bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of the
Eikenella corrodens pilA
Locus in Pilus Function and Phase Variation
Authors: Villar Maria T. , Hirschberg Rona L. , Schaefer Michael R.
Published: July 2002

The human pathogen
Eikenella corrodens
expresses type IV pili and exhibits a phase variation involving the irreversible transition from piliated to nonpiliated variants. On solid medium, piliated variants form small (S-phase), corroding colonies whereas nonpiliated variants form large (L-phase), noncorroding colonies. We are studying pilus structure and function in the clinical isolate
E. corrodens
VA1. Earlier work defined the
pilA
locus which includes
pilA1
,
pilA2
,
pilB
, and
hagA
. Both
pilA1
and
pilA2
predict a type IV pilin, whereas
pilB
predicts a putative pilus assembly protein. The role of
hagA
has not been clearly established. That work also confirmed that
pilA1
encodes the major pilus protein in this strain and showed that the phase variation involves a posttranslational event in pilus formation. In this study, the function of the individual genes comprising the
pilA
locus was examined using a recently developed protocol for targeted interposon mutagenesis of S-phase variant VA1-S1. Different
pilA
mutants were compared to S-phase and L-phase variants for several distinct aspects of phase variation and type IV pilus biosynthesis and function. S-phase cells were characterized by surface pili, competence for natural transformation, and twitching motility, whereas L-phase cells lacked these features. Inactivation of
pilA1
yielded a mutant that was phenotypically indistinguishable from L-phase variants, showing that native biosynthesis of the type IV pilus in strain VA1 is dependent on expression of
pilA1
and proper export and assembly of PilA1. Inactivation of
pilA2
yielded a mutant that was phenotypically indistinguishable from S-phase variants, indicating that
pilA2
is not essential for biosynthesis of functionally normal pili. A mutant inactivated for
pilB
was deficient for twitching motility, suggesting a role for PilB in this pilus-related phenomenon. Inactivation of
hagA
, which may encode a tellurite resistance protein, had no effect on pilus structure or function.
Cell Wall Core Galactofuran Synthesis Is Essential for Growth of Mycobacteria
Authors: Pan Fei , Jackson Mary , Ma Yufang , McNeil Michael
Published: July 2002

The mycobacterial cell wall core consists of an outer lipid (mycolic acid) layer attached to peptidoglycan via a galactofuranosyl-containing polysaccharide, arabinogalactan. This structural arrangement strongly suggests that galactofuranosyl residues are essential for the growth and viability of mycobacteria. Galactofuranosyl residues are formed in nature by a ring contraction of UDP-galactopyranose to UDP-galactofuranose catalyzed by the enzyme UDP-galactopyranose mutase (Glf). In
Mycobacterium tuberculosis
the
glf
gene overlaps, by 1 nucleotide, a gene, Rv3808c, that has been shown to encode a galactofuranosyl transferase. We demonstrate here that
glf
can be knocked out in
Mycobacterium smegmatis
by allelic replacement only in the presence of two rescue plasmids carrying functional copies of
glf
and Rv3808c. The
glf
rescue plasmid was designed with a temperature-sensitive origin of replication and the
M. smegmatis glf
knockout mutant is unable to grow at the higher temperature at which the
glf
-containing rescue plasmid is lost. In a separate experiment, the Rv3808c rescue plasmid was designed with a temperature-sensitive origin of replication and the
glf
-bearing plasmid was designed with a normal original of replication; this strain was also unable to grow at the nonpermissive temperature. Thus, both
glf
and Rv3808c are essential for growth. These findings and the fact that galactofuranosyl residues are not found in humans supports the development of UDP-galactopyranose mutase and galactofuranosyl transferase as important targets for the development of new antituberculosis drugs.
Regulatory Interactions of Csr Components: the RNA Binding Protein CsrA Activates
csrB
Transcription in
Escherichia coli
Authors: Gudapaty Seshagirirao , Suzuki Kazushi , Wang Xin , Babitzke Paul , Romeo Tony
Published: July 2002

The global regulator CsrA (carbon storage regulator) of
Escherichia coli
is a small RNA binding protein that represses various metabolic pathways and processes that are induced in the stationary phase of growth, while it activates certain exponential phase functions. Both repression and activation by CsrA involve posttranscriptional mechanisms, in which CsrA binding to mRNA leads to decreased or increased transcript stability, respectively. CsrA also binds to a small untranslated RNA, CsrB, forming a ribonucleoprotein complex, which antagonizes CsrA activity. We have further examined the regulatory interactions of CsrA and CsrB RNA. The 5′ end of the CsrB transcript was mapped, and a
csrB
::
cam
null mutant was constructed. CsrA protein and CsrB RNA levels were estimated throughout the growth curves of wild-type and isogenic
csrA
,
csrB
,
rpoS
, or
csrA rpoS
mutant strains. CsrA levels exhibited modest or negligible effects of these mutations. The intracellular concentration of CsrA exceeded the total CsrA-binding capacity of intracellular CsrB RNA. In contrast, CsrB levels were drastically decreased (∼10-fold) in the
csrA
mutants. CsrB transcript stability was unaffected by
csrA
. The expression of a
csrB-lacZ
transcriptional fusion containing the region from −242 to +4 bp of the
csrB
gene was decreased ∼20-fold by a
csrA
::
kanR
mutation in vivo but was unaffected by CsrA protein in vitro. These results reveal a significant, though most likely indirect, role for CsrA in regulating
csrB
transcription. Furthermore, our findings suggest that CsrA mediates an intriguing form of autoregulation, whereby its activity, but not its levels, is modulated through effects on an RNA antagonist, CsrB.
Campylobacter fetusUses Multiple Loci for DNA Inversion within the 5′ Conserved Regions ofsapHomologs
Authors: Tu Zheng-Chao , Ray Kevin C. , Thompson Stuart A. , Blaser Martin J.
Published: July 2002

Campylobacter fetuscells possess multiple promoterlesssaphomologs, each capable of expressing a surface layer protein (SLP) by utilizing a unique promoter present on a 6.2-kb invertible element. Eachsaphomolog includes a 626-bp 5′ conserved region (FCR) with 74 bp upstream and 552 bp within the open reading frame. After DNA inversion, the splice is seamless because the FCRs are identical. In mutant strain 23D:ACA2K101, in whichsapAandsapA2flanking the invertible element in opposite orientations were disrupted by promoterless chloramphenicol resistance (Cmr) and kanamycin resistance (Kmr) cassettes, respectively, the frequency of DNA inversion is 100-fold lower than that of wild-type strain 23D. To define the roles of a 15-bp inverted repeat (IR) and a Chi-like site (CLS) in the FCR, we mutagenized each upstream ofsapA2in 23D:ACA2K101 by introducingNotI andKpnI sites to create strains 23D:ACA2K101Nand 23D:ACA2K101K, respectively. Alternatively selecting colonies for Cmror Kmrshowed that mutagenizing the IR or CLS had no apparent effect on the frequency of the DNA inversion. However, mapping the uniqueNotI orKpnI site in relation to the Cmror Kmrcassette in the cells that changed phenotype showed that splices occurred both upstream and downstream of the mutated sites. PCR and sequence analyses also showed that the splice could occur in the 425-bp portion of the FCR downstream of the cassettes. In total, these data indicate thatC.fetuscan use multiple sites within the FCR for itssap-related DNA inversion.
Regulatory Architecture of the Iron-Regulated
fepD-ybdA
Bidirectional Promoter Region in
Escherichia coli
Authors: Christoffersen Catherine A. , Brickman Timothy J. , Hook-Barnard India , McIntosh Mark A.
Published: July 2002

The overlapping and opposing promoter elements for the
Escherichia coli fepDGC
operon and the
ybdA
gene (encoding a 43-kDa cytoplasmic membrane protein) within the enterobactin gene cluster were investigated by measuring the effects of site-specific mutations on transcript levels and on expression of reporter genes in a bidirectional transcriptional fusion vector. Primary promoter structures for the opposing transcripts overlapped extensively such that their −10 sequences were almost directly opposed on the two strands of the DNA helix and their +1 transcription start sites were only 23 bp apart. Relative to the
E. coli
consensus sequence, both promoters were poorly conserved at the −35 position and mutations which strengthened the −35 element of either promoter significantly enhanced its transcription, decreased that of the opposing promoter, and dramatically altered iron-mediated regulation of expression. Both the
fepD
and
ybdA
primary promoters were shown to require a 5′-TGn-3′ upstream extension of their −10 elements for optimal activities. Secondary promoters were identified for both
fepD
and
ybdA
, and their contributions to the overall expression levels were evaluated in these dual expression vector constructs. The data provided strong evidence that the architecture of the regulatory elements within the overlapping
fepD
and
ybdA
promoters is configured such that there is a direct competition for binding RNA polymerase and that the expression levels at these promoters are influenced not only by the activity of the opposing promoters but also by additional promoter sequence elements and perhaps accessory regulatory factors. Iron-mediated regulation of these promoters through the repressor protein Fur is a consequence of the relative promoter strengths and the position of an operator site that consists of two overlapping Fur-binding sequences in this compact regulatory region.
Thermoadaptation of α-Galactosidase AgaB1 in
Thermus thermophilus
Authors: Fridjonsson Olafur , Watzlawick Hildegard , Mattes Ralf
Published: July 2002

The evolutionary potential of a thermostable α-galactosidase, with regard to improved catalytic activity at high temperatures, was investigated by employing an in vivo selection system based on thermophilic bacteria. For this purpose, hybrid α-galactosidase genes of
agaA
and
agaB
from
Bacillus stearothermophilus
KVE39, designated
agaA1
and
agaB1
, were cloned into an autonomously replicating
Thermus
vector and introduced into
Thermus thermophilus
OF1053GD (Δ
agaT
) by transformation. This selector strain is unable to metabolize melibiose (α-galactoside) without recombinant α-galactosidases, because the native α-galactosidase gene,
agaT
, has been deleted. Growth conditions were established under which the strain was able to utilize melibiose as a single carbohydrate source when harboring a plasmid-encoded
agaA1
gene but unable when harboring a plasmid-encoded
agaB1
gene. With incubation of the
agaB1
plasmid-harboring strain under selective pressure at a restrictive temperature (67°C) in a minimal melibiose medium, spontaneous mutants as well as
N
-methyl-
N′
-nitro-
N
-nitrosoguanidine-induced mutants able to grow on the selective medium were isolated. The mutant α-galactosidase genes were amplified by PCR, cloned in
Escherichia coli
, and sequenced. A single-base substitution that replaces glutamic acid residue 355 with glycine or valine was found in the mutant
agaB1
genes. The mutant enzymes displayed the optimum hydrolyzing activity at higher temperatures together with improved catalytic capacity compared to the wild-type enzyme and furthermore showed an enhanced thermal stability. To our knowledge, this is the first report of an in vivo evolution of glycoside-hydrolyzing enzyme and selection within a thermophilic host cell.
Comparison of DNA Sequencing of the Protein A Gene Polymorphic Region with Other Molecular Typing Techniques for Typing Two Epidemiologically Diverse Collections of Methicillin-Resistant
Staphylococcus aureus
Authors: Oliveira Duarte C. , Crisóstomo Inês , Santos-Sanches Ilda , Major Peter , Alves C. Rute , Aires-de-Sousa Marta , Thege Marianne K. , de Lencastre Hermı́nia
Published: July 2002

The aim of this study was to compare the recently developed typing approach for methicillin-resistant
Staphylococcus aureus
(MRSA) based on the DNA sequencing of the protein A gene polymorphic region (
spaA
typing) with a combination of three well-established molecular typing techniques:
Cla
I-
mecA
vicinity polymorphisms,
Cla
I-Tn
554
insertion patterns, and
Sma
I pulsed-field gel electrophoresis (PFGE) profiles. In order to evaluate the applicability of this typing technique in different types of studies, two groups of MRSA clinical isolates were analyzed: a collection of 185 MRSA isolates circulating in Hungary recovered from 17 hospitals in seven cities during a 3-year period (1994 through 1996), and a selection of 53 MRSA strains isolated in a single hospital in Hungary between 1997 and 1998. The 238 MRSA clinical strains from Hungary were first classified in clonal types (defined as
Cla
I-
mecA
::
Cla
I-Tn
554
::
Sma
I-PFGE patterns), and 65 of the 238 strains, representing major MRSA clones and some sporadic clones, were further analyzed by
spaA
typing. Our results showed that the lineages most recently introduced in the hospital setting showed little variability in
spaA
types, whereas the MRSA clones circulating for a longer period of time and spread among several hospitals showed a higher degree of variability. The implementation of the
spaA
typing method was straightforward, and the results obtained were reproducible, unambiguous, and easily interpreted. This method seems to be adequate for outbreak investigations but should be complemented with other techniques in long-term surveillance or in studies comparing distant clonal lineages.
Routine Molecular Identification of Enterococci by Gene-Specific PCR and 16S Ribosomal DNA Sequencing
Authors: Angeletti Silvia , Lorino Giulia , Gherardi Giovanni , Battistoni Fabrizio , De Cesaris Marina , Dicuonzo Giordano
Published: July 2002

For 279 clinically isolated specimens identified by commercial kits as enterococci, genotypic identification was performed by two multiplex PCRs, one with

ddl
E. faecalis

and

ddl
E. faecium

primers and another with
vanC-1
and
vanC-2/3
primers, and by 16S ribosomal DNA (rDNA) sequencing. For 253 strains, phenotypic and genotypic results were the same. Multiplex PCR allowed for the identification of 13 discordant results. Six strains were not enterococci and were identified by 16S rDNA sequencing. For 5 discordant and 10 concordant enterococcal strains, 16S rDNA sequencing was needed. Because many supplementary tests are frequently necessary for phenotypic identification, the molecular approach is a good alternative.
Resistance-Associated Mutations in the Human Immunodeficiency Virus Type 1 Subtype C Protease Gene from Treated and Untreated Patients in the United Kingdom
Authors: Cane Patricia A. , de Ruiter Annemiek , Rice Philip , Wiselka Martin , Fox Ray , Pillay Deenan
Published: July 2002

This work reports the variability of human immunodeficiency virus type 1 (HIV-1) protease from treated and untreated patients infected with HIV-1 subtype C in the United Kingdom. The most common primary mutation observed in treated patients was L90M. D30N, M46I, V82A/F, and I84V were seen rarely. M36I and I93L mutations were observed in nearly all samples from both treated and untreated patients and so cannot be considered as resistance-associated mutations in this subtype.
Stx2 Subtyping of Shiga Toxin-Producing
Escherichia coli
Isolated from Cattle in France: Detection of a New Stx2 Subtype and Correlation with Additional Virulence Factors
Authors: Bertin Yolande , Boukhors Karima , Pradel Nathalie , Livrelli Valerie , Martin Christine
Published: July 2002

At least 11 Stx2 variants produced by Shiga toxin-producing
Escherichia coli
(STEC) isolated from patients and animals have been described. The Stx2 subtyping of STEC isolated from healthy cows positive for
stx
2
(
n
= 104) or
stx
2
and
stx
1
(
n
= 63) was investigated. Stx2vh-b, Stx2 (renamed Stx2-EDL933), and Stx2vh-a were the subtypes mostly detected among the bovine isolates (39.5, 39, and 25.5%, respectively). Stx2e was not present, and subtypes included in the Stx2d group (Stx2d-OX3a, Stx2d-O111, and Stx2d-Ount) were found infrequently among the isolates examined (8.5%). A combination of two distinct Stx2 subtypes was observed among 23.5% of the strains. For the first time, a combination of three subtypes (Stx2-EDL933/Stx2vh-b/Stx2d and Stx2vh-a/Stx2vh-b/Stx2d) was detected (3.5% of the isolates). In addition, bovine STEC harboring
stx
1
and one or two
stx
2
genes appeared highly cytotoxic toward Vero cells. A new Stx2 subtype (Stx2-NV206), present among 14.5% of the isolates, showed high cytotoxicity for Vero cells. Two amino acid residues (Ser-291 and Glu-297) important for the activation of Stx2 by human intestinal mucus were conserved on the Stx2-NV206 A subunit. The gene encoding Ehx enterohemolysin was prominent among STEC harboring
stx
2
-EDL933 alone (78%) or a combination of
stx
2
-EDL933 and
stx
2
vh-b (85%). In addition, Stx2-EDL933 and/or Stx2vh-b subtypes were highly associated with other putative virulence factors such as Stx1 and EspP extracellular serine protease, but not with EAST1 enterotoxin.
Immunoglobulin M (IgM)-Glycoinositolphospholipid Enzyme-Linked Immunosorbent Assay: an Immunoenzymatic Assay for Discrimination between Patients with Acute Toxoplasmosis and Those with Persistent Parasite-Specific IgM Antibodies
Authors: Giraldo Mónica , Portela Ricardo W. D. , Snege Mirian , Leser Paulo G. , Camargo Mário E. , Mineo José Roberto , Gazzinelli Ricardo T.
Published: July 2002

In the present study we developed an enzyme-linked immunosorbent assay (ELISA) to measure immunoglobulin M (IgM) specific for glycoinositolphospholipids (GIPL) derived from tachyzoite membrane (IgM-GIPL ELISA). The sensitivity and specificity of the assay were compared with those of commercially availableToxoplasma-specific IgM serological tests, namely, immunofluorescence assay (IFA) with fixed tachyzoites and capture ELISA employing tachyzoite extracts. Our results show that all patients with acute toxoplasmosis, as determined by clinical data and conventional serological tests, were also positive by the IgM-GIPL ELISA. Interestingly, many patients that were classified as indeterminate, who had IgG with high avidity but positive results in the IgM-specific IFA and capture ELISA, were negative by the IgM-GIPL ELISA. Finally, we tested the sera from patients with rheumatoid arthritis and various parasitic infections and found no evidence of false positives in the IgM-GIPL ELISA.
Identification of a Novel Methicillin-Resistant
Staphylococcus aureus
Epidemic Clone in Córdoba, Argentina, Involved in Nosocomial Infections
Authors: Sola Claudia , Gribaudo Germán , Vindel Ana , Patrito Luis , Bocco José Luis
Published: July 2002

Infections caused by methicillin-resistant
Staphylococcus aureus
(MRSA) are increasingly a main health concern worldwide for hospitalized patients. In addition, the prevalence of community-acquired infection has risen continuously during the last few years. Some MRSA clones spread easier than others within the hospital environment and therefore are frequently implicated in outbreaks. Thus, the spread of a unique epidemic multiresistant clone, the so-called South American clone, is the main cause of nosocomial infections produced by this bacterium in Brazil and in some regions of Argentina, Chile, and Uruguay. In the present work we describe the identification of a novel clone of MRSA that is involved in nosocomial infections and that shows a prevalence as high as that for the South American clone. A total of 53 consecutive single-patient MRSA isolates were recovered during a 3-month period (May to July 1999) from six different hospitals (955 beds) in Córdoba. The isolates were initially typed according the antibiotic resistance and phage susceptibility patterns, followed by genotyping using pulsed-field gel electrophoresis (PFGE). PFGE analysis of the 53 MRSA isolates revealed six major types (A to F) and 25 subtypes. The B-type DNA pattern was indistinguishable from that of the South American epidemic clone observed in 34% of the isolates. A novel highly prevalent clone, showing the A-type DNA pattern and representing 38% of the isolates, was also identified. Moreover, the most frequent subtype of the A clonal family triggered an outbreak in a hospital 2 months later, further confirming its epidemic feature.
Rubella Virus Nonstructural Protein Protease Domains Involved in
trans
- and
cis
-Cleavage Activities
Authors: Liang Yuying , Yao Jiansheng , Gillam Shirley
Published: July 2002

Rubella virus (RV) genomic RNA contains two large open reading frames (ORFs): a 5′-proximal ORF encoding nonstructural proteins (NSPs) that function primarily in viral RNA replication and a 3′-proximal ORF encoding the viral structural proteins. Proteolytic processing of the RV NSP ORF translation product p200 is essential for viral replication. Processing of p200 to two mature products (p150 and p90) in the order NH
2
-p150-p90-COOH is carried out by an RV-encoded protease residing in the C-terminal region of p150. The RV nonstructural protease (NS-pro) belongs to a viral papain-like protease family that cleaves the polyprotein both in
trans
and in
cis
. A conserved X domain of unknown function was found from previous sequence analysis to be associated with NS-pro. To define the domains responsible for
cis
- and
trans
-cleavage activities and the function of the X domain in terms of protease activity, an in vitro translation system was employed. We demonstrated that the NSP region from residue 920 to 1296 is necessary for
trans
-cleavage activity. The domain from residue 920 to 1020 is not required for
cis
-cleavage activity. The X domain located between residues 834 and 940, outside the regions responsible for both
cis
- and
trans
-cleavage activities of NS-pro, was found to be important for NS-pro
trans
-cleavage activity but not for
cis
-cleavage activity. Analysis of sequence homology and secondary structure of the RV NS-pro catalytic region reveals a folding structure similar to that of papain.
Functional Interaction between Pleiotropic Transactivator pUL69 of Human Cytomegalovirus and the Human Homolog of Yeast Chromatin Regulatory Protein SPT6
Authors: Winkler Michael , aus dem Siepen Thomas , Stamminger Thomas
Published: July 2002

The phosphoprotein pUL69 of human cytomegalovirus (HCMV), which is a herpesvirus of considerable medical importance in immunosuppressed patients and newborns, has previously been identified as an early-late viral protein that can stimulate several viral and cellular promoters and thus exerts a rather broad activation pattern. To gain insight into the mechanism of this transactivation process, we looked for cellular factors interacting with pUL69 in a yeast two-hybrid screen. Using a B-lymphocyte cDNA library fused to the GAL4 activation domain, we identified 34 clones, 11 of which comprised one distinct gene. Interaction with this gene turned out to be very strong, producing β-galactosidase levels 100-fold greater than the background as measured in an ONPG (
o
-nitrophenyl-β-
d
-galactopyranoside) assay. Sequencing identified this gene as the human homolog of the yeast factor SPT6, which is thought to be involved in the regulation of chromatin structure. A direct interaction of pUL69 and the carboxy terminus of hSPT6 could be demonstrated using in vitro pull-down experiments. After having generated a specific antiserum that is able to detect the endogenous hSPT6 protein, we were able to observe an in vivo interaction of both proteins by coimmunoprecipitation analysis. The interaction domain within pUL69 was mapped to a central domain of this viral protein that is conserved within the homologous proteins of other herpesviruses such as the ICP27 protein of herpes simplex virus. Internal deletions within this central domain, as well as a single amino acid exchange at position C495, resulted in a loss of interaction. This correlated with a loss of the transactivation potential of the respective mutants, suggesting that the hSPT6 interaction of pUL69 is essential for stimulating gene expression. Furthermore, we demonstrate that the carboxy terminus of hSPT6 also binds to histon H3 and that this interaction can be antagonized by pUL69. This allows the deduction of a model by which pUL69 acts as an antirepressor by competing for binding of histones to hSPT6, thereby antagonizing the chromatin remodeling function of this cellular protein.
Growth Factor-Independent Proliferation of Erythroid Cells Infected with Friend Spleen Focus-Forming Virus Is Protein Kinase C Dependent but Does Not Require Ras-GTP
Authors: Muszynski Karen W. , Thompson Delores , Hanson Charlotte , Lyons Rebecca , Spadaccini Angelo , Ruscetti Sandra K.
Published: July 2002

Interaction of erythropoietin (Epo) with its cell surface receptor activates signal transduction pathways which result in the proliferation and differentiation of erythroid cells. Infection of erythroid cells with the Friend spleen focus-forming virus (SFFV) leads to the interaction of the viral envelope glycoprotein with the Epo receptor and renders these cells Epo independent. We previously reported that SFFV induces Epo independence by constitutively activating components of several Epo signal transduction pathways, including the Jak-Stat and the Raf-1/mitogen-activated protein kinase (MAPK) pathways. To further evaluate the mechanism by which SFFV activates the Raf-1/MAPK pathway, we investigated the effects of SFFV on upstream components of this pathway, and our results indicate that SFFV activates Shc and Grb2 and that this leads to Ras activation. While studies with a dominant-negative Ras indicated that Ras was required for Epo-induced proliferation of normal erythroid cells, the Epo-independent growth of SFFV-infected cells can still occur in the absence of Ras, although at reduced levels. In contrast, protein kinase C (PKC) was shown to be required for the Epo-independent proliferation of SFFV-infected cells. Further studies indicated that PKC, which is thought to be involved in the activation of both Raf-1 and MAPK, was required only for the activation of MAPK, not Raf-1, in SFFV-infected cells. Our results indicate that Ras and PKC define two distinct signals converging on MAPK in both Epo-stimulated and SFFV-infected erythroid cells and that activation of only PKC is sufficient for the Epo-independent proliferation of SFFV-infected cells.
Replication of Herpes Simplex Virus Type 1 within Trigeminal Ganglia Is Required for High Frequency but Not High Viral Genome Copy Number Latency
Authors: Thompson Richard L. , Sawtell N. M.
Published: July 2002

The replication properties of a thymidine kinase-negative (TK
−
) mutant of herpes simplex virus type 1 (HSV-1) were exploited to examine the relative contributions of replication at the body surface and within trigeminal ganglia (TG) on the establishment of latent infections. The replication of a TK
−
mutant, 17/tBTK
−
, was reduced by ∼12-fold on the mouse cornea compared to the rescued isolate 17/tBRTK
+
, and no replication of 17/tBTK
−
in the TG of these mice was detected. About 1.8% of the TG neurons of mice infected with 17/tBTK
−
harbored the latent viral genome compared to 23% of those infected with 17/tBRTK
+
. In addition, the latent sites established by the TK
−
mutant contained fewer copies of the HSV-1 genome (average, 2.3/neuron versus 28/neuron). On the snout, sustained robust replication of 17tBTK
−
in the absence of significant replication within the TG resulted in a modest increase in the number of latent sites. Importantly, these latently infected neurons displayed a wild-type latent-genome copy number profile, with some neurons containing hundreds of copies of the TK
−
mutant genome. As expected, the replication of the TK
−
mutant appeared to be blocked prior to DNA replication in most ganglionic neurons in that (i) virus replication was severely restricted in ganglia, (ii) the number of neurons expressing HSV proteins was reduced 30-fold compared to the rescued isolate, (iii) cell-to-cell spread of virus was not detected within ganglia, and (iv) the proportion of infected neurons expressing late proteins was reduced by 89% compared to the rescued strain. These results demonstrate that the viral TK gene is required for the efficient establishment of latency. This requirement appears to be primarily for efficient replication within the ganglion, which leads to a sixfold increase in the number of latent sites established. Further, latent sites with high genome copy number can be established in the absence of significant virus genome replication in neurons. This suggests that neurons can be infected by many HSV virions and still enter the latent state.
Development of a Rubella Virus Vaccine Expression Vector: Use of a Picornavirus Internal Ribosome Entry Site Increases Stability of Expression
Authors: Pugachev Konstantin V. , Tzeng Wen-Pin , Frey Teryl K.
Published: July 2002

Rubella virus
(RUB) is a small plus-strand RNA virus classified in the
Rubivirus
genus of the family
Togaviridae
. Live, attenuated RUB vaccines have been successfully used in vaccination programs for over 25 years, making RUB an attractive vaccine vector. In this study, such a vector was constructed using a recently developed RUB infectious cDNA clone (Robo). Using a standard strategy employed to produce expression and vaccine vectors with other togaviruses, the subgenomic promoter was duplicated to produce a recombinant construct (termed dsRobo) that expressed reporter genes such as chloramphenicol acetyltransferase and green fluorescent protein (GFP) under control of the second subgenomic promoter. However, expression of the reporter genes, as exemplified by GFP expression by dsRobo/GFP virus, was unstable during passaging, apparently due to homologous recombination between the subgenomic promoters leading to deletion of the GFP gene. To improve the stability of the vector, the internal ribosome entry site (IRES) of a picornavirus, encephalomyocarditis virus, was used instead of the second subgenomic promoter to eliminate homology. Construction was initiated by first replacing the subgenomic promoter in the parent Robo infectious clone with the IRES. Surprisingly, viable virus resulted; this virus did not synthesize a subgenomic RNA. The subgenomic promoter was then reintroduced in an orientation such that a single subgenomic RNA was produced, GFP was the initial gene on this RNA, while the RUB structural protein open reading frame was downstream and under control of the IRES element. GFP expression by this vector was significantly improved in comparison to dsRobo/GFP. This strategy should be applicable to increase the stability of other togavirus vectors.
Comparative Analysis of Translation Efficiencies of Hepatitis C Virus 5′ Untranslated Regions among Intraindividual Quasispecies Present in Chronic Infection: Opposite Behaviors Depending on Cell Type
Authors: Laporte Julien , Malet Isabelle , Andrieu Thibault , Thibault Vincent , Toulme Jean-Jacques , Wychowski Czeslaw , Pawlotsky Jean-Michel , Huraux Jean-Marie , Agut Henri , Cahour Annie
Published: July 2002

Hepatitis C virus (HCV) RNA translation initiation is dependent on the presence of an internal ribosome entry site (IRES) that is found mostly in its 5′ untranslated region (5′ UTR). While exhibiting the most highly conserved sequence within the genome, the 5′ UTR accumulates small differences, which may be of biological and clinical importance. In this study, using a bicistronic dual luciferase expression system, we have examined the sequence of 5′ UTRs from quasispecies characterized in the serum of a patient chronically infected with HCV genotype 1a and its corresponding translational activity. Sequence heterogeneity between IRES elements led to important changes in their translation efficiency both in vitro and in different cell cultures lines, implying that interactions of RNA with related transacting factors may vary according to cell type. These data suggest that variants occasionally carried by the serum prior to reinfection could be selected toward different compartments of the same infected organism, thus favoring the hypothesis of HCV multiple tropism.
Proteolytic Cleavage of the Fusion Protein but Not Membrane Fusion Is Required for Measles Virus-Induced Immunosuppression In Vitro
Authors: Weidmann Armin , Maisner Andrea , Garten Wolfgang , Seufert Marion , ter Meulen Volker , Schneider-Schaulies Sibylle
Published: July 2002

Immunosuppression induced by measles virus (MV) is associated with unresponsiveness of peripheral blood lymphocytes (PBL) to mitogenic stimulation ex vivo and in vitro. In mixed lymphocyte cultures and in an experimental animal model, the expression of the MV glycoproteins on the surface of UV-inactivated MV particles, MV-infected cells, or cells transfected to coexpress the MV fusion (F) and the hemagglutinin (H) proteins was found to be necessary and sufficient for this phenomenon. We now show that MV fusion-inhibitory peptides do not interfere with the induction of immunosuppression in vitro, indicating that MV F-H-mediated fusion is essentially not involved in this process. Proteolytic cleavage of MV F0protein by cellular proteases, such as furin, into the F1-F2subunits is, however, an absolute requirement, since (i) the inhibitory activity of MV-infected BJAB cells was significantly impaired in the presence of a furin-inhibitory peptide and (ii) cells expressing or viruses containing uncleaved F0proteins revealed a strongly reduced inhibitory activity which was improved following trypsin treatment. The low inhibitory activity of effector structures containing mainly F0proteins was not due to an impaired F0-H interaction, since both surface expression and cocapping efficiencies were similar to those found with the authentic MV F and H proteins. These results indicate that the fusogenic activity of the MV F-H complexes can be uncoupled from their immunosuppressive activity and that the immunosuppressive domains of these proteins are exposed only after proteolytic activation of the MV F0protein.
Characterization and Sequencing of Prototypic Human T-Lymphotropic Virus Type 1 (HTLV-1) from an HTLV-1/2 Seroindeterminate Patient
Authors: Waziri Allen , Soldan Samantha S. , Graf Michael D. , Nagle Jim , Jacobson Steven
Published: July 2002

Serological screening for human T-lymphotropic virus type 1 (HTLV-1) parallels the standard screening process for human immunodeficiency virus (HIV), in which samples found positive by enzyme-linked immunosorbent assay (ELISA) are confirmed with a modified Western blot procedure. There are a significant number of cases in which HTLV-1/2 ELISA-positive specimens demonstrate an incomplete banding pattern on this Western blot. Individuals providing these atypical antibody responses are categorized as seroindeterminate for HTLV-1/2. Although HTLV-1 genomic sequences are readily detectable in the peripheral blood lymphocytes (PBL) of seropositive individuals, previous studies have repeatedly demonstrated that PBL from the vast majority of HTLV-1/2 seroindeterminate individuals are PCR negative for HTLV-1. As a result, identification of the agent responsible for this indeterminate reactivity has been of interest. We have generated an HTLV-1-positive B-cell line (SI-1 B) from one of these seroindeterminate individuals. Previous screening for HTLV-1 in PBL from this patient had been routinely negative by primary PCR; however, HTLV-1
tax
had been periodically detected by nested PCR. DNA sequence data generated with genomic DNA from the SI-1 B cell line and HTLV-1-specific primers demonstrated the presence of a full-length viral genome with >97% homology to the Cosmopolitan form of HTLV-1. A 12-bp deletion was identified in the 3′-
gag
/5′-
prot
region, which would predict translation of altered or nonfunctional proteins from these genes. We propose that this HTLV-1/2-seroindeterminate patient is infected with a prototypic form of HTLV-1 at an extremely low viral load and that this finding may explain HTLV-1/2 seroindeterminate reactivity in at least a subset of these individuals.
Interaction of the Adenovirus IVa2 Protein with Viral Packaging Sequences
Authors: Zhang Wei , Imperiale Michael J.
Published: July 2002

We have demonstrated previously that the adenovirus L1 52/55-kDa protein binds to the viral IVa2 protein in infected cells. The significance of this interaction was unclear, however, based on the known functions of these two proteins: the 52/55-kDa protein is required for viral DNA packaging, while the IVa2 protein is a transactivator of the major late promoter (MLP). In this report, we have attempted to elucidate a role for each of the two proteins in the other's known function. There is no apparent effect of the 52/55-kDa protein on the interaction of the IVa2 protein with the MLP. Surprisingly, however, we found that the IVa2 protein can interact with the adenoviral packaging signal and that this interaction involves DNA sequences that have previously been demonstrated to be required for packaging.
Pseudopackaging of Adenovirus Type 5 Genomes into Capsids Containing the Hexon Proteins of Adenovirus Serotypes B, D, or E
Authors: Ostapchuk Philomena , Hearing Patrick
Published: July 2002

Adenoviruses (Ad) show promise as a vector system for gene delivery in vivo. However, a major challenge in the development of Ad vectors is the circumvention of the host immune responses to Ad infection, including both the host cytotoxic T-cell response and the humoral response resulting in neutralizing antibodies. One method to circumvent the effect of neutralizing antibodies against an Ad vector is to use different Ad serotypes to deliver the transgene of interest. This approach has been demonstrated with Ad genomes of highly related members of subgroup C. However, it is not known whether an Ad5-based vector DNA molecule can be packaged into capsids of evolutionarily more divergent adenoviruses. The aim of these studies was to determine if capsids containing hexon proteins from other Ad subgroups could package the Ad5 genome. A genetic approach utilizing an Ad5 temperature-sensitive (ts) mutant with a mutation in the hexon protein was used. When grown at the nonpermissive temperature, Ad5ts147 replicates normally, providing a source of Ad5 DNA for virus assembly, but does not produce virus particles due to the hexon protein mutation. Coinfection of Ad5ts147 with a wild-type virus of other Ad serotypes (Ad3, Ad4, or Ad9), which supply functional hexon proteins, resulted in the pseudopackaging of the Ad5 DNA genome. Furthermore, the pseudopackaged Ad5 DNA virions obtained in the coinfections were infectious. Therefore, switching hexons did not impair the infectivity or uncoating process of the pseudopackaged virion. Since hexon protein is a major antigenic determinant of the Ad capsid, this approach may prove useful to reduce the antigenicity of therapeutic Ad vectors and allow repeated vector administration.
During Readaptation In Vivo, a Tissue Culture-Adapted Strain of Feline Immunodeficiency Virus Reverts to Broad Neutralization Resistance at Different Times in Individual Hosts but through Changes at the Same Position of the Surface Glycoprotein
Authors: Bendinelli Mauro , Pistello Mauro , Del Mauro Daniela , Cammarota Giancarlo , Maggi Fabrizio , Leonildi Alessandro , Giannecchini Simone , Bergamini Carlo , Matteucci Donatella
Published: July 2002

The broad resistance to antibody-mediated neutralization of lentiviruses recently isolated from infected hosts is a poorly understood feature which might contribute to the ability of these viruses to persist and to the failure of experimental vaccines to protect against virulent viruses. We studied the underlying molecular mechanisms by examining the evolution of a neutralization-sensitive, tissue culture-adapted strain of feline immunodeficiency virus upon reinoculation into specific-pathogen-free cats. Reversion to broad neutralization resistance was observed in seven of seven inoculated animals and, in individual hosts, started to develop between less than 4 and more than 15 months from infection. After comparison of the envelope sequences of the inoculum virus, of an additional 4 neutralization-sensitive in vitro variants, and of 14 ex vivo-derived variants (6 neutralization sensitive, 5 resistant, and 3 with intermediate phenotype), a Lys→Asn or →Glu change at position 481 in the V4 region of the surface glycoprotein appeared as a key player in the reversion. This conclusion was confirmed by mutagenesis of molecularly cloned virus. Analysis of viral quasispecies and biological clones showed that the intermediate phenotype was due to transient coexistence of neutralization-sensitive and -resistant variants. Since the amino acid position involved was the same in four of four recent revertants, it is suggested that the number of residues that control reversion to broad neutralization resistance in FIV might be very limited. Amino acid 481 was found to be changed only in one of three putative long-term revertants. These variants shared a Ser→Asn change at position 557 in region V5, which probably collaborated with other mutations in long-term maintenance of neutralization resistance, as suggested by the study of mutagenized virus.
Analysis of a Novel Strain of Murine Gammaherpesvirus Reveals a Genomic Locus Important for Acute Pathogenesis
Authors: Macrae Alastair I. , Dutia Bernadette M. , Milligan Steven , Brownstein David G. , Allen Deborah J. , Mistrikova Jela , Davison Andrew J. , Nash Anthony A. , Stewart James P.
Published: July 2002

Infection of mice by murine gammaherpesvirus 68 (MHV-68) is an excellent small-animal model of gammaherpesvirus pathogenesis in a natural host. We have carried out comparative studies of another herpesvirus, murine herpesvirus 76 (MHV-76), which was isolated at the same time as MHV-68 but from a different murid host, the yellow-necked mouse (
Apodemus flavicollis)
. Molecular analyses revealed that the MHV-76 genome is essentially identical to that of MHV-68, except for deletion of 9,538 bp at the left end of the unique region. MHV-76 is therefore a deletion mutant that lacks four genes unique to MHV-68 (
M1
,
M2
,
M3,
and
M4
) as well as the eight viral tRNA-like genes. Replication of MHV-76 in cell culture was identical to that of MHV-68. However, following infection of mice, MHV-76 was cleared more rapidly from the lungs. In line with this, there was an increased inflammatory response in lungs with MHV-76. Splenomegaly was also significantly reduced following MHV-76 infection, and much less latent MHV-76 was detected in the spleen. Nevertheless, MHV-76 maintained long-term latency in the lungs and spleen. We utilized a cosmid containing the left end of the MHV-68 genome to reinsert the deleted sequence into MHV-76 by recombination in infected cells, and we isolated a rescuant virus designated MHV-76(cA8+)4 which was ostensibly genetically identical to MHV-68. The growth properties of the rescuant in infected mice were identical to those of MHV-68. These results demonstrate that genetic elements at the left end of the unique region of the MHV-68 genome play vital roles in host evasion and are critical to the development of splenic pathology.
Promoter-Proximal Regulatory Elements Involved in
oriP
-EBNA1-Independent and -Dependent Activation of the Epstein-Barr Virus C Promoter in B-Lymphoid Cell Lines
Authors: Nilsson Tina , Zetterberg Henrik , Wang Yuyan Camilla , Rymo Lars
Published: July 2002

The identification of the cellular factors that control the transcription regulatory activity of the Epstein-Barr virus C promoter (Cp) is fundamental to the understanding of the molecular mechanisms that control virus latent gene expression. Using transient transfection of reporter plasmids in group I phenotype B-lymphoid cells, we have previously shown that the −248 to −55 region (−248/−55 region) of Cp contains elements that are essential for
oriPI
-EBNA1-dependent as well as
oriPI
-EBNA1-independent activation of the promoter. We now establish the importance of this region by a detailed mutational analysis of reporter plasmids carrying Cp regulatory sequences together with or without
oriPI
. The reporter plasmids were transfected into group I phenotype Rael cells and group III phenotype cbc-Rael cells, and the Cp activity measured was correlated with the binding of candidate transcription factors in electrophoretic mobility shift assays and further assessed in cotransfection experiments. We show that the NF-Y transcription factor interacts with the previously identified CCAAT box in the −71/−63 Cp region (M. T. Puglielli, M. Woisetschlaeger, and S. H. Speck, J. Virol. 70:5758–5768, 1996). We also show that members of the C/EBP transcription factor family interact with a C/EBP consensus sequence in the −119/−112 region of Cp and that this interaction is important for promoter activity. A central finding is the identification of a GC-rich sequence in the −99/−91 Cp region that is essential for
oriPI
-EBNA1-independent as well as
oriPI
-EBNA1-dependent activity of the promoter. This region contains overlapping binding sites for Sp1 and Egr-1, and our results suggest that Sp1 is a positive and Egr-1 is a negative regulator of Cp activity. Furthermore, we demonstrate that a reporter plasmid that in addition to
oriPI
contains only the −111/+76 region of Cp still retains the ability to be activated by EBNA1.
Expression and Functional Characterization of Bluetongue Virus VP5 Protein: Role in Cellular Permeabilization
Authors: Hassan S. H. , Wirblich C. , Forzan M. , Roy P.
Published: July 2002

Segment 5 of bluetongue virus (BTV) serotype 10, which encodes the outer capsid protein VP5, was tagged with glutathione
S
-transferase and expressed by a recombinant baculovirus. The recombinant protein was subsequently purified to homogeneity, and its possible biological role in virus infection was investigated. Purified VP5 was able to bind mammalian cells but was not internalized, which indicates it is not involved in receptor-mediated endocytosis. The purified VP5 protein was shown to be able to permeabilize mammalian and
Culicoides
insect cells, inducing cytotoxicity. Sequence analysis revealed that VP5 possesses characteristic structural features (including two amino-terminal amphipathic helices) compatible with virus penetration activity. To assess the role of each feature in the observed cytotoxicity, a series of deleted VP5 molecules were generated, and their expression and biological activity was compared with the parental molecule. VP5 derivatives that included the two amphipathic helices exhibited cytotoxicity, while those that omitted these sequences did not. To confirm their role in membrane destabilization two synthetic peptides (amino acids [aa] 1 to 20 and aa 22 to 41) encompassing the two helices and an additional peptide representing the adjacent downstream sequences were also assessed for their effect on the cell membrane. Both helices, but not the downstream VP5 sequence, exhibited cytotoxicity with the most-amino-terminal helix (aa 1 to 20) showing a higher activity than the adjacent peptide (aa 22 to 41). Purified VP5 was shown to readily form trimers in solution, a feature of many proteins involved in membrane penetration. Taken together, these data support a role for VP5 in virus-cell penetration consistent with its revelation in the entry vesicle subsequent to cell binding and endocytosis.