synaptic markers, which was followed by a significant loss of cortical neurons. Our findings suggest that BDV persistent infection interferes with neuroplasticity processes in specific cell populations. This, in turn, could affect the proper supply of growth factors and other molecules required for survival of selective neuronal populations within the cortex and limbic system structures.
Role of the Cytoplasmic Tail of Pseudorabies Virus Glycoprotein E in Virion Formation
Authors: Brack Alexandra R. , Klupp Barbara G. , Granzow Harald , Tirabassi Rebecca , Enquist Lynn W. , Mettenleiter Thomas C.
Published: July 2002

Glycoproteins M (gM), E (gE), and I (gI) of pseudorabies virus (PrV) are required for efficient formation of mature virions. The simultaneous absence of gM and the gE/gI complex results in severe deficiencies in virion morphogenesis and cell-to-cell spread, leading to drastically decreased virus titers and a small-plaque phenotype (A. Brack, J. Dijkstra, H. Granzow, B. G. Klupp, and T. C. Mettenleiter, J. Virol. 73:5364–5372, 1999). Serial passaging in noncomplementing cells of a virus mutant unable to express gM, gE, and gI resulted in a reversion of the small-plaque phenotype and restoration of infectious virus formation to the level of a gM
−
mutant. Genetic analyses showed that reversion of the phenotype was accompanied by a genomic rearrangement which led to the fusion of a portion of the gE gene encoding the cytoplasmic domain to the 3′ end of the glycoprotein D gene, resulting in expression of a chimeric gD-gE protein. Since this indicated that the intracytoplasmic domain of gE was responsible for the observed phenotypic alterations, the UL10 (gM) gene was deleted in a PrV mutant, PrV-107, which specifically lacked the cytoplasmic tail of gE. Regarding one-step growth, plaque size, and virion formation as observed under the electron microscope, the mutant lacking gM and the gE cytoplasmic tail proved to be very similar to the gE/I/M triple mutant. Thus, our data indicate that it is the cytoplasmic tail of gE which is responsible for the observed phenotypic effects in conjunction with deletion of gM. We hypothesize that the cytoplasmic domain of gE specifically interacts with components of the capsid and/or tegument, leading to efficient secondary envelopment of intracytoplasmic capsids.
Mutagenic Analysis of the 5′ Arm of the Influenza A Virus Virion RNA Promoter Defines the Sequence Requirements for Endonuclease Activity
Authors: Leahy Michael B. , Pritlove David C. , Poon Leo L. M. , Brownlee George G.
Published: July 2002

Short synthetic influenza virus-like RNAs derived from influenza virus promoter sequences were examined for their ability to stimulate the endonuclease activity of recombinant influenza virus polymerase complexes in vitro, an activity that is required for the cap-snatching activity of primers from host pre-mRNA. An extensive set of point mutants of the 5′ arm of the influenza A virus viral RNA (vRNA) was constructed to determine the
cis
-acting elements which influenced endonuclease activity. Activity was found to be dependent on three features of the conserved vRNA termini: (i) the presence of the 5′ hairpin loop structure, (ii) the identity of residues at positions 5 and 10 bases from the 5′ terminus, and (iii) the presence of base pair interactions between the 5′ and 3′ segment ends. Further experiments discounted a role for the vRNA U track in endonuclease activation. This study represents the first mutagenic analysis of the influenza virus promoter with regard to endonuclease activity.
Identification of the Regions of Fv1 Necessary for Murine Leukemia Virus Restriction
Authors: Bishop Kate N. , Bock Michael , Towers Greg , Stoye Jonathan P.
Published: July 2002

The
Fv1
gene restricts murine leukemia virus replication via an interaction with the viral capsid protein. To study this interaction, a number of mutations, including a series of N-terminal and C-terminal deletions, internal deletions, and a number of single-amino-acid substitutions, were introduced into the n and b alleles of the
Fv1
gene and the effects of these changes on virus restriction were measured. A significant fraction of the Fv1 protein was not required for restriction; however, retention of an intact major homology region as well as of domains toward the N and C termini was essential. Binding specificity appeared to be a combinatorial property of a number of residues within the C-terminal portion of Fv1.
Characterization of the Infectious Salmon Anemia Virus Genomic Segment That Encodes the Putative Hemagglutinin
Authors: Rimstad Espen , Mjaaland Siri , Snow Michael , Mikalsen Aase B. , Cunningham Carey O.
Published: July 2002

The genomic segment encoding the putative hemagglutinin of infectious salmon anemia virus (ISAV) is described. Expression of the putative hemagglutinin in a salmon cell line demonstrated hemadsorptive properties of the protein for salmon erythrocytes. The polypeptide was recognized by an ISAV-specific monoclonal antibody. Nucleotide sequencing indicated the occurrence of a variable region in the hemagglutinin gene.
Epstein-Barr Virus Immediate-Early Protein BRLF1 Interacts with CBP, Promoting Enhanced BRLF1 Transactivation
Authors: Swenson Jennifer J. , Holley-Guthrie Elizabeth , Kenney Shannon C.
Published: July 2002

The Epstein-Barr virus (EBV) immediate-early protein BRLF1 is a transcriptional activator that mediates the switch from latent to lytic viral replication. Many transcriptional activators function, in part, due to an interaction with histone acetylases, such as CREB-binding protein (CBP). Here we demonstrate that BRLF1 interacts with the amino and carboxy termini of CBP and that multiple domains of the BRLF1 protein are necessary for this interaction. Furthermore, we show that the interaction between BRLF1 and CBP is important for BRLF1-induced activation of the early lytic EBV gene SM in Raji cells.
Interleukin-1 Receptor Antagonist Gene Polymorphism and Circulating Levels of Human Immunodeficiency Virus Type 1 RNA in Brazilian Women
Authors: Witkin Steven S. , Linhares Iara M. , Gerber Stefan , Caetano Marie E. , Segurado Alusio C.
Published: July 2002

Interleukin-1 receptor antagonist (IL-1ra) gene polymorphisms in 83 human immunodeficiency virus (HIV)-seropositive women were evaluated. Fourteen of the subjects (16.9%) were homozygous for IL-1ra allele 2 (IL-1RN*2). These women had a lower median level of HIV RNA than did women homozygous for allele 1 (IL-1RN*1) (P= 0.01) or heterozygous for both alleles (P= 0.04). Among 46 subjects not receiving antiretroviral treatment, HIV levels were also reduced in IL-1RN*2 homozygous individuals (P< 0.05). There was no relation between IL-1ra alleles and CD4 levels.
HERV-K(OLD): Ancestor Sequences of the Human Endogenous Retrovirus Family HERV-K(HML-2)
Authors: Reus Katrin , Mayer Jens , Sauter Marlies , Zischler Hans , Müller-Lantzsch Nikolaus , Meese Eckart
Published: July 2002

Sequences homologous to the human endogenous retrovirus (HERV) family HERV-K(HML-2) are present in all Old World primate species. A previous study showed that a central region of the HERV-K(HML-2)
gag
genes in
Hominoidea
species displays a 96-bp deletion compared to the
gag
genes in lower Old World primates. The more ancient HERV-K(HML-2) sequences present in lower Old World primates were apparently not conserved during hominoid evolution, as opposed to the deletion variants. To further clarify the evolutionary origin of the HERV-K(HML-2) family, we screened GenBank with the 96-bp
gag
-sequence characteristic of lower Old World primates and identified, to date, 10 human sequence entries harboring either full-length or partially deleted proviral structures, probably representing remnants of a more ancient HERV-K(HML-2) variant. The high degree of mutations demonstrates the long-time presence of these HERV-K(OLD) proviruses in the genome. Nevertheless, they still belong to the HML-2 family as deduced from dot matrix and phylogenetic analyses. We estimate, based on the family ages of integrated
Alu
elements and on long terminal repeat (LTR) divergence data, that the average age of HERV-K(OLD) proviruses is ca. 28 million years, supporting an integration time before the evolutionary split of
Hominoidea
from lower Old World primates. Analysis of HERV-K(OLD) LTR sequences led to the distinction of two subgroups, both of which cluster with LTRs belonging to an evolutionarily older cluster. Taken together, our data give further insight into the evolutionary history of the HERV-K(HML-2) family during primate evolution.
Characterization of the AdoMet-Dependent Guanylyltransferase Activity That Is Associated with the N Terminus of Bamboo Mosaic Virus Replicase
Authors: Li Yi-Ija , Chen Yi-Jun , Hsu Yau-Heiu , Meng Menghsiao
Published: July 2002

Bamboo mosaic virus (BaMV), a member of the potexvirus group, infects primarily members of the
Bambusoideae
. Open reading frame 1 (ORF1) of BaMV encodes a 155-kDa polypeptide that has long been postulated to be a replicase involved in the replication and formation of the cap structure at the 5′ end of the viral genome. To identify and characterize the enzymatic activities associated with the N-terminal domain of the BaMV ORF1 protein, the intact replicase and two C-terminally truncated proteins were expressed in
Saccharomyces cerevisiae
. All three versions of BaMV ORF1 proteins could be radiolabeled by [α-
32
P]GTP, which is a characteristic of guanylyltransferase activity. The presence of
S
-adenosylmethionine (AdoMet) was essential for this enzymatic activity. Thin-layer chromatography analysis suggests that the radiolabeled moiety linked to the N-terminal domain of the BaMV ORF1 protein is m
7
GMP. The N-terminal domain also exhibited methyltransferase activity that catalyzes the transfer of the [
3
H]methyl group from AdoMet to GTP or guanylylimidodiphosphate. Therefore, during cap structure formation in BaMV, methylation of GTP may occur prior to transguanylation as for alphaviruses and brome mosaic virus. This study establishes the association of RNA capping activity with the N-terminal domain of the replicase of potexviruses and further supports the idea that the reaction sequence of RNA capping is conserved throughout the alphavirus-like superfamily of RNA viruses.
Strict Control of Human Immunodeficiency Virus Type 1 Replication by a Genetic Switch: Tet for Tat
Authors: Verhoef Koen , Marzio Giuseppe , Hillen Wolfgang , Bujard Hermann , Berkhout Ben
Published: July 2002

Live-attenuated human immunodeficiency virus type 1 (HIV-1) variants have shown great promise as AIDS vaccines, but continued replication can lead to the selection of faster-replicating variants that are pathogenic. We therefore designed HIV-1 genomes that replicate exclusively upon addition of the nontoxic effector doxycycline (dox). This was achieved by replacement of the viral TAR-Tat system for transcriptional activation by the
Escherichia coli
-derived Tet system for inducible gene expression. These designer “HIV-rtTA” viruses replicate in a strictly dox-dependent manner both in a T-cell line and in primary blood cells, and the rate of replication can be fine-tuned by simple variation of the dox concentration. These HIV-rtTA viruses provide a tool to perform genetics, e.g., selection and optimization experiments, with the
E. coli
-derived Tet reagents in a eukaryotic background. Furthermore, such viruses may represent improved vaccine candidates because their replication can be turned on and off at will.