bluuebunny/crossref_metadata_2025 huggingface dataset:
SpoIIB Localizes to Active Sites of Septal Biogenesis and Spatially Regulates Septal Thinning during Engulfment in
Bacillus subtilis
Authors: Perez Ana R. , Abanes-De Mello Angelica , Pogliano Kit
Published: July 2002

A key step in the
Bacillus subtilis
spore formation pathway is the engulfment of the forespore by the mother cell, a phagocytosis-like process normally accompanied by the loss of peptidoglycan within the sporulation septum. We have reinvestigated the role of SpoIIB in engulfment by using the fluorescent membrane stain FM 4-64 and deconvolution microscopy. We have found that
spoIIB
mutant sporangia display a transient engulfment defect in which the forespore pushes through the septum and bulges into the mother cell, similar to the situation in
spoIID
,
spoIIM
, and
spoIIP
mutants. However, unlike the sporangia of those three mutants,
spoIIB
mutant sporangia are able to complete engulfment; indeed, by time-lapse microscopy, sporangia with prominent bulges were found to complete engulfment. Electron micrographs showed that in
spoIIB
mutant sporangia the dissolution of septal peptidoglycan is delayed and spatially unregulated and that the engulfing membranes migrate around the remaining septal peptidoglycan. These results demonstrate that mother cell membranes will move around septal peptidoglycan that has not been completely degraded and suggest that SpoIIB facilitates the rapid and spatially regulated dissolution of septal peptidoglycan. In keeping with this proposal, a SpoIIB-myc fusion protein localized to the sporulation septum during its biogenesis, discriminating between the site of active septal biogenesis and the unused potential division site within the same cell.
The CcrM DNA Methyltransferase of
Agrobacterium tumefaciens
Is Essential, and Its Activity Is Cell Cycle Regulated
Authors: Kahng Lyn Sue , Shapiro Lucy
Published: July 2002

DNA methylation is now recognized as a regulator of multiple bacterial cellular processes. CcrM is a DNA adenine methyltransferase found in the alpha subdivision of the proteobacteria. Like the Dam enzyme, which is found primarily in
Escherichia coli
and other gamma proteobacteria, it does not appear to be part of a DNA restriction-modification system. The CcrM homolog of
Agrobacterium tumefaciens
was found to be essential for viability. Overexpression of CcrM is associated with significant abnormalities of cell morphology and DNA ploidy. Mapping of the transcriptional start site revealed a conserved binding motif for the global response regulator CtrA at the −35 position; this motif was footprinted by purified
Caulobacter crescentus
CtrA protein in its phosphorylated state. We have succeeded in isolating synchronized populations of
Agrobacterium
cells and analyzing their progression through the cell cycle. We demonstrate that DNA replication and cell division can be followed in an orderly manner and that flagellin expression is cyclic, consistent with our observation that motility varies during the cell cycle. Using these synchronized populations, we show that CcrM methylation of the chromosome is restricted to the late S phase of the cell cycle. Thus, within the alpha subdivision, there is a conserved cell cycle dependence and regulatory mechanism controlling
ccrM
expression.
umuDC-dnaQ
Interaction and Its Implications for Cell Cycle Regulation and SOS Mutagenesis in
Escherichia coli
Authors: Sutton Mark D. , Murli Sumati , Opperman Timothy , Klein Carly , Walker Graham C.
Published: July 2002

The
Escherichia coli
SOS-regulated
umuDC
gene products participate in a DNA damage checkpoint control and in translesion DNA synthesis. Specific interactions involving the UmuD and UmuD′ proteins, both encoded by the
umuD
gene, and components of the replicative DNA polymerase, Pol III, appear to be important for regulating these two biological activities of the
umuDC
gene products. Here we show that overproduction of the ɛ proofreading subunit of Pol III suppresses the cold sensitivity normally associated with overexpression of the
umuDC
gene products. Our results suggest that this suppression is attributable to specific interactions between UmuD or UmuD′ and the C-terminal domain of ɛ.
Fnr Is Required for NifL-Dependent Oxygen Control of
nif
Gene Expression in
Klebsiella pneumoniae
Authors: Grabbe Roman , Klopprogge Kai , Schmitz Ruth A.
Published: July 2002

In
Klebsiella pneumoniae,
NifA-dependent transcription of nitrogen fixation (
nif
) genes is inhibited by NifL in response to molecular oxygen and combined nitrogen. We recently showed that
K. pneumoniae
NifL is a flavoprotein, which apparently senses oxygen through a redox-sensitive, conformational change. We have now studied the oxygen regulation of NifL activity in
Escherichia coli
and
K. pneumoniae
strains by monitoring its inhibition of NifA-mediated expression of
K. pneumoniae
ø(
nifH
′
-
′
lacZ
) fusions in different genetic backgrounds. Strains of both organisms carrying
fnr
null mutations failed to release NifL inhibition of NifA transcriptional activity under oxygen limitation:
nif
induction was similar to the induction under aerobic conditions. When the transcriptional regulator Fnr was synthesized from a plasmid, it was able to complement, i.e., to relieve NifL inhibition in the
fnr
mutant backgrounds. Hence, Fnr appears to be involved, directly or indirectly, in NifL-dependent oxygen regulation of
nif
gene expression in
K. pneumoniae
. The data indicate that in the absence of Fnr, NifL apparently does not receive the signal for anaerobiosis. We therefore hypothesize that in the absence of oxygen, Fnr, as the primary oxygen sensor, activates transcription of a gene or genes whose product or products function to relieve NifL inhibition by reducing the flavin adenine dinucleotide cofactor under oxygen-limiting conditions.
Drosophila
as a Model Host for
Pseudomonas aeruginosa
Infection
Authors: D'Argenio David A. , Gallagher Larry A. , Berg Celeste A. , Manoil Colin
Published: July 2002

Using the fruit fly
Drosophila melanogaster
as model host, we have identified mutants of the bacterium
Pseudomonas aeruginosa
with reduced virulence. Strikingly, all strains strongly impaired in fly killing also lacked twitching motility; most such strains had a mutation in
pilGHIJKL chpABCDE
, a gene cluster known to be required for twitching motility and potentially encoding a signal transduction system. The
pil chp
genes appear to control the expression of additional virulence factors, however, since the wild-type fly-killing phenotype of a subset of mutants isolated on the basis of their compact colony morphology indicated that twitching motility itself was not required for full virulence in the fly.
Expression of Individual Copies of
Methylococcus capsulatus
Bath Particulate Methane Monooxygenase Genes
Authors: Stolyar Sergei , Franke Marion , Lidstrom Mary E.
Published: July 2002

The expression of the two gene clusters encoding the particulate methane monooxygenase (pMMO) in
Methylococcus capsulatus
Bath was assessed by analysis of transcripts and by use of chromosomal gene fusions. The results suggest that the two clusters are functionally redundant but that relative expression alters depending on the copper levels available for growth.
Characterization of Interactions between the Transcriptional Repressor PhlF and Its Binding Site at the
phlA
Promoter in
Pseudomonas fluorescens
F113
Authors: Abbas Abdelhamid , Morrissey John P. , Marquez Pilar Carnicero , Sheehan Michelle M. , Delany Isabel R. , O'Gara Fergal
Published: July 2002

The
phlACBD
genes responsible for the biosynthesis of the antifungal metabolite 2,4-diacetylphloroglucinol (PHL) by the biocontrol strain
Pseudomonas fluorescens
F113 are regulated at the transcriptional level by the pathway-specific repressor PhlF. Strong evidence suggests that this regulation occurs mainly in the early logarithmic phase of growth. First, the expression of the
phlF
gene is relatively high between 3 and 13 h of growth and relatively low thereafter, with the
phlACBD
operon following an opposite expression profile. Second, the kinetics of PHL biosynthesis are specifically altered in the logarithmic phase in a
P. fluorescens
F113
phlF
mutant. The
phlA-phlF
intergenic region presents a complex organization in that
phlACBD
is transcribed from a σ
70
RNA polymerase-dependent promoter that is likely to overlap the promoter of the divergently transcribed
phlF
gene. The repression by PhlF is due to its interaction with an inverted repeated sequence,
phO
, located downstream of the
phlA
transcriptional start site. Cross-linking experiments indicate that PhlF can dimerize in solution, and thus PhlF may bind
phO
as a dimer or higher-order complex. Furthermore, it is now demonstrated that certain regulators of PHL synthesis act by modulating PhlF binding to
phO
. PHL, which has previously been shown to be an autoinducer of PHL biosynthesis, interacts with PhlF to destabilize the PhlF-
phO
complex. Conversely, the PhlF-
phO
complex is stabilized by the presence of salicylate, which has been shown to be an inhibitor of
phlA
expression.
The Type IV Pilus Assembly Complex: Biogenic Interactions among the Bundle-Forming Pilus Proteins of Enteropathogenic
Escherichia coli
Authors: Ramer Sandra W. , Schoolnik Gary K. , Wu Cheng-Yen , Hwang Jaiweon , Schmidt Sarah A. , Bieber David
Published: July 2002

Production of type IV bundle-forming pili (BFP) by enteropathogenic
Escherichia coli
(EPEC) requires the protein products of 12 genes of the 14-gene
bfp
operon. Antisera against each of these proteins were used to demonstrate that in-frame deletion of individual genes within the operon reduces the abundance of other
bfp
operon-encoded proteins. This result was demonstrated not to be due to downstream polar effects of the mutations but rather was taken as evidence for protein-protein interactions and their role in the stabilization of the BFP assembly complex. These data, combined with the results of cell compartment localization studies, suggest that pilus formation requires the presence of a topographically discrete assembly complex that is composed of BFP proteins in stoichiometric amounts. The assembly complex appears to consist of an inner membrane component containing three processed, pilin-like proteins, BfpI, -J, and -K, that localize with BfpE, -L, and -A (the major pilin subunit); an outer membrane, secretin-like component, BfpB and -G; and a periplasmic component composed of BfpU. Of these, only BfpL consistently localizes with both the inner and outer membranes and thus, together with BfpU, may articulate between the Bfp proteins in the inner membrane and outer membrane compartments.
Complete Polar Lipid Composition of
Thermoplasma acidophilum
HO-62 Determined by High-Performance Liquid Chromatography with Evaporative Light-Scattering Detection
Authors: Shimada Haruo , Nemoto Naoki , Shida Yasuo , Oshima Tairo , Yamagishi Akihiko
Published: July 2002

Polar ether lipids of
Thermoplasma acidophilum
HO-62 were purified by high-performance liquid chromatography with an evaporative light-scattering detector. Structures of purified lipids were investigated by capillary gas chromatography, mass spectrometry, and nuclear magnetic resonance. Three types of ether lipids were found: phospholipids, glycolipids, and phosphoglycolipids. The two phospholipids had glycerophosphate as the phosphoester moiety. The seven glycolipids had different combinations of gulose, mannose, and glucose, which formed mono- or oligosaccharides. The eight phosphoglycolipids with two polar head groups contained glycerophosphate as the phosphoester moiety and gulose alone or gulose and mannose, which formed mono- or oligosaccharides, as the sugar moiety. Although gulose is an unusual sugar in nature, several glyco- and phosphoglycolipids contained gulose as one of the sugar moieties in
Thermoplasma acidophilum
. All the ether lipids had isopranoid chains of C
40
or C
20
with zero to three cyclopentane rings. The structures of these lipids including four new glycolipids and three new phosphoglycolipids were determined, and a glycosylation process for biosynthesis of these glycolipids was suggested.
Mutations in
Flavobacterium johnsoniae gldF
and
gldG
Disrupt Gliding Motility and Interfere with Membrane Localization of GldA
Authors: Hunnicutt David W. , Kempf Michael J. , McBride Mark J.
Published: July 2002

Flavobacterium johnsoniae
moves rapidly over surfaces by a process known as gliding motility. The mechanism of this form of motility is not known. Four genes that are required for
F. johnsoniae
gliding motility,
gldA
,
gldB
,
gldD
, and
ftsX
, have recently been described. GldA is similar to the ATP-hydrolyzing components of ATP binding cassette (ABC) transporters. Tn
4351
mutagenesis was used to identify two additional genes,
gldF
and
gldG
, that are required for cell movement.
gldF
and
gldG
appear to constitute an operon, and a Tn
4351
insertion in
gldF
was polar on
gldG
. pMK314, which carries the entire
gldFG
region, restored motility to each of the
gldF
and
gldG
mutants. pMK321, which expresses GldG but not GldF, restored motility to each of the
gldG
mutants but did not complement the
gldF
mutant. GldF has six putative membrane-spanning segments and is similar in sequence to channel-forming components of ABC transporters. GldG is similar to putative accessory proteins of ABC transporters. It has two apparent membrane-spanning helices, one near the amino terminus and one near the carboxy terminus, and a large intervening loop that is predicted to reside in the periplasm. GldF and GldG are involved in membrane localization of GldA, suggesting that GldA, GldF, and GldG may interact to form a transporter.
F. johnsoniae gldA
is not closely linked to
gldFG
, but the
gldA
,
gldF
, and
gldG
homologs of the distantly related gliding bacterium
Cytophaga hutchinsonii
are arranged in what appears to be an operon. The exact roles of
F. johnsoniae
GldA, GldF, and GldG in gliding are not known. Sequence similarities of GldA to components of other ABC transporters suggest that the Gld transporter may be involved in export of some material to the periplasm, outer membrane, or beyond.
Quantitation of Porcine Cytomegalovirus in Pig Tissues by PCR
Authors: Fryer Jacqueline F. L. , Griffiths Paul D. , Fishman Jay A. , Emery Vincent C. , Clark Duncan A.
Published: July 2002

A quantitative-competitive PCR for the quantification of porcine cytomegalovirus (PCMV) was developed. The virus was detected in a variety of pig organs (including potential xenotransplant donations), with viral loads ranging from <10 to 97 genome copies/μg of DNA. This assay will have significant utility for studying the activation and replication of PCMV and in swine models for allo- and xenotransplantation.
Streptococcal Meningitis Resulting from Contact with an Infected Horse
Authors: Downar James , Willey Barbara M. , Sutherland Jeffrey W. , Mathew Kelly , Low Donald E.
Published: July 2002

We report a case of group C streptococcal meningitis in a woman with a history of close animal contact as well as head trauma as a result of a kick by a horse. Blood and cerebrospinal fluid cultures grew
Streptococcus equi
subsp.
zooepidemicus
, as did a throat culture taken from the colt that had kicked her 2 weeks prior to admission.
Highly Sensitive Assay for Human Herpesvirus 8 Antibodies That Uses a Multiple Antigenic Peptide Derived from Open Reading Frame K8.1
Authors: Lam Lee L. , Pau Chou-Pong , Dollard Sheila C. , Pellett Philip E. , Spira Thomas J.
Published: July 2002

The immunodominant region of the human herpesvirus 8 (HHV-8), the antibody-binding site of glycoprotein K8.1A, was mapped to the N-terminal region by using overlapping peptides and a residue replacement method. The main epitope was located within residues 44 to 56 (GQVYQDWL----C). Based on this information, we developed an enzyme immunoassay to detect HHV-8 antibodies in human sera using a four-branch multiple antigenic peptide as the antigen. The sensitivity and specificity of the assay were 96 and 99.4%, respectively. This assay should be useful for population-based, epidemiological studies of HHV-8 infection.
The Human Immunodeficiency Virus Type 1 gp120 V2 Domain Mediates gp41-Independent Intersubunit Contacts
Authors: Center Rob J. , Earl Patricia L. , Lebowitz Jacob , Schuck Peter , Moss Bernard
Published: July 2002

The envelope protein of human immunodeficiency virus type 1 HIV-1 undergoes proteolytic cleavage in the Golgi complex to produce subunits designated gp120 and gp41, which remain noncovalently associated. While gp41 has a well-characterized oligomeric structure, the maintenance of gp41-independent gp120 intersubunit contacts remains a contentious issue. Using recombinant vaccinia virus to achieve high-level expression of gp120 in mammalian cells combined with gel filtration analysis, we were able to isolate a discrete oligomeric form of gp120. Oligomerization of gp120 occurred intracellularly between 30 and 120 min after synthesis. Analysis by sedimentation equilibrium unequivocally identified the oligomeric species as a dimer. In order to identify the domains involved in the intersubunit contact, we expressed a series of gp120 proteins lacking various domains and assessed the effects of mutation on oligomeric structure. Deletion of the V1 or V3 loops had little effect on the relative amounts of monomer and dimer in comparison to wild-type gp120. In contrast, deletion of either all or part of the V2 loop drastically reduced dimer formation, indicating that this domain is required for intersubunit contact formation. Consistent with this, the V2 loop of the dimer was less accessible than that of the monomer to a specific monoclonal antibody. Previous studies have shown that while the V2 loop is not an absolute requirement for viral entry, the absence of this domain reduces viral resistance to neutralization by monoclonal antibodies or sera. We propose that the quaternary structure of gp120 may contribute to resistance to neutralization by limiting the exposure of conserved epitopes.
Identifying the Determinants in the Equatorial Domain of
Buchnera
GroEL Implicated in Binding
Potato Leafroll Virus
Authors: Hogenhout Saskia A. , van der Wilk Frank , Verbeek Martin , Goldbach Rob W. , van den Heuvel Johannes F. J. M.
Published: July 2002

Luteoviruses avoid degradation in the hemolymph of their aphid vector by interacting with a GroEL homolog from the aphid's primary endosymbiotic bacterium (
Buchnera
sp.). Mutational analysis of GroEL from the primary endosymbiont of
Myzus persicae
(MpB GroEL) revealed that the amino acids mediating binding of
Potato leafroll virus
(PLRV;
Luteoviridae
) are located within residues 9 to 19 and 427 to 457 of the N-terminal and C-terminal regions, respectively, of the discontinuous equatorial domain. Virus overlay assays with a series of overlapping synthetic decameric peptides and their derivatives demonstrated that R13, K15, L17, and R18 of the N-terminal region and R441 and R445 of the C-terminal region of the equatorial domain of GroEL are critical for PLRV binding. Replacement of R441 and R445 by alanine in full-length MpB GroEL and in MpB GroEL deletion mutants reduced but did not abolish PLRV binding. Alanine substitution of either R13 or K15 eliminated the PLRV-binding capacity of the other and those of L17 and R18. In the predicted tertiary structure of GroEL, the determinants mediating virus binding are juxtaposed in the equatorial plain.
Isolation of Borna Disease Virus from Human Brain Tissue
Authors: Nakamura Yurie , Takahashi Hirokazu , Shoya Yuko , Nakaya Takaaki , Watanabe Makiko , Tomonaga Keizo , Iwahashi Kazuhiko , Ameno Kiyoshi , Momiyama Noriko , Taniyama Hiroyuka , Sata Tetsutaro , Kurata Takeshi , de la Torre Juan Carlos , Ikuta Kazuyoshi
Published: July 2002

Serological and molecular epidemiological studies indicate that Borna disease virus (BDV) can infect humans and is possibly associated with certain neuropsychiatric disorders. We examined brain tissue collected at autopsy from four schizophrenic patients and two healthy controls for the presence of BDV markers in 12 different brain regions. BDV RNA and antigen was detected in four brain regions of a BDV-seropositive schizophrenic patient (P2) with a very recent (2 years) onset of disease. BDV markers exhibited a regionally localized distribution. BDV RNA was found in newborn Mongolian gerbils intracranially inoculated with homogenates from BDV-positive brain regions of P2. Human oligodendroglia (OL) cells inoculated with brain homogenates from BDV-positive gerbils allowed propagation and isolation of BDVHuP2br, a human brain-derived BDV. Virus isolation was also possible by transfection of Vero cells with ribonucleoprotein complexes prepared from BDV-positive human and gerbil brain tissues. BDVHuP2br was genetically closely related to but distinct from previously reported human- and animal-derived BDV sequences.
Rotavirus-Induced Structural and Functional Alterations in Tight Junctions of Polarized Intestinal Caco-2 Cell Monolayers
Authors: Obert Guillaume , Peiffer Isabelle , Servin Alain L.
Published: July 2002

We provide here new insights into rotavirus (RRV) pathogenicity by showing that RRV infection promotes structural and functional injuries localized at the tight junctions (TJ) in the cell-cell junctional complex of cultured polarized human intestinal Caco-2 cells forming monolayers. RRV infection resulted in a progressive increase in the paracellular permeability to [
3
H]mannitol as a function of the time postinfection. We observed a disorganization of the TJ-associated protein occludin as a function of the time postinfection, whereas distribution of the zonula adherens associated E-cadherin was not affected. These structural and functional RRV-induced TJ injuries were not accompanied by alteration in cell and monolayer integrity, as assessed by the lack of change in transepithelial membrane resistance and lactate dehydrogenase release. Finally, using the stabilizer of actin filaments Jasplakinolide, we demonstrated that the RRV-induced structural and functional alterations in TJ are independent of the RRV-induced apical F-actin rearrangements.
Functional Analyses of the EBNA1 Origin DNA Binding Protein of Epstein-Barr Virus
Authors: Ceccarelli Derek F. J. , Frappier Lori
Published: July 2002

The EBNA1 protein of Epstein-Barr virus (EBV) governs the replication and segregation of the viral episomes in latently infected cells and transactivates the expression of other EBV latency proteins through direct interactions with DNA sequences in the EBV latent origin of replication,
oriP
. To better understand how EBNA1 controls these processes, we have assessed the contribution of various EBNA1 sequences to its replication, segregation, and transactivation functions. Here we show that EBNA1 residues 325 to 376 are responsible for the transactivation activity of EBNA1. This region coincides with the DNA looping domain previously shown to mediate interactions at a distance between DNA-bound EBNA1 molecules. The same residues mediate DNA segregation but have no apparent role in DNA replication, indicating that the replication and transcription activation activities of EBNA1 are distinct. The acidic C-terminal tail of EBNA1 was not found to contribute to replication, transactivation, or segregation. We have also investigated the functional significance of two structural motifs within the DNA binding and dimerization domains of EBNA1, the proline loop and the WF motif. Although the amino acids in these motifs do not directly contact the DNA, both of these motifs were found to contribute to EBNA1 functions by increasing the DNA-binding ability of EBNA1. Mechanisms by which DNA binding is stimulated by these motifs are discussed.
Assembly of the Coronavirus Envelope: Homotypic Interactions between the M Proteins
Authors: de Haan Cornelis A. M. , Vennema Harry , Rottier Peter J. M.
Published: July 2002

The viral membrane proteins M and E are the minimal requirements for the budding of coronavirus particles. Since the E protein occurs in particles only in trace amounts, the lateral interactions between the M proteins apparently generate the major driving force for envelope formation. By using coimmunoprecipitation and envelope incorporation assays, we provide extensive evidence for the existence of such M-M interactions. In addition, we determined which domains of the M protein are involved in this homotypic association, using a mutagenetic approach. Mutant M proteins which were not able to assemble into viruslike particles (VLPs) by themselves (C. A. M. de Haan, L. Kuo, P. S. Masters, H. Vennema, and P. J. M. Rottier, J. Virol. 72:6838–6850, 1998) were tested for the ability to associate with other M proteins and to be rescued into VLPs formed by assembly-competent M proteins. We found that M proteins lacking parts of the transmembrane cluster, of the amphipathic domain, or of the hydrophilic carboxy-terminal tail, or M proteins that had their luminal domain replaced by heterologous ectodomains, were still able to associate with assembly-competent M proteins, resulting in their coincorporation into VLPs. Only a mutant M protein in which all three transmembrane domains had been replaced lost this ability. The results indicate that M protein molecules interact with each other through multiple contact sites, particularly at the transmembrane level. Finally, we tested the stringency with which membrane proteins are selected for incorporation into the coronavirus envelope by probing the coassembly of some foreign proteins. The observed efficient exclusion from budding of the vesicular stomatitis virus G protein and the equine arteritis virus M protein indicates that envelope assembly is indeed a highly selective sorting process. The low but detectable incorporation of CD8 molecules, however, demonstrated that this process is not perfect.
Antiapoptotic Herpesvirus Bcl-2 Homologs Escape Caspase-Mediated Conversion to Proapoptotic Proteins
Authors: Bellows David S. , Chau B. Nelson , Lee Percy , Lazebnik Yuri , Burns William H. , Hardwick J. Marie
Published: July 2002

The antiapoptotic Bcl-2 and Bcl-x
L
proteins of mammals are converted into potent proapoptotic factors when they are cleaved by caspases, a family of apoptosis-inducing proteases (E. H.-Y. Cheng, D. G. Kirsch, R. J. Clem, R. Ravi, M. B. Kastan, A. Bedi, K. Ueno, and J. M. Hardwick, Science 278:1966–1968, 1997; R. J. Clem, E. H.-Y. Cheng, C. L. Karp, D. G. Kirsch, K. Ueno, A. Takahashi, M. B. Kastan, D. E. Griffin, W. C. Earnshaw, M. A. Veliuona, and J. M. Hardwick, Proc. Natl. Acad. Sci. USA 95:554–559, 1998). Gamma herpesviruses also encode homologs of the Bcl-2 family. All tested herpesvirus Bcl-2 homologs possess antiapoptotic activity, including the more distantly related homologs encoded by murine gammaherpesvirus 68 (γHV68) and bovine herpesvirus 4 (BHV4), as described here. To determine if viral Bcl-2 proteins can be converted into death factors, similar to their cellular counterparts, five herpesvirus Bcl-2 homologs from five different viruses were tested for their susceptibility to caspases. Only the viral Bcl-2 protein encoded by γHV68 was susceptible to caspase digestion. However, unlike the caspase cleavage products of cellular Bcl-2, Bcl-x
L
, and Bid, which are potent inducers of apoptosis, the cleavage product of γHV68 Bcl-2 lacked proapoptotic activity. KSBcl-2, encoded by the Kaposi's sarcoma-associated herpesvirus, was the only viral Bcl-2 homolog that was capable of killing cells when expressed as an N-terminal truncation. However, because KSBcl-2 was not cleavable by caspases, the latent proapoptotic activity of KSBcl-2 apparently cannot be released. The Bcl-2 homologs encoded by herpesvirus saimiri, Epstein-Barr virus, and BHV4 were not cleaved by apoptotic cell extracts and did not possess latent proapoptotic activities. Thus, herpesvirus Bcl-2 homologs escape negative regulation by retaining their antiapoptotic activities and/or failing to be converted into proapoptotic proteins by caspases during programmed cell death.
Loss of G
1
/S Checkpoint in Human Immunodeficiency Virus Type 1-Infected Cells Is Associated with a Lack of Cyclin-Dependent Kinase Inhibitor p21/Waf1
Authors: Clark Elizabeth , Santiago Francisco , Deng Longwen , Chong Siew yen , de la Fuente Cynthia , Wang Lai , Fu Peng , Stein Dana , Denny Thomas , Lanka Venkata , Mozafari Fariba , Okamoto Takashi , Kashanchi Fatah
Published: July 2002

Productive high-titer infection by human immunodeficiency virus type 1 (HIV-1) requires the activation of target cells. Infection of quiescent peripheral CD4 lymphocytes by HIV-1 results in incomplete, labile reverse transcripts and lack of viral progeny formation. An interplay between Tat and p53 has previously been reported, where Tat inhibited the transcription of the p53 gene, which may aid in the development of AIDS-related malignancies, and p53 expression inhibited HIV-1 long terminal repeat transcription. Here, by using a well-defined and -characterized stress signal, gamma irradiation, we find that upon gamma irradiation, HIV-1-infected cells lose their G
1
/S checkpoints, enter the S phase inappropriately, and eventually apoptose. The loss of the G
1
/S checkpoint is associated with a loss of p21/Waf1 protein and increased activity of a major G
1
/S kinase, namely, cyclin E/cdk2. The p21/Waf1 protein, a known cyclin-dependent kinase inhibitor, interacts with the cdk2/cyclin E complex and inhibits progression of cells into S phase. We find that loss of the G
1
/S checkpoint in HIV-1-infected cells may in part be due to Tat's ability to bind p53 (a known activator of the p21/Waf1 promoter) and sequester its transactivation activity, as seen in both in vivo and in vitro transcription assays. The loss of p21/Waf1 in HIV-1-infected cells was specific to p21/Waf1 and did not occur with other KIP family members, such as p27 (KIP1) and p57 (KIP2). Finally, the advantage of a loss of the G
1
/S checkpoint for HIV-1 per se may be that it pushes the host cell into the S phase, which may then allow subsequent virus-associated processes, such as RNA splicing, transport, translation, and packaging of virion-specific genes, to occur.
Characterization and Construction of Functional cDNA Clones of Pariacoto Virus, the First
Alphanodavirus
Isolated outside Australasia
Authors: Johnson Karyn N. , Zeddam Jean-Louis , Ball L. Andrew
Published: July 2002

Pariacoto virus (PaV) was recently isolated in Peru from the Southern armyworm (
Spodoptera eridania
). PaV particles are isometric, nonenveloped, and about 30 nm in diameter. The virus has a bipartite RNA genome and a single major capsid protein with a molecular mass of 39.0 kDa, features that support its classification as a
Nodavirus
. As such, PaV is the first
Alphanodavirus
to have been isolated from outside Australasia. Here we report that PaV replicates in wax moth larvae and that PaV genomic RNAs replicate when transfected into cultured baby hamster kidney cells. The complete nucleotide sequences of both segments of the bipartite RNA genome were determined. The larger genome segment, RNA1, is 3,011 nucleotides long and contains a 973-amino-acid open reading frame (ORF) encoding protein A, the viral contribution to the RNA replicase. During replication, a 414-nucleotide long subgenomic RNA (RNA3) is synthesized which is coterminal with the 3′ end of RNA1. RNA3 contains a small ORF which could encode a protein of 90 amino acids similar to the B2 protein of other alphanodaviruses. RNA2 contains 1,311 nucleotides and encodes the 401 amino acids of the capsid protein precursor α. The amino acid sequences of the PaV capsid protein and the replicase subunit share 41 and 26% identity with homologous proteins of
Flock house virus
, the best characterized of the alphanodaviruses. These and other sequence comparisons indicate that PaV is evolutionarily the most distant of the alphanodaviruses described to date, consistent with its novel geographic origin. Although the PaV capsid precursor is cleaved into the two mature capsid proteins β and γ, the amino acid sequence at the cleavage site, which is Asn/Ala in all other alphanodaviruses, is Asn/Ser in PaV. To facilitate the investigation of PaV replication in cultured cells, we constructed plasmids that transcribed full-length PaV RNAs with authentic 5′ and 3′ termini. Transcription of these plasmids in cells recreated the replication of PaV RNA1 and RNA2, synthesis of subgenomic RNA3, and translation of viral proteins A and α.
Yin Yang 1 Negatively Regulates the Differentiation-Specific E1 Promoter of Human Papillomavirus Type 6
Authors: Ai Wandong , Narahari Janaki , Roman Ann
Published: July 2002

Human papillomavirus type 6 (HPV-6) is a low-risk HPV whose replication cycle, like that of all HPVs, is differentiation dependent. We have previously shown that CCAAT displacement protein (CDP) binds the differentiation-induced HPV-6 E1 promoter and negatively regulates its activity in undifferentiated cells (W. Ai, E. Toussaint, and A. Roman, J. Virol. 73:4220–4229, 1999). Using electrophoretic mobility shift assays (EMSAs), we now report that Yin Yang 1 (YY1), a multifunctional protein that can act as a transcriptional activator or repressor and that can also inhibit HPV replication in vitro, binds the HPV-6 E1 promoter. EMSAs, using subfragments of the promoter as competitors, showed that the YY1 binding site is located at the 5′ end of the E1 promoter. When a putative YY1 site was mutated, the ability of YY1 to bind was greatly decreased. The activity of the mutated E1 promoter, monitored with the reporter gene
luciferase
, was threefold greater than that of the wild-type promoter, suggesting that YY1 negatively regulates HPV-6 E1 promoter activity. Nuclear extracts from differentiated keratinocytes showed decreased binding of YY1 to the wild-type promoter. Consistent with this, in differentiated keratinocytes, the activity of the transfected
luciferase
gene transcribed from the mutated promoter was comparable to that of the wild-type promoter; both promoters were up-regulated in differentiated keratinocytes compared to undifferentiated cells. These data suggest that YY1 functions in undifferentiated keratinocytes but not in differentiated keratinocytes. Both the wild-type and mutated promoters could be negatively regulated by overexpression of a plasmid encoding CDP. Thus, both YY1 and CDP appear to be negative regulators of the differentiation-induced HPV-6 E1 promoter and thereby the HPV life cycle. In contrast, only binding of CDP was detected using the E1 promoter of the high-risk HPV-31.
Protective Immunity to Rotavirus Shedding in the Absence of Interleukin-6: Th1 Cells and Immunoglobulin A Develop Normally
Authors: VanCott John L. , Franco Manuel A. , Greenberg Harry B. , Sabbaj Steffanie , Tang Baozhing , Murray Richard , McGhee Jerry R.
Published: July 2002

We investigated whether interleukin-6 (IL-6) was required for the development of immunoglobulin A (IgA)- and T-helper 1 (Th1)-associated protective immune responses to rotavirus by using adult IL-6-deficient mice [BALB/c and (C57BL/6 × O1a)F2backgrounds]. Naive IL-6−mice had normal frequencies of IgA plasma cells in the gastrointestinal tract. Consistent with this, total levels of IgA in fecal extracts, saliva, and sera were unaltered. In specific response to oral infection with rhesus rotavirus, IL-6−and IL-6+mice exhibited efficient Th1-type gamma interferon responses in Peyer's patches with high levels of serum IgG2a and intestinal IgA. Although there was an increase in Th2-type IL-4 in CD4+T cells from IL-6−mice following restimulation with rotavirus antigen in the presence of irradiated antigen-presenting cells, unfractionated Peyer's patch cells failed to produce a significant increase in IL-4. Moreover, virus-specific IgG1 in serum was not significantly increased in IL-6−mice in comparison with IL-6+mice. Following oral inoculation with murine rotavirus, IL-6−and IL-6+mice mediated clearance of rotavirus and mounted a strong IgA response. When IL-6−and IL-6+mice [(C57BL/6 × O1a)F2background] were orally inoculated with rhesus rotavirus and later challenged with murine rotavirus, all of the mice maintained high levels of IgA in feces and were protected against reinfection. Thus, IL-6 failed to provide unique functions in the development of IgA-secreting B cells and in the establishment of Th1-associated protective immunity against rotavirus infection in adult mice.
Persistence and Reactivation of Bovine Herpesvirus 1 in the Tonsils of Latently Infected Calves
Authors: Winkler M. T. C. , Doster A. , Jones C.
Published: July 2002

Bovine herpesvirus 1 (BHV-1), like other members of theAlphaherpesvirinaesubfamily, establishes latent infection in sensory neurons. Reactivation from latency can occur after natural or corticosteroid-induced stress culminating in recurrent disease and/or virus transmission to uninfected animals. Our previous results concluded that CD4+T cells in the tonsil and other adjacent lymph nodes are infected and undergo apoptosis during acute infection (M. T. Winkler, A. Doster, and C. Jones, J. Virol. 73:8657–8668, 1999). To test whether BHV-1 persisted in lymphoreticular tissue, we analyzed tonsils of latently infected calves for the presence of viral DNA and gene expression. BHV-1 DNA was consistently detected in the tonsils of latently infected calves. Detection of the latency-related transcript (LRT) in tonsils of latently infected calves required nested reverse transcription-PCR (RT-PCR) suggesting that only a few cells contained viral DNA or that LRT is not an abundant transcript. bICP0 (immediate-early and early transcripts), ribonucleotide reductase (early transcript), and glycoprotein C (late transcript) were not detected by RT-PCR in latently infected calves. When reactivation was initiated by dexamethasone, bICP0 and ribonucleotide reductase transcripts were detected. Following dexamethasone treatment, viral nucleic acid was detected simultaneously in trigeminal ganglionic neurons and lymphoid follicles of tonsil. LRT was detected at 6 and 24 h after dexamethasone treatment but not at 48 h. Dexamethasone-induced reactivation led to apoptosis that was localized to tonsillar lymphoid follicles. Taken together, these findings suggest that the tonsil is a site for persistence or latency from which virus can be reactivated by dexamethasone. We further hypothesize that the shedding of virus from the tonsil during reactivation plays a role in virus transmission.
Human Immunodeficiency Virus Type 1 Coreceptor Preferences Determine Target T-Cell Depletion and Cellular Tropism in Human Lymphoid Tissue
Authors: Grivel Jean-Charles , Penn Michael L. , Eckstein Daniel A. , Schramm Birgit , Speck Roberto F. , Abbey Nancy W. , Herndier Brian , Margolis Leonid , Goldsmith Mark A.
Published: July 2002

The present study sought to determine how usage of coreceptors by human immunodeficiency virus type 1 dictates cell tropism and depletion of CD4
+
T cells in human lymphoid tissues cultured ex vivo. We found that coreceptor preferences control the marked, preferential depletion of coreceptor-expressing CD4
+
lymphocytes. In addition, there was a strong, but not absolute, preference shown by CXCR4-using strains for lymphocytes and by CCR5-using strains for macrophages.
Evaluation of a Neonatal Rat Model for Prediction of Mumps Virus Neurovirulence in Humans
Authors: Rubin Steven A. , Pletnikov Mikhail , Taffs Rolf , Snoy Phil J. , Kobasa Darwyn , Brown Earl G. , Wright Kathryn E. , Carbone Kathryn M.
Published: July 2002

Neurovirulence of several mumps virus strains was assessed in a prototype rat neurovirulence test and compared to results obtained in the monkey neurovirulence test. The relative human neurovirulence of these strains was proportional to the severity of hydrocephalus in rats but not to lesion scores in the monkeys.
Estimating Relative Fitness in Viral Competition Experiments
Authors: Marée Athanasius F. M. , Keulen Wilco , Boucher Charles A. B. , De Boer Rob J.
Published: July 2002

The relative fitness of viral variants has previously been defined as the slope of the logarithmic ratio of the genotype or phenotype frequencies in time plots of pairwise competition experiments. Developing mathematical models for such experiments by employing the conventional coefficient of selection
s
, we demonstrate that this logarithmic ratio gives the fitness difference, rather than the relative fitness. This fitness difference remains proportional to the actual replication rate realized in the particular experimental setup and hence cannot be extrapolated to other situations. Conversely, the conventional relative fitness (1 +
s
) should be more generic. We develop an approach to compute the generic relative fitness in conventional competition experiments. This involves an estimation of the total viral replication during the experiment and requires an estimate of the average lifetime of productively infected cells. The novel approach is illustrated by estimating the relative fitness, i.e., the relative replication rate, of a set of zidovudine-resistant human immunodeficiency virus type 1 variants. A tool for calculating the relative fitness from observed changes in viral load and genotype (or phenotype) frequencies is publically available on the website at

http://www-binf.bio.uu.nl/
∼
rdb/fitness.html

.
Complete Nucleotide Sequence and Genome Organization of Hibiscus Chlorotic Ringspot Virus, a New Member of the Genus
Carmovirus
: Evidence for the Presence and Expression of Two Novel Open Reading Frames
Authors: Huang Mei , Koh Dora Chin-Yen , Weng Li-Juan , Chang Min-Li , Yap Yun-Kiam , Zhang Lee , Wong Sek-Man
Published: July 2002

The complete nucleotide sequence of hibiscus chlorotic ringspot virus (HCRSV) was determined. The genomic RNA (gRNA) is 3,911 nucleotides long and has the potential to encode seven viral proteins in the order of 28 (p28), 23 (p23), 81 (p81), 8 (p8), 9 (p9), 38 (p38), and 25 (p25) kDa. Excluding two unique open reading frames (ORFs) encoding p23 and p25, the ORFs encode proteins with high amino acid similarity to those of carmoviruses. In addition to gRNA, two 3′-coterminated subgenomic RNA (sgRNA) species were identified. Full-length cDNA clones derived from gRNA and sgRNA were constructed under the control of a T7 promoter. Both capped and uncapped transcripts derived from the full-length genomic cDNA clone were infectious. In vitro translation and mutagenesis assays confirmed that all the predicted ORFs except the ORF encoding p8 are translatable, and the two novel ORFs (those encoding p23 and p25) may be functionally indispensable for the viral infection cycle. Based on virion morphology and genome organization, we propose that HCRSV be classified as a new member of the genus
Carmovirus
in family
Tombusviridae
.
Regression of Established Human Papillomavirus Type 16 (HPV-16) Immortalized Tumors In Vivo by Vaccinia Viruses Expressing Different Forms of HPV-16 E7 Correlates with Enhanced CD8+T-Cell Responses That Home to the Tumor Site
Authors: Lamikanra Abigail , Pan Zhen-Kun , Isaacs Stuart N. , Wu Tzyy-Choou , Paterson Yvonne
Published: July 2002

Using vaccinia virus as a live vector, we show that the expression of human papillomavirus type 16 (HPV-16) E7 fused to a nonhemolytic portion of theListeriamonocytogenesvirulence factor, listeriolysin O (LLO), induces an immune response that causes the regression of established HPV-16 immortalized tumors in C57BL/6 mice. The vaccinia virus construct expressing LLO fused to E7 (VacLLOE7) was compared with two previously described vaccinia virus constructs: one that expresses unmodified E7 (VacE7) and another that expresses E7 in a form designed to direct it to intracellular lysosomal compartments and improve major histocompatibility complex class II-restricted responses (VacSigE7LAMP-1). C57BL/6 mice bearing established HPV-16 immortalized tumors of 5 or 8 mm were treated with each of these vaccines. Fifty percent of the mice treated with VacLLOE7 remained tumor free 2 months after tumor inoculation, whereas 12 to 25% of the mice were tumor free after treatment with VacSigE7LAMP-1 (depending on the size of the tumor). No mice were tumor free in the group given VacE7. Compared to VacE7, VacSigE7LAMP-1 and VacLLOE7 resulted in increased numbers of H2-Db-specific tetramer-positive CD8+T cells in mouse spleens that produced gamma interferon and tumor necrosis factor alpha upon stimulation with RAHYNIVTF peptide. In addition, the highest frequency of tetramer-positive T cells was seen in the tumor sites of mice treated with VacLLOE7. An increased efficiency of E7-specific lysis by splenocytes from mice immunized with VacLLOE7 was also observed. These results indicate that the fusion of E7 with LLO not only enhances antitumor therapy by improving the tumoricidal function of E7-specific CD8+T cells but may also increase the number of antigen-specific CD8+T cells in the tumor, the principle site of antigen expression.
Novel
cis
-Acting Replication Element in the Adeno-Associated Virus Type 2 Genome Is Involved in Amplification of Integrated
rep-cap
Sequences
Authors: Nony Pascale , Tessier Jacques , Chadeuf Gilliane , Ward Peter , Giraud Aurélie , Dugast Marc , Linden R. Michael , Moullier Philippe , Salvetti Anna
Published: July 2002

This study identifies a region of the adeno-associated virus type 2 (AAV-2)
rep
gene (nucleotides 190 to 540 of wild-type AAV-2) as a
cis
-acting Rep-dependent element able to promote the replication of transiently transfected plasmids. This viral element is also shown to be involved in the amplification of integrated sequences in the presence of adenovirus and Rep proteins.
Elicitation of High-Frequency Cytotoxic T-Lymphocyte Responses against both Dominant and Subdominant Simian-Human Immunodeficiency Virus Epitopes by DNA Vaccination of Rhesus Monkeys
Authors: Barouch Dan H. , Craiu Abie , Santra Sampa , Egan Michael A. , Schmitz Jörn E. , Kuroda Marcelo J. , Fu Tong-Ming , Nam Jae-Hwan , Wyatt Linda S. , Lifton Michelle A. , Krivulka Georgia R. , Nickerson Christine E. , Lord Carol I. , Moss Bernard , Lewis Mark G. , Hirsch Vanessa M. , Shiver John W. , Letvin Norman L.
Published: July 2002

Increasing evidence suggests that the generation of cytotoxic T-lymphocyte (CTL) responses specific for a diversity of viral epitopes will be needed for an effective human immunodeficiency virus type 1 (HIV-1) vaccine. Here, we determine the frequencies of CTL responses specific for the simian immunodeficiency virus Gag p11C and HIV-1 Env p41A epitopes in simian-human immunodeficiency virus (SHIV)-infected and vaccinated rhesus monkeys. The p11C-specific CTL response was high frequency and dominant and the p41A-specific CTL response was low frequency and subdominant in both SHIV-infected monkeys and in monkeys vaccinated with recombinant modified vaccinia virus Ankara vectors expressing these viral antigens. Interestingly, we found that plasmid DNA vaccination led to high-frequency CTL responses specific for both of these epitopes. These data demonstrate that plasmid DNA may be useful in eliciting a broad CTL response against multiple epitopes.