bluuebunny/crossref_metadata_2025 huggingface dataset:
Establishing Lysogenic Transcription in the Temperate Coliphage 186
Authors: Neufing Petra J. , Shearwin Keith E. , Egan J. Barry
Published: July 2002

A single-copy chromosomal reporter system was used to measure the intrinsic strengths and interactions between the three promoters involved in the establishment of lysogeny by coliphage 186. The maintenance lysogenic promoter
p
L
for the immunity repressor gene
c
I is intrinsically ∼20-fold weaker than the lytic promoter
p
R
. These promoters are arranged face-to-face, and transcription from
p
L
is further weakened some 14-fold by the activity of
p
R
. Efficient establishment of lysogeny requires the
p
E
promoter, which lies upstream of
p
L
and is activated by the phage CII protein to a level comparable to that of
p
R
. Transcription of
p
E
is less sensitive to converging
p
R
transcription and raises
c
I transcription at least 55-fold. The
p
E
promoter does not occlude
p
L
but inhibits lytic transcription by 50%. This interference is not due to bound CII preventing elongation of the lytic transcript. The
p
E
RNA is antisense to the anti-immune repressor gene
apl
, but any role of this in the establishment of lysogeny appears to be minimal.
Dissection of the Functional and Structural Domains of Phosphorelay Histidine Kinase A of
Bacillus subtilis
Authors: Wang Ling , Fabret Céline , Kanamaru Kyoko , Stephenson Keith , Dartois Veronique , Perego Marta , Hoch James A.
Published: July 2002

The initiation of sporulation in
Bacillus subtilis
results primarily from phosphoryl group input into the phosphorelay by histidine kinases, the major kinase being kinase A. Kinase A is active as a homodimer, the protomer of which consists of an approximately 400-amino-acid N-terminal putative signal-sensing region and a 200-amino-acid C-terminal autokinase. On the basis of sequence similarity, the N-terminal region may be subdivided into three PAS domains: A, B, and C, located from the N- to the C-terminal end. Proteolysis experiments and two-hybrid analyses indicated that dimerization of the N-terminal region is accomplished through the PAS-B/PAS-C region of the molecule, whereas the most amino-proximal PAS-A domain is not dimerized. N-terminal deletions generated with maltose binding fusion proteins showed that an intact PAS-A domain is very important for enzymatic activity. Amino acid substitution mutations in PAS-A as well as PAS-C affected the in vivo activity of kinase A, suggesting that both PAS domains are required for signal sensing. The C-terminal autokinase, when produced without the N-terminal region, was a dimer, probably because of the dimerization required for formation of the four-helix-bundle phosphotransferase domain. The truncated autokinase was virtually inactive in autophosphorylation with ATP, whereas phosphorylation of the histidine of the phosphotransfer domain by back reactions from Spo0F∼P appeared normal. The phosphorylated autokinase lost the ability to transfer its phosphoryl group to ADP, however. The N-terminal region appears to be essential both for signal sensing and for maintaining the correct conformation of the autokinase component domains.
Bordetella
Interspecies Allelic Variation in AlcR Inducer Requirements: Identification of a Critical Determinant of AlcR Inducer Responsiveness and Construction of an
alcR
(Con) Mutant Allele
Authors: Brickman Timothy J. , Armstrong Sandra K.
Published: July 2002

Previous studies established the critical roles of AlcR and alcaligin inducer in positive regulation of alcaligin siderophore biosynthesis and transport genes in
Bordetella pertussis
and
Bordetella bronchiseptica
. Transcriptional analyses using plasmid-borne
alcR
genes of
B. pertussis
UT25 and
B. bronchiseptica
B013N to complement the
alcR
defect of
B. bronchiseptica
strain BRM13 (Δ
alcR1 alcA
::mini-Tn
5 lacZ1
) revealed interspecies differences in AlcR inducer requirements for activation of
alcABCDER
operon transcription. Whereas the
B. pertussis
UT25 AlcR protein retained strong inducer dependence when produced from multicopy plasmids,
B. bronchiseptica
B013N
alcR
partially suppressed the alcaligin requirement for transcriptional activation. Functional analysis of AlcR chimeras produced by interspecies domain swapping and interspecies reciprocal site-specific mutagenesis determined that the phenotypic difference in AlcR inducer dependence was due to a single amino acid difference within the proposed inducer-binding and multimerization domain of AlcR. Structural predictions guided the design of a mutant AlcR protein with a single amino acid substitution at this critical position, AlcR(S103T), that was fully constitutive not only when produced from multicopy plasmids but also at a single-copy gene dosage. These results indicate that AlcR residue 103 affects a critical determinant of alcaligin inducer dependence of AlcR-mediated transcriptional activation. The
alcR
(S103T) mutant allele is the first
alcR
(Con) mutant allele identified.
Rhodospirillum rubrum
Possesses a Variant of the
bchP
Gene, Encoding Geranylgeranyl-Bacteriopheophytin Reductase
Authors: Addlesee Hugh A. , Hunter C. Neil
Published: July 2002

The
bchP
gene product of
Rhodobacter sphaeroides
is responsible for the reduction of the isoprenoid moiety of bacteriochlorophyll (Bchl) from geranylgeraniol (GG) to phytol; here, we show that this enzyme also catalyzes the reduction of the isoprenoid moiety of bacteriopheophytin (Bphe). In contrast, we demonstrate that a newly identified homolog of this gene in
Rhodospirillum rubrum
encodes an enzyme, GG-Bphe reductase, capable of reducing the isoprenoid moiety of Bphe only. We propose that
Rhodospirillum rubrum
is a naturally occurring
bchP
mutant and that an insertion mutation may have been the initial cause of a partial loss of function. Normal BchP function can be restored to
Rhodospirillum rubrum
, creating a new transconjugant strain possessing Bchl esterified with phytol. We speculate on the requirement of
Rhodospirillum rubrum
for phytylated Bphe and on a potential link between the absence of LH2 and of phytylated Bchl from the wild-type bacterium. The identification of a second role for the fully functional BchP in catalyzing the synthesis of phytylated Bphe strongly suggests that homologs of this enzyme may be similarly responsible for the synthesis of phytylated pheophytin in organisms possessing photosystem 2. In addition to
bchP
, other members of a photosynthesis gene cluster were identified in
Rhodospirillum rubrum
, including a
bchG
gene, demonstrated to encode a functional Bchl synthetase by complementation of a
Rhodobacter sphaeroides
mutant.
A MecA Paralog, YpbH, Binds ClpC, Affecting both Competence and Sporulation
Authors: Persuh Marjan , Mandic-Mulec Ines , Dubnau David
Published: July 2002

ComK, the master regulator of competence, is degraded by the general stress-related protease ClpCP but must be targeted to this protease by binding to the adapter protein MecA. The genome of
Bacillus subtilis
contains a paralog of
mecA
,
ypbH
. We show in the present study that YpbH, like MecA, binds ClpC and that its elimination or overproduction affects competence and sporulation.
Sensitive and Specific Method for Rapid Identification of
Streptococcus pneumoniae
Using Real-Time Fluorescence PCR
Authors: McAvin James C. , Reilly Patricia A. , Roudabush Robert M. , Barnes William J. , Salmen Ann , Jackson Glen W. , Beninga Kathleen K. , Astorga Alicia , McCleskey Ferne K. , Huff William B. , Niemeyer Debra , Lohman Kenton L.
Published: July 2002

Molecular surveillance of pathogens has shown the need for rapid and dependable methods for the identification of organisms of clinical and epidemiological importance. As the leading cause of community-acquired pneumonia,
Streptococcus pneumoniae
was used as a model organism to develop and refine a real-time fluorescence PCR assay and enhanced DNA purification method. Seventy clinical isolates of
S. pneumoniae
, verified by latex agglutination
,
were screened against 26 negative control clinical isolates employing a TaqMan assay on a thermocycler (LightCycler). The probe, constructed from the
lytA
gene, correctly detected all
S. pneumoniae
genomes without cross-reaction to negative controls. The speed and ease of this approach will make it adaptable to identification of many bacterial pathogens and provide potential for adaptation to direct detection from patient specimens.
Turicella Otitidis
as an Unusual Agent Causing a Posterior Auricular Abscess
Authors: Reynolds Steven J. , Behr Marcel , McDonald Jane
Published: July 2002

A posterior auricular abscess in a 3-year-old girl was confirmed to have been caused by an unusual organism,
Turicella otitidis.
Improved Detection of Methicillin-Resistant
Staphylococcus aureus
Using Phenyl Mannitol Broth Containing Aztreonam and Ceftizoxime
Authors: Wertheim Heiman , Verbrugh Henri A. , van Pelt Cindy , de Man Peter , van Belkum Alex , Vos Margreet C.
Published: July 2002

We tested a phenyl mannitol broth containing ceftizoxime and aztreonam (PHMB
+
) for detection of methicillin-resistant
Staphylococcus aureus
(MRSA) with reference MRSA strains and, subsequently, with clinical samples (
n
= 1,098). All reference MRSA strains induced color change in PHMB
+
after 24 to 72 h of incubation. In a clinical setting, 40 MRSA strains were detected with PHMB
+
, compared with only 23 detected with a routine method. Thus, this selective broth significantly (
P
< 0.001) improved the rate of MRSA detection.
Genetic Diversity of
Pneumocystis carinii
Isolated from Human Immunodeficiency Virus-Positive Patients in Turin, Italy
Authors: Volpe Gisella , Sbaiz Luca , Avanzini Claudio , Caramello Pietro , Savoia Dianella
Published: July 2002

By DNA sequence analysis we identified two new strain types and five novel sporadic variations among 25 isolates of
Pneumocystis carinii
f. sp.
hominis
obtained from 19 human immunodeficiency virus-positive patients. Of these, 13 were infected with a single strain and 6 were coinfected. Fifteen different combination types were identified among the 18 strains for which complete molecular typing was accomplished.
Characterization of
Mycobacterium tuberculosis
Complex Isolates from the Cerebrospinal Fluid of Meningitis Patients at Six Fever Hospitals in Egypt
Authors: Cooksey Robert C. , Abbadi Said H. , Woodley Charles L. , Sikes David , Wasfy Momtaz , Crawford Jack T. , Mahoney Frank
Published: July 2002

Mycobacterium tuberculosis
complex isolates from cerebrospinal fluid of 67 meningitis patients were obtained from six fever hospitals in Egypt. One
M. bovis
and 66
M. tuberculosis
isolates were identified by PCR-restriction fragment length polymorphism (RFLP) analysis of
oxyR
. Among the
M. tuberculosis
isolates, 53 unique strain types (with 3 to 16 copies of IS
6110
) were found by RFLP analyses. Nine clusters (eight with two isolates each and one with six isolates) were also found. Thirty-six spoligotypes, including at least 10 that have been previously reported from other countries, were also observed. Forty-one (62.1%) of the isolates were in spoligotype clusters, and 22 (33%) of the isolates were in RFLP clusters. Fifty-one of the isolates were susceptible in vitro to all of the antituberculosis drugs tested, 11 were monoresistant to capreomycin, rifampin, isoniazid (INH), pyrazinamide, or streptomycin (STR), 4 were resistant to STR and INH, and 1 was resistant to STR, INH, and ethambutol.
Human Immunodeficiency Virus (HIV)-Positive Sera Obtained Shortly after Seroconversion Neutralize Autologous HIV Type 1 Isolates on Primary Macrophages but Not on Lymphocytes
Authors: Ruppach Horst , Nara Peter , Raudonat Ina , Elanjikal Ziju , Rübsamen-Waigmann Helga , Dietrich Ursula
Published: July 2002

The aim of this study was to analyze the role of humoral immunity in early human immunodeficiency virus (HIV) infection. As neutralizing activities in HIV-positive sera are rarely detectable earlier than 9 to 12 months after infection using primary lymphocytes as target cells in neutralization assays, humoral immunity is generally thought not to contribute significantly to early virus control in the patients. Besides lymphocytes, cells of the monocyte/macrophage lineage are known to be important target cells for HIV in vivo during the establishment of the infection. Therefore, we studied the neutralization of early primary HIV isolates by autologous serum samples using primary macrophages as target cells in the neutralization assays. We analyzed neutralizing activities against the autologous HIV-1 isolates in 10 patients' sera taken shortly after seroconversion, both on primary macrophages and, for comparison, on lymphocytes. Viruses were isolated and expanded in primary mixed cultures containing macrophages and lymphocytes in order to avoid selection for one particular cell type. All viruses replicated to different degrees in macrophages and lymphocytes; nine had a nonsyncytium-inducing phenotype, and one was syncytium inducing. The detection of neutralizing antibodies in acute primary HIV infection depended on the target cells used. Confirming previous studies, we did not find neutralizing activities on lymphocytes at this early time point. In contrast, neutralizing activities were detectable in the same sera if primary macrophages were used as target cells. Differences in neutralizing activities on macrophages and lymphocytes were not due to different virus variants being present in the different cell systems, as gp120 sequences derived from both cell types were homogeneous. Neutralization activities on macrophages did not correlate with the amount of β-chemokines in the sera. As affinity-purified immunoglobulin G preparations from an early patient serum also exhibited neutralization of the autologous virus isolate on primary macrophages, but not on lymphocytes, neutralization is very likely due to antibodies against viral epitopes necessary for infection of macrophages but not for infection of lymphocytes. Our data suggest that, along with cell-mediated immunity, humoral immunity may contribute to the reduction of primary viremia in the patient. This was further supported by a certain association between neutralizing antibody titers on macrophages and viral load in the patients.
Avian Retrovirus DNA Internal Attachment Site Requirements for Full-Site Integration In Vitro
Authors: Chiu Roger , Grandgenett Duane P.
Published: July 2002

Concerted integration of retrovirus DNA termini into the host chromosome in vivo requires specific interactions between the
cis
-acting attachment (
att
) sites at the viral termini and the viral integrase (IN) in
trans
. In this study, reconstruction experiments with purified avian myeloblastosis virus (AMV) IN and retrovirus-like donor substrates containing wild-type and mutant termini were performed to map the internal
att
DNA sequence requirements for concerted integration, here termed full-site integration. The avian retrovirus mutations were modeled after internal
att
site mutations studied at the in vivo level with human immunodeficiency virus type 1 (HIV-1) and murine leukemia virus (MLV). Systematic overlapping 4-bp deletions starting at nucleotide positions 7, 8, and 9 in the U3 terminus had a decreasing detrimental gradient effect on full-site integration, while more internal 4-bp deletions had little or no effect. This decreasing detrimental gradient effect was measured by the ability of mutant U3 ends to interact with wild-type U3 ends for full-site integration in
trans
. Modification of the highly conserved C at position 7 on the catalytic strand to either A or T resulted in the same severe decrease in full-site integration as the 4-bp deletion starting at this position. These studies suggest that nucleotide position 7 is crucial for interactions near the active site of IN for integration activity and for communication in
trans
between ends bound by IN for full-site integration. The ability of AMV IN to interact with internal
att
sequences to mediate full-site integration in vitro is similar to the internal
att
site requirements observed with MLV and HIV-1 in vivo and with their preintegration complexes in vitro.
Human Immunodeficiency Virus Type 1 Nucleocapsid Protein Can Prevent Self-Priming of Minus-Strand Strong Stop DNA by Promoting the Annealing of Short Oligonucleotides to Hairpin Sequences
Authors: Driscoll Mark D. , Hughes Stephen H.
Published: July 2002

Understanding how viral components collaborate to convert the human immunodeficiency virus type 1 genome from single-stranded RNA into double-stranded DNA is critical to the understanding of viral replication. Not only must the correct reactions be carried out, but unwanted side reactions must be avoided. After minus-strand strong stop DNA (−sssDNA) synthesis, degradation of the RNA template by the RNase H domain of reverse transcriptase (RT) produces single-stranded DNA that has the potential to self-prime at the imperfectly base-paired TAR hairpin, making continued DNA synthesis impossible. Although nucleocapsid protein (NC) interferes with −sssDNA self-priming in reverse transcription reactions in vitro, NC alone did not prevent self-priming of a synthetic −sssDNA oligomer. NC did not influence DNA bending and therefore cannot inhibit self-priming at hairpins by directly blocking hairpin formation. Using DNA oligomers as a model for genomic RNA fragments, we found that a 17-base DNA fragment annealed to the 3′ end of the −sssDNA prevented self-priming in the presence of NC. This implies that to avoid self-priming, an RNA-DNA hybrid that is more thermodynamically stable than the hairpin must remain within the hairpin region. This suggests that NC prevents self-priming by generating or stabilizing a thermodynamically favored RNA-DNA heteroduplex instead of the kinetically favored TAR hairpin. In support of this idea, sequence changes that increased base pairing in the DNA TAR hairpin resulted in an increase in self-priming in vitro. We present a model describing the role of NC-dependent inhibition of self-priming in first-strand transfer.
Intracellular Forms of Human
NOTCH1
Functionally Activate Essential Epstein-Barr Virus Major Latent Promoters in the Burkitt's Lymphoma BJAB Cell Line but Repress These Promoters in Jurkat Cells
Authors: Cotter Murray , Callahan Jennifer , Aster Jon , Robertson Erle
Published: July 2002

We have demonstrated that intracellular forms of
NOTCH1
transactivate two major Epstein-Barr virus (EBV) latent promoters, the LMP1 and Cp1 promoters in an EBV-negative B-cell line, BJAB. Truncated intracellular
NOTCH1
associated with the nuclear membrane (ΔE) transactivates the LMP1 promoter fivefold; however, the intranucleus localized form of
NOTCH1
(ICN) transactivates this promoter approximately twofold in chloroamphenicol acetyltransferase (CAT) reporter assays in BJAB cells. Additionally, ΔE activated the major Cp1 promoter 12-fold, whereas the ICN form of
NOTCH1
activates at only about half that level when compared to that of ΔE membrane-bound
NOTCH1
. This result differs from previously observed data, where intracellular
NOTCH1
bound to the nuclear membrane, ΔE, and nucleus-localized
NOTCH1
, ICN, all had similar levels of activation in 293 cells. This suggests distinct transcriptional activities in different cell types. Moreover, in Jurkat cells, a T-cell line, intranucleus localized
NOTCH1
molecules demonstrated a repressive activity against the two EBV major latent promoters. Only ΔE activated the Cp1 and LMP1 promoters at a level slightly above background, whereas intranucleus localized
NOTCH1
ICN, or the form of
NOTCH1
lacking the ankyrin repeats, ΔE
TAR
, surprisingly resulted in the repression of these promoters in Jurkat cells. Similarly, another truncated form of
NOTCH1
, referred to as ICNW, which contains the tryptophan residue W
1767
within one of the RBP-Jκ interacting domains, repressed the LMP1 promoter approximately twofold. Further analysis of the truncated
NOTCH1
molecules on the LMP1 promoter element, lacking the two RBP-Jκ binding sites, suggests that repression in Jurkat cells may be affected by the presence of the two RBP-Jκ binding sites. These studies indicate that intracellular
NOTCH1
can activate the EBV major latent promoters in BJAB cells. However, in Jurkat cells, intracellular truncated forms of
NOTCH1
lacking the RBP-Jκ binding sites repress these EBV latent promoters. Only the membrane-bound form of
NOTCH1
, ΔE, activated the EBV major latent promoters in Jurkat cells, albeit at a lower level than that seen in BJAB cells. Our data suggest that EBNA2 and truncated intracellular nuclear localized forms of
NOTCH1
may be functionally similar in their interactions with RBP-Jκ; however, these molecules may have distinctly different transcriptional partners in BJAB and Jurkat cells. Moreover, these truncated
NOTCH1
molecules may not represent the normal processed forms of
NOTCH1
in cells and may exhibit dominant negative phenotypes in the absence of the required posttranslational modifications. Further investigations are necessary to determine the similarity and differences occurring with intracellular
NOTCH1
in other B- and T-cell lines.
Disruption of the Murine Gammaherpesvirus 68 M1 Open Reading Frame Leads to Enhanced Reactivation from Latency
Authors: Clambey Eric T. , Virgin Herbert W. , Speck Samuel H.
Published: July 2002

Murine gammaherpesvirus 68 (γHV68, or MHV-68) is a genetically tractable, small animal model for the analysis of gammaherpesvirus pathogenesis. The γHV68 genome is colinear with the genomes of other sequence gammaherpesviruses, containing large blocks of conserved genes interspersed by a number of putative genes without clear homologs in the other gammaherpesviruses. One of these putative unique genes, the M1 open reading frame (ORF), exhibits sequence homology to a poxvirus serine protease inhibitor, SPI-1, as well as to another γHV68 gene, M3, which we have recently shown encodes an abundantly secreted chemokine binding protein. To assess the contribution of the M1 ORF to γHV68 pathogenesis, we have generated a recombinant γHV68 in which the M1 ORF has been disrupted through targeted insertion of a
lacZ
expression cassette (M1.LacZ). Although M1.LacZ replicated normally in tissue culture, it exhibited decreased splenic titers at days 4 and 9 postinfection in both immunocompetent and immunodeficient mice. Despite decreased levels of acute virus replication, M1.LacZ established a latent infection comparable to wild-type (wt) γHV68, but exhibited an approximately fivefold increase in efficiency of reactivation from latency. M1.LacZ also caused severe vasculitis of the great elastic arteries in gamma interferon receptor (IFN-γR)-deficient mice with a frequency comparable to wt γHV68, but did not cause the mortality or splenic pathology observed with wt γHV68 infection of IFN-γR-deficient mice. Restoration of M1 ORF sequences into M1.LacZ (M1 marker rescue, or M1.MR) demonstrated that M1.LacZ phenotypic alterations in growth in vivo and latency were not due to the presence of additional mutations located elsewhere in the M1.LacZ genome. Generation of a second M1 mutant virus containing a deletion at the 5′ end of the M1 ORF (M1Δ511), but lacking the LacZ expression cassette, revealed the same latency phenotype observed with the M1.LacZ mutant. However, M1Δ511 was not attenuated for acute virus replication in the spleen. We conclude that (i) the induction of arteritis in γHV68-infected IFN-γR-deficient mice can occur in the absence of splenic pathology and mortality, (ii) replication during acute infection is not the primary determinant for the establishment of latent infection, and (iii) the M1 ORF, or a closely linked gene, encodes a gene product that functions to suppress virus reactivation.
The Extracellular Domain of Herpes Simplex Virus gE Is Sufficient for Accumulation at Cell Junctions but Not for Cell-to-Cell Spread
Authors: Wisner Todd , Brunetti Craig , Dingwell Kevin , Johnson David C.
Published: July 2002

Herpes simplex virus (HSV) expresses a number of membrane glycoproteins, including gB, gD, and gH/gL, that function in both entry of virus particles and movement of virus from an infected cell to an uninfected cell (cell-to-cell spread). However, a complex of HSV glycoproteins gE and gI (gE/gI) is required for efficient cell-to-cell spread, especially between cells that form extensive cell junctions, yet it is not necessary for entry of extracellular virions. We previously showed that gE/gI has the capacity to localize specifically to cell junctions; the glycoprotein complex was found at lateral surfaces of cells in contact with other cells but not at those lateral surfaces not forming junctions or at apical surfaces. By virtue of these properties, gE/gI is an important molecular handle on the poorly understood process of cell-to-cell spread. Here, we show that the cytoplasmic domain of gE is important for the proper delivery of gE/gI to lateral surfaces of cells. Without this domain, gE/gI is found on the apical surface of epithelial cells, and more uniformly in the cytoplasm, although incorporation into the virion envelope is unaffected. However, even without proper trafficking signals, a substantial fraction of gE/gI retained the capacity to accumulate at cell junctions. Therefore, the extracellular domain of gE can mediate accumulation of gE/gI at cell junctions, if the glycoprotein can be delivered there, probably through interactions with ligands on the opposing cell. The role of phosphorylation of the cytoplasmic domain of gE was also studied. A second mutant HSV type 1 was constructed in which three serine residues that form a casein kinase II phosphorylation site were changed to alanine residues, reducing phosphorylation by 70 to 80%. This mutation did not affect accumulation at cell junctions or cell-to-cell spread.
Sequence Heterogeneity of TT Virus and Closely Related Viruses
Authors: Khudyakov Yury E. , Cong Mian-er , Nichols Barbara , Reed Deoine , Dou Xiao-Guang , Viazov Sergei O. , Chang Joy , Fried Michael W. , Williams Ian , Bower William , Lambert Stephen , Purdy Michael , Roggendorf Michael , Fields Howard A.
Published: July 2002

TT virus (TTV) is a recently discovered infectious agent originally obtained from transfusion-related hepatitis. However, the causative link between the TTV infection and liver disease remains uncertain. Recent studies demonstrated that genome sequences of different TTV strains are significantly divergent. To assess genetic heterogeneity of the TTV genome in more detail, a sequence analysis of PCR fragments (271 bp) amplified from open reading frame 1 (ORF1) was performed. PCR fragments were amplified from 5 to 40% of serum specimens obtained from patients with different forms of hepatitis who reside in different countries (e.g., China, Egypt, Vietnam, and the United States) and from normal human specimens obtained from U.S. residents. A total of 170 PCR fragments were sequenced and compared to sequences derived from the corresponding TTV genome region deposited in GenBank. Genotypes 2 and 3 were found to be significantly more genetically related than any other TTV genotype. Moreover, three sequences were shown to be almost equally related to both genotypes 2 and 3. These observations suggest a merger of genotypes 2 and 3 into one genotype, 2/3. Additionally, five new groups of TTV sequences were identified. One group represents a new genotype, whereas the other four groups were shown to be more evolutionary distant from all known TTV sequences. The evolutionary distances between these four groups were also shown to be greater than between TTV genotypes. The phylogenetic analysis suggested that these four new genetic groups represent closely related yet different viral species. Thus, TTV exists as a “swarm” of at least five closely related but different viruses. These observations suggest a high degree of genetic complexity within the TTV population. The finding of the additional TTV-related species should be taken into consideration when the association between TTV infections and human diseases of unknown etiology is studied.
Protective Immunity in Macaques Vaccinated with a Modified Vaccinia Virus Ankara-Based Measles Virus Vaccine in the Presence of Passively Acquired Antibodies
Authors: Stittelaar Koert J. , Wyatt Linda S. , de Swart Rik L. , Vos Helma W. , Groen Jan , van Amerongen Geert , van Binnendijk Robert S. , Rozenblatt Shmuel , Moss Bernard , Osterhaus Albert D. M. E.
Published: July 2002

Recombinant modified vaccinia virus Ankara (MVA), encoding the measles virus (MV) fusion (F) and hemagglutinin (H) (MVA-FH) glycoproteins, was evaluated in an MV vaccination-challenge model with macaques. Animals were vaccinated twice in the absence or presence of passively transferred MV-neutralizing macaque antibodies and challenged 1 year later intratracheally with wild-type MV. After the second vaccination with MVA-FH, all the animals developed MV-neutralizing antibodies and MV-specific T-cell responses. Although MVA-FH was slightly less effective in inducing MV-neutralizing antibodies in the absence of passively transferred antibodies than the currently used live attenuated vaccine, it proved to be more effective in the presence of such antibodies. All vaccinated animals were effectively protected from the challenge infection. These data suggest that MVA-FH should be further tested as an alternative to the current vaccine for infants with maternally acquired MV-neutralizing antibodies and for adults with waning vaccine-induced immunity.
Natural 2′,5′-Phosphodiester Bonds Found at the Ligation Sites of Peach Latent Mosaic Viroid
Authors: Côté Fabien , Lévesque Dominique , Perreault Jean-Pierre
Published: July 2002

Peach latent mosaic viroid (PLMVd) is a circular RNA pathogen that replicates in a DNA-independent fashion via a rolling circle mechanism. PLMVd has been shown to self-ligate in vitro primarily via the formation of 2′,5′-phosphodiester bonds; however, in vivo the occurrence and necessity of this nonenzymatic mechanism are not evident. Here, we unequivocally report the presence of 2′,5′-phosphodiester bonds at the ligation site of circular PLMVd strands isolated from infected peach leaves. These bonds serve to close the linear conformers (i.e., intermediates), yielding circular ones. Furthermore, these bonds are shown to stabilize the replicational circular templates, resulting in a significant advantage in terms of viroid viability. Although the mechanism responsible for the formation of these 2′,5′-phosphodiester bonds remains to be elucidated, a hypothesis describing in vivo nonenzymatic self-ligation is proposed. Most significantly, our results clearly show that 2′,5′-phosphodiester bonds are still present in nature and that they are of biological importance.
Sequences in Influenza A Virus PB2 Protein That Determine Productive Infection for an Avian Influenza Virus in Mouse and Human Cell Lines
Authors: Yao Yongxiu , Mingay Louise J. , McCauley John W. , Barclay Wendy S.
Published: July 2002

Reverse genetics was used to analyze the host range of two avian influenza viruses which differ in their ability to replicate in mouse and human cells in culture. Engineered viruses carrying sequences encoding amino acids 362 to 581 of PB2 from a host range variant productively infect mouse and human cells.
Experimental Preemptive Immunotherapy of Murine Cytomegalovirus Disease with CD8 T-Cell Lines Specific for ppM83 and pM84, the Two Homologs of Human Cytomegalovirus Tegument Protein ppUL83 (pp65)
Authors: Holtappels Rafaela , Podlech Jürgen , Grzimek Natascha K. A. , Thomas Doris , Pahl-Seibert Marcus-Folker , Reddehase Matthias J.
Published: July 2002

CD8 T cells are the principal antiviral effectors controlling cytomegalovirus (CMV) infection. For human CMV, the virion tegument protein ppUL83 (pp65) has been identified as a source of immunodominant peptides and is regarded as a candidate for cytoimmunotherapy and vaccination. Two sequence homologs of ppUL83 are known for murine CMV, namely the virion protein ppM83 (pp105) expressed late in the viral replication cycle and the nonstructural protein pM84 (p65) expressed in the early phase. Here we show that ppM83, unlike ppUL83, is not delivered into the antigen presentation pathway after virus penetration before or in absence of viral gene expression, while other virion proteins of murine CMV are processed along this route. In cytokine secretion-based assays, ppM83 and pM84 appeared to barely contribute to the acute immune response and to immunological memory. Specifically, the frequencies of M83 and M84 peptide-specific CD8 T cells were low and undetectable, respectively. Nonetheless, in a murine model of cytoimmunotherapy of lethal CMV disease, M83 and M84 peptide-specific cytolytic T-cell lines proved to be highly efficient in resolving productive infection in multiple organs of cell transfer recipients. These findings demonstrate that proteins which fail to prime a quantitatively dominant immune response can nevertheless represent relevant antigens in the effector phase. We conclude that quantitative and qualitative immunodominance are not necessarily correlated. As a consequence of these findings, there is no longer a rationale for considering T-cell abundance as the key criterion for choosing specificities to be included in immunotherapy and immunoprophylaxis of CMV disease and of viral infections in general.
The R Region Found in the Human Foamy Virus Long Terminal Repeat Is Critical for both Gag and Pol Protein Expression
Authors: Russell Rebecca A. , Zeng Yan , Erlwein Otto , Cullen Bryan R. , McClure Myra O.
Published: July 2002

It has been suggested that sequences located within the 5′ noncoding region of human foamy virus (HFV) are critical for expression of the viral Gag and Pol structural proteins. Here, we identify a discrete ∼151-nucleotide sequence, located within the R region of the HFV long terminal repeat, that activates HFV Gag and Pol expression when present in the 5′ noncoding region but that is inactive when inverted or when placed in the 3′ noncoding region. Sequences that are critical for the expression of both Gag and Pol include not only the 5′ splice site positioned at +51 in the R region, which is used to generate the spliced
pol
mRNA, but also intronic R sequences located well 3′ to this splice site. Analysis of total cellular
gag
and
pol
mRNA expression demonstrates that deletion of the R region has little effect on
gag
mRNA levels but that R deletions that would be predicted to leave the
pol
5′ splice site intact nevertheless inhibit the production of the spliced
pol
mRNA. Gag expression can be largely rescued by the introduction of an intron into the 5′ noncoding sequence in place of the R region but not by an intron or any one of several distinct retroviral nuclear RNA export sequences inserted into the mRNA 3′ noncoding sequence. Neither the R element nor the introduced 5′ intron markedly affects the cytoplasmic level of HFV
gag
mRNA. The poor translational utilization of these cytoplasmic mRNAs when the R region is not present in
cis
also extended to a
cat
indicator gene linked to an internal ribosome entry site introduced into the 3′ noncoding region. Together these data imply that the HFV R region acts in the nucleus to modify the cytoplasmic fate of target HFV mRNA. The close similarity between the role of the HFV R region revealed in this study and previous data (M. Butsch, S. Hull, Y. Wang, T. M. Roberts, and K. Boris-Lawrie, J. Virol. 73:4847–4855, 1999) demonstrating a critical role for the R region in activating gene expression in the unrelated retrovirus spleen necrosis virus suggests that several distinct retrovirus families may utilize a common yet novel mechanism for the posttranscriptional activation of viral structural protein expression.
Stability of the Human Papillomavirus Type 18 E2 Protein Is Regulated by a Proteasome Degradation Pathway through Its Amino-Terminal Transactivation Domain
Authors: Bellanger Sophie , Demeret Caroline , Goyat Sylvain , Thierry Françoise
Published: July 2002

The E2 proteins of papillomaviruses regulate both viral transcription and DNA replication. The human papillomavirus type 18 (HPV18) E2 protein has been shown to repress transcription of the oncogenic E6 and E7 genes, inducing growth arrest in HeLa cells. Using HPV18 E2 fused to the green fluorescent protein (GFP), we showed that this protein was short-lived in transfected HeLa cells. Real-time microscopy experiments indicated that the E2-dependent signal increased for roughly 24 h after transfection and then rapidly disappeared, indicating that E2 was unstable in HeLa cells and could confer instability to GFP. Similar studies done with a protein lacking the transactivation domain indicated that this truncation strongly stabilizes the E2 protein. In vitro, full-length E2 or the transactivation domain alone was efficiently ubiquitinated, whereas deletion of the transactivation domain strongly decreased the ubiquitination of the E2 protein. Proteasome inhibition in cells expressing E2 increased its half-life about sevenfold, which was comparable to the half-life of the amino-terminally truncated protein. These characteristics of E2 instability were independent of the E2-mediated G
1
growth arrest in HeLa cells, as they were reproduced in MCF7 cells, where E2 does not affect the cell cycle. Altogether, these experiments showed that the HPV18 E2 protein was degraded by the ubiquitin-proteasome pathway through its amino-terminal transactivation domain. Tight regulation of the stability of the HPV 18 E2 protein may be essential to avoid accumulation of a potent transcriptional repressor and antiproliferative agent during the viral vegetative cycle.
Neutralizing Antibodies Associated with Viremia Control in a Subset of Individuals after Treatment of Acute Human Immunodeficiency Virus Type 1 Infection
Authors: Montefiori David C. , Hill Tanya S. , Vo Ha T. T. , Walker Bruce D. , Rosenberg Eric S.
Published: July 2002

Immediate treatment of acute human immunodeficiency virus type 1 (HIV-1) infection has been associated with subsequent control of viremia in a subset of patients after therapy cessation, but the immune responses contributing to control have not been fully defined. Here we examined neutralizing antibodies as a correlate of viremia control following treatment interruption in HIV-1-infected individuals in whom highly active antiretriviral therapy (HAART) was initiated during early seroconversion and who remained on therapy for 1 to 3 years. Immediately following treatment interruption, neutralizing antibodies were undetectable with T-cell-line adapted strains and the autologous primary HIV-1 isolate in seven of nine subjects. Env- and Gag-specific antibodies as measured by enzyme-linked immunosorbent assay were also low or undetectable at this time. Despite this apparent poor maturation of the virus-specific B-cell response during HAART, autologous neutralizing antibodies emerged rapidly and correlated with a spontaneous downregulation in rebound viremia following treatment interruption in three subjects. Control of rebound viremia was seen in other subjects in the absence of detectable neutralizing antibodies. The results indicate that virus-specific B-cell priming occurs despite the early institution of HAART, allowing rapid secondary neutralizing-antibody production following treatment interruption in a subset of individuals. Since early HAART limits viral diversification, we hypothesize that potent neutralizing-antibody responses to autologous virus are able to mature and that in some persons these responses contribute to the control of plasma viremia after treatment cessation.
The Transgenic ICP4 Promoter Is Activated in Schwann Cells in Trigeminal Ganglia of Mice Latently Infected with Herpes Simplex Virus Type 1
Authors: Taus Naomi S. , Mitchell William J.
Published: July 2002

Herpes simplex virus type 1 (HSV-1) establishes a latent infection in neurons of sensory ganglia, including those of the trigeminal ganglia. Latent viral infection has been hypothesized to be regulated by restriction of viral immediate-early gene expression in neurons. Numerous in situ hybridization studies in mice and in humans have shown that transcription from the HSV-1 genome in latently infected neurons is limited to the latency-associated transcripts. In other studies, immediate-early gene (ICP4) transcripts have been detected by reverse transcription-PCR (RT-PCR) in homogenates of latently infected trigeminal ganglia of mice. We used reporter transgenic mice containing the HSV-1(F) ICP4 promoter fused to the coding sequence of the β-galactosidase gene to determine whether neurons in latently infected trigeminal ganglia activated the ICP4 promoter. Mice were inoculated via the corneal route with HSV-1(F). At 5, 11, 23, and 37 days postinfection (dpi), trigeminal ganglia were examined for β-galactosidase-positive cells. The numbers of β-galactosidase-positive neurons and nonneuronal cells were similar at 5 dpi. The number of positive neurons decreased at 11 dpi and returned to the level of mock-inoculated transgenic controls at 23 and 37 dpi. The number of positive nonneuronal cells increased at 11 and 23 dpi and remained elevated at 37 dpi. Viral proteins were detected in neurons and nonneuronal cells in acutely infected ganglia, but were not detected in latently infected ganglia. Colabeling experiments confirmed that the transgenic ICP4 promoter was activated in Schwann cells during latent infection. These findings suggest that the cells that express the HSV-1 ICP4 gene in latently infected ganglia are not neurons.
Role for the Adenovirus IVa2 Protein in Packaging of Viral DNA
Authors: Zhang Wei , Low Jonathan A. , Christensen Joan B. , Imperiale Michael J.
Published: July 2002

Although it has been demonstrated that the adenovirus IVa2 protein binds to the packaging domains on the viral chromosome and interacts with the viral L1 52/55-kDa protein, which is required for viral DNA packaging, there has been no direct evidence demonstrating that the IVa2 protein is involved in DNA packaging. To understand in greater detail the DNA packaging mechanisms of adenovirus, we have asked whether DNA packaging is serotype or subgroup specific. We found that Ad7 (subgroup B), Ad12 (subgroup A), and Ad17 (subgroup D) cannot complement the defect of an Ad5 (subgroup C) mutant,
pm
8001, which does not package its DNA due to a mutation in the L1 52/55-kDa gene. This indicates that the DNA packaging systems of different serotypes cannot interact productively with Ad5 DNA. Based on this, a chimeric virus containing the Ad7 genome except for the inverted terminal repeats and packaging sequence from Ad5 was constructed. This chimeric virus replicates its DNA and synthesizes Ad7 proteins, but it cannot package its DNA in 293 cells or 293 cells expressing the Ad5 L1 52/55-kDa protein. However, this chimeric virus packages its DNA in 293 cells expressing the Ad5 IVa2 protein. These results indicate that the IVa2 protein plays a role in viral DNA packaging and that its function is serotype specific. Since this chimeric virus cannot package its own DNA, but produces all the components for packaging Ad7 DNA, it may be a more suitable helper virus for the growth of Ad7 gutted vectors for gene transfer.
Poliovirus 5′-Terminal Cloverleaf RNA Is Required in
cis
for VPg Uridylylation and the Initiation of Negative-Strand RNA Synthesis
Authors: Lyons Traci , Murray Kenneth E. , Roberts Allan W. , Barton David J.
Published: July 2002

Chimeric poliovirus RNAs, possessing the 5′ nontranslated region (NTR) of hepatitis C virus in place of the 5′ NTR of poliovirus, were used to examine the role of the poliovirus 5′ NTR in viral replication. The chimeric viral RNAs were incubated in cell-free reaction mixtures capable of supporting the sequential translation and replication of poliovirus RNA. Using preinitiation RNA replication complexes formed in these reactions, we demonstrated that the 3′ NTR of poliovirus RNA was insufficient, by itself, to recruit the viral replication proteins required for negative-strand RNA synthesis. The 5′-terminal cloverleaf of poliovirus RNA was required in
cis
to form functional preinitiation RNA replication complexes capable of uridylylating VPg and initiating the synthesis of negative-strand RNA. These results are consistent with a model in which the 5′-terminal cloverleaf and 3′ NTRs of poliovirus RNA interact via temporally dynamic ribonucleoprotein complexes to coordinately mediate and regulate the sequential translation and replication of poliovirus RNA.
Transduction of Cellular Sequence by a Human Immunodeficiency Virus Type 1-Derived Vector
Authors: Sun Guoli , O'Neil Patrick K. , Yu Hong , Ron Yacov , Preston Bradley D. , Dougherty Joseph P.
Published: July 2002

During studies examining the rate of human immunodeficiency virus type 1 (HIV-1) mutation in a single cycle of replication, the 5′ long terminal repeat of one progeny provirus was found to contain an insertion of 147 bp including an entire tRNA

3

Lys

sequence as well as an additional 66 bp insertion of nonviral origin. Database searches revealed that 65 of 66 bp aligned with the human CpG island sequence found on chromosomes 6, 14, and 17. Therefore it seems probable that it is of human cellular sequence origin and was transduced by HIV-1. This is the first demonstration that HIV-1 can capture a cellular sequence. The site of integration of the parental provirus was mapped to chromosome 1p32.1. Sequence with homology to the transduced CpG island was not found on chromosome 1, suggesting that the transduced cellular sequence was not linked to the site of viral integration.
Nucleocytoplasmic Shuttling by Human Immunodeficiency Virus Type 1 Vpr
Authors: Sherman Michael P. , de Noronha Carlos M. C. , Heusch Marina I. , Greene Spencer , Greene Warner C.
Published: July 2002

Human immunodeficiency virus type 1 (HIV-1) is capable of infecting nondividing cells such as macrophages because the viral preintegration complex is able to actively traverse the limiting nuclear pore due to the redundant and possibly overlapping nuclear import signals present in Vpr, matrix, and integrase. We have previously recognized the presence of at least two distinct and novel nuclear import signals residing within Vpr that, unlike matrix and integrase, bypass the classical importin α/β-dependent signals and do not require energy or a RanGTP gradient. We now report that the carboxy-terminal region of Vpr (amino acids 73 to 96) contains a bipartite nuclear localization signal (NLS) composed of multiple arginine residues. Surprisingly, when the leucine-rich Vpr(1–71) fragment, previously shown to harbor an NLS, or full-length Vpr is fused to the C terminus of a green fluorescent protein-pyruvate kinase (GFP-PK) chimera, the resultant protein is almost exclusively detected in the cytoplasm. However, the addition of leptomycin B (LMB), a potent inhibitor of CRM1-dependent nuclear export, produces a shift from a cytoplasmic localization to a nuclear pattern, suggesting that these Vpr fusion proteins shuttle into and out of the nucleus. Studies of nuclear import with GFP-PK–Vpr fusion proteins in the presence of LMB reveals that both of the leucine-rich α-helices are required for effective nuclear uptake and thus define a unique NLS. Using a modified heterokaryon analysis, we have localized the Vpr nuclear export signal to the second leucine-rich helix, overlapping a portion of the amino-terminal nuclear import signal. These studies thus define HIV-1 Vpr as a nucleocytoplasmic shuttling protein.