bluuebunny/crossref_metadata_2025_split huggingface dataset:
Functional Divergence of Kaposi's Sarcoma-Associated Herpesvirus and Related Gamma-2 Herpesvirus Thymidine Kinases: Novel Cytoplasmic Phosphoproteins That Alter Cellular Morphology and Disrupt Adhesion
Authors: Gill Michael B. , Murphy Jo-Ellen , Fingeroth Joyce D. 
Published: November 2005

ABSTRACT
The
nucleoside kinase encoded by Kaposi's sarcoma-associated herpesvirus
(KSHV) is a relatively inefficient enzyme with substrate specificity
for thymidine alone, unlike alphaherpesvirus thymidine kinases (TKs).
Similar to all gammaherpesvirus TKs, KSHV TK is composed of
two distinct domains, a conserved C-terminal kinase and a novel and
uncharacterized N terminus. Ectopic expression of KSHV TK in adherent
cells induced striking morphological changes and anchorage independence
although cells survived, a property shared with the related
rhadinovirus TKs of rhesus monkey rhadinovirus and herpesvirus saimiri.
To determine whether KSHV TK served alternate functions relevant to the
rhadinovirus life cycle and to reveal the contribution of the N
terminus, an enhanced green fluorescent protein-tagged fusion protein
and serial mutants were generated for investigation of intracellular
localization and cell biology. Analysis of truncation mutants showed
that a proline-rich region located within the N terminus cooperated
with the conserved C-terminal kinase to tether KSHV TK to a reticular
network in the cytoplasm and to induce morphological change. Fusion of
the KSHV N terminus to herpes simplex virus type 1 TK, a
nucleus-localized enzyme, similarly resulted in cytoplasmic
redistribution of the chimeric protein but did not alter cell shape or
adhesion. Unlike other human herpesvirus TKs, KSHV TKs and related
rhadinovirus TKs are constitutively tyrosine phosphorylated; a KSHV TK
mutant that was hypophosphorylated failed to detach and grow in
suspension. Loss of adhesion may enhance terminal differentiation,
viral replication, and egress at the cellular level and at the organism
level may facilitate detachment and distant migration of
KSHV-replicating cells within body fluids—promoting
oropharyngeal transmission and perhaps contributing to the multifocal
lesions that characterize KS.
Interaction of the Putative Human Cytomegalovirus Portal Protein pUL104 with the Large Terminase Subunit pUL56 and Its Inhibition by Benzimidazole-
d
-Ribonucleosides
Authors: Dittmer Alexandra , Drach John C. , Townsend Leroy B. , Fischer Anke , Bogner Elke 
Published: November 2005

ABSTRACT

Herpesvirus DNA replication leads to unit length genomes that are translocated into preformed procapsids through a unique portal vertex. The translocation is performed by the terminase that cleaves the DNA and powers the insertion by its ATPase activity. Recently, we demonstrated that the putative human cytomegalovirus (HCMV) portal protein, pUL104, also forms high-molecular-weight complexes. Analyses now have been performed to determine the intracellular localization and identification of interaction partners of pUL104. In infected cells, HCMV pUL104 was found to be predominantly localized throughout the nucleus as well as in cytoplasmic clusters at late times of infection. The latter localization was abolished by phosphonoacetic acid, an inhibitor of viral DNA replication. Immunofluorescence revealed that pUL104 colocalized with pUL56, the large subunit of the HCMV terminase. Specific association of in vitro translated pUL104 with the carboxy-terminal half of GST-UL56C was detected. By using coimmunoprecipitations a direct interaction with pUL56 was confirmed. In addition, this interaction was no longer detected when the benzimidazole-
d
-nucleosides BDCRB or Cl
4
RB were added, thus indicating that these HCMV inhibitors block the insertion of the DNA into the capsid by preventing a necessary interaction of pUL56 with the portal. Electron microscopy revealed that in the presence of Cl
4
RB DNA is not packaged into capsids and these capsids failed to egress from the nucleus. Furthermore, pulsed-field gel electrophoresis showed that DNA concatemers synthesized in the presence of the compound failed to be processed.
Invasion and Maintenance of Dengue Virus Type 2 and Type 4 in the Americas
Authors: Carrington Christine V. F. , Foster Jerome E. , Pybus Oliver G. , Bennett Shannon N. , Holmes Edward C. 
Published: November 2005

ABSTRACT
Dengue virus type 4 (DENV-4) was first reported in the Americas in 1981, where it caused epidemics of dengue fever throughout the region. In the same year, the region's first epidemic of dengue hemorrhagic fever was reported, caused by an Asian strain of dengue virus type 2 (DENV-2) that was distinct from the American subtype circulating previously. Despite the importance of these epidemics, little is known about the rates or determinants of viral spread among island and mainland populations or their directions of movement. We employed a Bayesian coalescent approach to investigate the transmission histories of DENV-2 and DENV-4 since their introduction in 1981 and a parsimony method to assess patterns of strain migration. For both viruses there was an initial invasion phase characterized by an exponential increase in the number of DENV lineages, after which levels of genetic diversity remained constant despite reported fluctuations in DENV-2 and DENV-4 activity. Strikingly, viral lineage numbers increased far more rapidly for DENV-4 than DENV-2, indicative of a more rapid rate of exponential population growth in DENV-4 or a higher rate of geographic dispersal, allowing this virus to move more effectively among localities. We propose that these contrasting dynamics may reflect underlying differences in patterns of host immunity. Despite continued gene flow along particular transmission routes, the overall extent of viral traffic was less than expected under panmixis. Hence, DENV in the Americas has a clear geographic structure that maintains viral diversity between outbreaks.
Antigen Expression Kinetics and Immune Responses of Mice Immunized with Noninfectious Simian-Human Immunodeficiency Virus DNA
Authors: Hegde Ramakrishna , Liu ZhenQian , Mackay Glenn , Smith Marilyn , Chebloune Yahia , Narayan Opendra , Singh Dinesh K. 
Published: November 2005

ABSTRACT

In a previous report we demonstrated that three injections of an
rt
-deleted noninfectious genome of the simian-human immunodeficiency virus SHIV
KU2
induced protection against AIDS in macaques (D. K. Singh, Z. Liu, D. Sheffer, G. A. Mackay, M. Smith, S. Dhillon, R. Hegde, F. Jia, I. Adany, and O. Narayan, J. Virol 79:3419-3428, 2005). To make this DNA safer, we deleted two more genes, the integrase gene and
vif
, along with the 3′ long terminal repeat. We also replaced the
gag
,
pro
, and
nef
genes (SIVmac239 origin) with those of human immunodeficiency virus (HIV) type 1 strain SF2. The resultant construct, designated Δ4SHIV
KU2
DNA, was used in this study to evaluate gene expression and immunogenicity in BALB/c mice. DNA-transfected human embryonic kidney epithelial cells (HEK 293) produced all of the major viral proteins and released p24 in the supernatant for 12 days. Inoculation of the vaccine DNA into the gastrocnemius muscles resulted in intense mononuclear cell infiltration at the inoculated sites and the production of viral p24 in myocytes, in infiltrating mononuclear cells, and in cells in the spleen and draining lymph nodes between 3 and 10 days postinoculation. Expression of p24 in the muscle cells peaked at day 7 and became undetectable after day 12. The same 12-day period of expression of p24 was observed in mice that were given a second injection 4 weeks after the first. Evaluation of immune responses in BALB/c mice revealed that the DNA induced enzyme-linked immunospot and antigen-specific proliferative cell-mediated immunity responses. The responses were stronger in mice that were coinjected with a second plasmid expressing granulocyte-macrophage colony-stimulating factor. Since new waves of viral antigen production could be induced with each boosting injection of the vaccine DNA, this DNA could be a safe and efficient agent to induce long-term protection against HIV.
MysTR: an Endogenous Retrovirus Family in Mammals That Is Undergoing Recent Amplifications to Unprecedented Copy Numbers
Authors: Cantrell Michael A. , Ederer Martina M. , Erickson Issac K. , Swier Vicki J. , Baker Robert J. , Wichman Holly A. 
Published: November 2005

ABSTRACT

A large percentage of the repetitive elements in mammalian genomes are retroelements, which have been moved primarily by LINE-1 retrotransposons and endogenous retroviruses. Although LINE-1 elements have remained active throughout the mammalian radiation, specific groups of endogenous retroviruses generally remain active for comparatively shorter periods of time. Identification of an unusual extinction of LINE-1 activity in a group of South American rodents has opened a window for examination of the interplay in mammalian genomes between these ubiquitous retroelements. In the course of a search for any type of repetitive sequences whose copy numbers have substantially changed in
Oryzomys palustris
, a species that has lost LINE-1 activity, versus
Sigmodon hispidus
, a closely related species retaining LINE-1 activity, we have identified an endogenous retrovirus family differentially amplified in these two species. Analysis of three full-length, recently transposed copies, called mysTR elements, revealed
gag
,
pro
, and
pol
coding regions containing stop codons which may have accumulated either before or after retrotransposition. Isolation of related sequences in
S. hispidus
and the LINE-1 active outgroup species,
Peromyscus maniculatus
, by PCR of a
pro-pol
region has allowed determination of copy numbers in each species. Unusually high copy numbers of approximately 10,000 in
O. palustris
versus 1,000 in
S. hispidus
and 4,500 in the more distantly related
P.maniculatus
leave open the question of whether there is a connection between endogenous retrovirus activity and LINE-1 inactivity. Nevertheless, these independent expansions of mysTR represent recent amplifications of this endogenous retrovirus family to unprecedented levels.
The Conserved Poxvirus L3 Virion Protein Is Required for Transcription of Vaccinia Virus Early Genes
Authors: Resch Wolfgang , Moss Bernard 
Published: November 2005

ABSTRACT

We provide the initial characterization of the product of the vaccinia virus L3L open reading frame (VACWR090), which is conserved in all sequenced members of the poxvirus family. The predicted polypeptide contains no motifs or other features that provided a clue to the role of the L3 protein, and no functional information was available regarding a homolog discovered in
Plasmodium falciparum
. The L3 protein was expressed following viral DNA replication, a finding consistent with a putative late promoter sequence, and was packaged as a non-membrane protein in mature virus particles. A recombinant virus, in which the L3L gene was regulated by the
Escherichia coli lac
operator/repressor system, had a conditional lethal phenotype. The virus replicated in the presence of inducer, but in its absence, the yields of infectious virus were reduced by 99%. When cells were infected without inducer, however, no defect in gene expression or morphogenesis was noted. Virus particles lacking L3, which assembled in the absence of inducer, were indistinguishable from wild-type virions with regard to morphology, major structural proteins, and DNA content but were noninfectious. L3-deficient virions were able to bind and penetrate cells but produced extremely small amounts of viral early mRNA. A defect in transcription was demonstrated by in vitro studies with permeabilized virions, but soluble extracts of L3-deficient virions showed normal levels of template-dependent transcriptional activity, indicating that only transcription of the packaged genome is impaired.
A Mutation in the First Ligand-Binding Repeat of the Human Very-Low-Density Lipoprotein Receptor Results in High-Affinity Binding of the Single V1 Module to Human Rhinovirus 2
Authors: Nizet Stephane , Wruss Juergen , Landstetter Nathalie , Snyers Luc , Blaas Dieter 
Published: November 2005

ABSTRACT

Minor group human rhinoviruses (HRVs) bind members of the low-density lipoprotein receptor family for cell entry. The ligand-binding domains of these membrane proteins are composed of various numbers of direct repeats of about 40 amino acids in length. Residues involved in binding of module 3 (V3) of the very-low-density lipoprotein receptor (VLDLR) to HRV2 have been identified by X-ray crystallography (N. Verdaguer, I. Fita, M. Reithmayer, R. Moser, and D. Blaas, Nat. Struct. Mol. Biol.
11:
429-434, 2004). Sequence comparisons of the eight repeats of VLDLR with respect to the residues implicated in the interaction between V3 and HRV2 suggested that (in addition to V3) V1, V2, V5, and V6 also fulfill the requirements for interacting with the virus. Using a highly sensitive binding assay employing phage display, we demonstrate that single modules V2, V3, and V5 indeed bind HRV2. However, V1 does not. A single mutation from threonine 17 to proline converted the nonbinding wild-type form of V1 into a very strong binder. We interpret the dramatic increase in affinity by the generation of a hydrophobic patch between virus and receptor; in the presence of threonine, the contact area might be disturbed. This demonstrates that the interaction between virus and its natural receptors can be strongly enhanced by mutation.
Interactions with Pocket Proteins Contribute to the Role of Human Papillomavirus Type 16 E7 in the Papillomavirus Life Cycle
Authors: Collins Asha S. , Nakahara Tomomi , Do Anh , Lambert Paul F. 
Published: November 2005

ABSTRACT
Human papillomaviruses (HPVs), most commonly the HPV16 genotype, are the principle etiological determinant for cervical cancer, a common cancer worldwide resulting in over 200,000 deaths annually. The oncogenic properties of HPVs are attributable in part to the virally encoded protein E7, best known for its ability to bind to and induce the degradation of the retinoblastoma tumor suppressor, pRb, and related “pocket proteins” p107 and p130. Previously, we defined a role for E7 in the productive stage of the HPV16 life cycle, which takes place in stratified squamous epithelia. HPV perturbs the normal processes of cell growth and differentiation of stratified squamous epithelia. HPVs reprogram cells to support continued DNA synthesis and inhibit their differentiation in the suprabasal compartment of the epithelia, where cells normally have withdrawn from the cell cycle and initiated a well-defined pattern of terminal differentiation. These virus-induced perturbations, which contribute to the production of progeny HPVs, are dependent on E7. In this study, we define the mechanism of action by which E7 contributes to the productive stage of the HPV16 life cycle. We found that the ability of HPV16 to reprogram suprabasal cells to support DNA synthesis correlates with E7's ability to bind pocket proteins but not its ability to induce their degradation. In contrast, the ability of HPV16 to perturb differentiation correlated with both E7's binding to and degradation of pocket proteins. These data indicate that different hallmarks of the productive stage of the HPV16 life cycle rely upon different sets of requirements for E7.
Molecular Characterization of Adeno-Associated Viruses Infecting Children
Authors: Chen Chun-Liang , Jensen Ryan L. , Schnepp Bruce C. , Connell Mary J. , Shell Richard , Sferra Thomas J. , Bartlett Jeffrey S. , Clark K. Reed , Johnson Philip R. 
Published: November 2005

ABSTRACT

Although adeno-associated virus (AAV) infection is common in humans, the biology of natural infection is poorly understood. Since it is likely that many primary AAV infections occur during childhood, we set out to characterize the frequency and complexity of circulating AAV isolates in fresh and archived frozen human pediatric tissues. Total cellular DNA was isolated from 175 tissue samples including freshly collected tonsils (
n
= 101) and archived frozen samples representing spleen (
n
= 21), lung (
n
= 16), muscle (
n
= 15), liver (
n
= 19), and heart (
n
= 3). Samples were screened for the presence of AAV and adenovirus sequences by PCR using degenerate primers. AAV DNA was detected in 7 of 101 (7%) tonsil samples and two of 74 other tissues (one spleen and one lung). Adenovirus sequences were identified in 19 of 101 tonsils (19%), but not in any other tissues. Complete capsid gene sequences were recovered from all nine AAV-positive tissues. Sequence analyses showed that eight of the capsid sequences were AAV2-like (∼98% amino acid identity), while the single spleen isolate was intermediate between serotypes 2 and 3. Comparison to the available AAV2 crystal structure revealed that the majority of the amino acid substitutions mapped to surface-exposed hypervariable domains. To further characterize the AAV capsid structure in these samples, we used a novel linear rolling-circle amplification method to amplify episomal AAV DNA and isolate infectious molecular clones from several human tissues. Serotype 2-like viruses were generated from these DNA clones and interestingly, failed to bind to a heparin sulfate column. Inspection of the capsid sequence from these two clones (and the other six AAV2-like isolates) revealed that they lacked arginine residues at positions 585 and 588 of the capsid protein, which are thought to be essential for interaction with the heparin sulfate proteoglycan coreceptor. These data provide a framework with which to explore wild-type AAV persistence in vivo and provide additional tools to further define the biodistribution and form of AAV in human tissues.
Characterization of Adeno-Associated Virus Genomes Isolated from Human Tissues
Authors: Schnepp Bruce C. , Jensen Ryan L. , Chen Chun-Liang , Johnson Philip R. , Clark K. Reed 
Published: November 2005

ABSTRACT
Infection with wild-type adeno-associated virus (AAV) is common in humans, but very little is known about the in vivo biology of AAV. On a molecular level, it has been shown in cultured cells that AAV integrates in a site-specific manner on human chromosome 19, but this has never been demonstrated directly in infected human tissues. To that end, we tested 175 tissue samples for the presence of AAV DNA, and when present, examined the specific form of the viral DNA. AAV was detected in 7 of 101 tonsil-adenoid samples and in 2 of 74 other tissue samples (spleen and lung). In these nine samples, we were unable to detect AAV integration in the AAVS1 locus using a sensitive PCR assay designed to amplify specific viral-cellular DNA junctions. Additionally, we used a second complementary assay, linear amplification-mediated-PCR (LAM-PCR) to widen our search for integration events. Analysis of individual LAM-PCR products revealed that the AAV genomes were arranged predominantly in a head-to-tail array, with deletions and extensive rearrangements in the inverted terminal repeat sequences. A single AAV-cellular junction was identified from a tonsil sample and it mapped to a highly repetitive satellite DNA element on chromosome 1. Given these data, we entertained the possibility that instead of integrated forms, AAV genomes were present as extrachromosomal forms. We used a novel amplification assay (linear rolling-circle amplification) to show that the majority of wild-type AAV DNA existed as circular double-stranded episomes in our tissues. Thus, following naturally acquired infection, AAV DNA can persist mainly as circular episomes in human tissues. These findings are consistent with the circular episomal forms of recombinant AAV vectors that have been isolated and characterized from in vivo transduced tissues.
Effect of Cell Cycle Arrest on the Activity of Nucleoside Analogues against Human Immunodeficiency Virus Type 1
Authors: Wurtzer Sebastien , Compain Séverine , Benech Henri , Hance Allan J. , Clavel François 
Published: November 2005

ABSTRACT

Human immunodeficiency virus (HIV) reverse transcription can be notably affected by cellular activation, differentiation, and division. We hypothesized that changes in the cell cycle could also affect HIV susceptibility to nucleoside analogues, which compete with natural nucleotides for incorporation into viral DNA and inhibit viral replication through premature termination of reverse transcription. Proliferating HeLa-derived indicator cells were arrested in the S/G
2
phase with etoposide, a topoisomerase II inhibitor, or in the G
1
/S phase with aphidicolin, a polymerase α inhibitor. Cell cycle arrest by both agents induced a remarkable decrease in HIV susceptibility to zidovudine (AZT). This decrease was seen both with a single-cycle infectivity assay and with a viral DNA quantitation assay, indicating that the effect of cell cycle arrest was exerted at the reverse transcription stage. The increase in the 50% inhibitory concentration (IC
50
) seen with arrested cells was strongest for AZT (23-fold) and stavudine (21-fold) but more modest for other drugs (lamivudine, 11-fold; dideoxyinosine, 7-fold; and nevirapine, 3-fold). In drug-resistant reverse transcriptase mutants, the increase in AZT IC
50
(relative to that in dividing cells) was most prominent with a Q151M mutant and was comparable to the wild type in other drug-resistant mutants. Quantitation of intracellular pools of dTTP and AZT 5′-triphosphate (AZTTP) showed that etoposide treatment induced a significant increase in intracellular dTTP and consequently a decrease in AZTTP/dTTP ratios, suggesting that the decrease in viral susceptibility to AZT was caused by reduced incorporation of the analogue into nascent viral DNA. These results emphasize the importance of cellular proliferation and deoxynucleoside triphosphate metabolism in HIV susceptibility to nucleoside analogues and underscore the need to study the activities of drugs of this class with natural target cells under physiological conditions of activation and proliferation.
Latent Infection with Herpes Simplex Virus Is Associated with Ongoing CD8+T-Cell Stimulation by Parenchymal Cells within Sensory Ganglia
Authors: van Lint Allison L. , Kleinert Lauren , Clarke Sally R. M. , Stock Angus , Heath William R. , Carbone Francis R. 
Published: November 2005

ABSTRACTCD8+T-cell persistence can be seen in ganglia harboring latent herpes simplex virus (HSV) infection. While there is some evidence that these cells suppress virus reactivation, this view remains controversial. Given that maintenance of latency by CD8+T cells would necessitate ongoing exposure to antigen within this site, we sought evidence for such chronic stimulation. Initial experiments showed infiltration by activated but not naïve CD8+T cells into ganglia harboring latent HSV infection. While such infiltration was independent of T-cell specificity, once recruited, only virus-specific T cells expressed high levels of preformed granzyme B, a marker of ongoing activation. Moreover, bone marrow replacement chimeras showed that these elevated granzyme levels were totally dependent on presentation by parenchymal cells within the ganglia. Overall, this study argues that activated CD8+T cells are nonspecifically recruited into latently infected ganglia, and in this site they are exposed to ongoing antigen stimulation, most likely by infected neuronal cells.