bluuebunny/crossref_metadata_2025_split huggingface dataset:
Rolling-Circle Replication of an Animal Circovirus Genome in a Theta-Replicating Bacterial Plasmid in
Escherichia coli
Authors: Cheung Andrew K. 
Published: August 2006

ABSTRACT

A bacterial plasmid containing 1.75 copies of double-stranded porcine circovirus (PCV) DNA in tandem (0.8 copy of PCV type 1 [PCV1], 0.95 copy of PCV2) with two origins of DNA replication (Ori) yielded three different DNA species when transformed into
Escherichia coli
: the input construct, a unit-length chimeric PCV1
Rep
/PCV2
Cap
genome with a composite Ori but lacking the plasmid vector, and a molecule consisting of the remaining 0.75 copy PCV1
Cap
/PCV2
Rep
genome with a different composite Ori together with the bacterial plasmid. Replication of the input construct was presumably via the theta replication mechanism utilizing the ColE
1
Ori, while characteristics of the other two DNA species, including a requirement of two PCV Oris and the virus-encoded replication initiator Rep protein, suggest they were generated via the rolling-circle copy-release mechanism. Interestingly, the PCV-encoded Rep′ protein essential for PCV DNA replication in mammalian cells was not required in bacteria. The fact that the Rep′ protein function(s) can be compensated by the bacterial replication machinery to support the PCV DNA replication process echoes previous suggestions that circular single-stranded DNA animal circoviruses, plant geminiviruses, and nanoviruses may have evolved from prokaryotic episomal replicons.
Purified Feline and Canine Transferrin Receptors Reveal Complex Interactions with the Capsids of Canine and Feline Parvoviruses That Correspond to Their Host Ranges
Authors: Palermo Laura M. , Hafenstein Susan L. , Parrish Colin R. 
Published: August 2006

ABSTRACT
The cell infection processes and host ranges of canine parvovirus (CPV) and feline panleukopenia virus (FPV) are controlled by their capsid interactions with the transferrin receptors (TfR) on their host cells. Here, we expressed the ectodomains of wild-type and mutant TfR and tested those for binding to purified viral capsids and showed that different naturally variant strains of the viruses were associated with variant interactions with the receptors which likely reflect the optimization of the viral infection processes in the different hosts. While all viruses bound the feline TfR, reflecting their tissue culture host ranges, a naturally variant mutant of CPV (represented by the CPV type-2b strain) that became the dominant virus worldwide in 1979 showed significantly lower levels of binding to the feline TfR. The canine TfR ectodomain did not bind to a detectable level in the in vitro assays, but this appears to reflect the naturally low affinity of that interaction, as only low levels of binding were seen when the receptor was expressed on mammalian cells; however, that was sufficient to allow endocytosis and infection. The apical domain of the canine TfR controls the specific interaction with CPV capsids, as a canine TfR mutant altering a glycosylation site in that domain bound FPV, CPV-2, and CPV-2b capsids efficiently. Enzymatic removal of the N-linked glycans did not allow FPV binding to the canine TfR, suggesting that the protein sequence difference is itself important. The purified feline TfR inhibited FPV and CPV-2 binding and infection of feline cells but not CPV-2b, indicating that the receptor binding may be able to prevent the attachment to the same receptor on cells.
Crystal Structure of the Moloney Murine Leukemia Virus RNase H Domain
Authors: Lim David , Gregorio G. Glenn , Bingman Craig , Martinez-Hackert Erik , Hendrickson Wayne A. , Goff Stephen P. 
Published: August 2006

ABSTRACT

A crystallographic study of the Moloney murine leukemia virus (Mo-MLV) RNase H domain was performed to provide information about its structure and mechanism of action. These efforts resulted in the crystallization of a mutant Mo-MLV RNase H lacking the putative helix C (ΔC). The 1.6-Å resolution structure resembles the known structures of the human immunodeficiency virus type 1 (HIV-1) and
Escherichia coli
RNase H. The structure revealed the coordination of a magnesium ion within the catalytic core comprised of the highly conserved acidic residues D524, E562, and D583. Surface charge mapping of the Mo-MLV structure revealed a high density of basic charges on one side of the enzyme. Using a model of the Mo-MLV structure superimposed upon a structure of HIV-1 reverse transcriptase bound to an RNA/DNA hybrid substrate, Mo-MLV RNase H secondary structures and individual amino acids were examined for their potential roles in binding substrate. Identified regions included Mo-MLV RNase H β1-β2, αA, and αB and residues from αB to αD and its following loop. Most of the identified substrate-binding residues corresponded with residues directly binding nucleotides in an RNase H from
Bacillus halodurans
as observed in a cocrystal structure with RNA/DNA. Finally, superimposition of RNases H of Mo-MLV,
E. coli
, and HIV-1 revealed that a loop of the HIV-1 connection domain resides within the same region of the Mo-MLV and
E. coli
C-helix. The HIV-1 connection domain may serve to recognize and bind the RNA/DNA substrate major groove.
Crimean-Congo Hemorrhagic Fever Virus Genomics and Global Diversity
Authors: Deyde Varough M. , Khristova Marina L. , Rollin Pierre E. , Ksiazek Thomas G. , Nichol Stuart T. 
Published: August 2006

ABSTRACT
Crimean-Congo hemorrhagic fever (CCHF) is a severe illness with high case fatality that occurs in Africa, Europe, the Middle East, and Asia. The complete genomes of 13 geographically and temporally diverse virus strains were determined, and CCHF viruses were found to be highly variable with 20 and 8%, 31 and 27%, and 22 and 10% nucleotide and deduced amino acid differences detected among virus S (nucleocapsid), M (glycoprotein), and L (polymerase) genome segments, respectively. Distinct geographic lineages exist, but with multiple exceptions indicative of long-distance virus movement. Discrepancies among the virus S, M, and L phylogenetic tree topologies document multiple RNA segment reassortment events. An analysis of individual segment datasets suggests genetic recombination also occurs. For an arthropod-borne virus, the genomic plasticity of CCHF virus is surprisingly high.
West Nile Virus 5′-Cap Structure Is Formed by Sequential Guanine N-7 and Ribose 2′-O Methylations by Nonstructural Protein 5
Authors: Ray Debashish , Shah Aaloki , Tilgner Mark , Guo Yi , Zhao Yiwei , Dong Hongping , Deas Tia S. , Zhou Yangsheng , Li Hongmin , Shi Pei-Yong 
Published: August 2006

ABSTRACT

Many flaviviruses are globally important human pathogens. Their plus-strand RNA genome contains a 5′-cap structure that is methylated at the guanine N-7 and the ribose 2′-OH positions of the first transcribed nucleotide, adenine (m
7
GpppAm). Using West Nile virus (WNV), we demonstrate, for the first time, that the nonstructural protein 5 (NS5) mediates both guanine N-7 and ribose 2′-O methylations and therefore is essential for flavivirus 5′-cap formation. We show that a recombinant full-length and a truncated NS5 protein containing the methyltransferase (MTase) domain methylates GpppA-capped and m
7
GpppA-capped RNAs to m
7
GpppAm-RNA, using
S
-adenosylmethionine as a methyl donor. Furthermore, methylation of GpppA-capped RNA sequentially yielded m
7
GpppA- and m
7
GpppAm-RNA products, indicating that guanine N-7 precedes ribose 2′-O methylation. Mutagenesis of a K
61
-D
146
-K
182
-E
218
tetrad conserved in other cellular and viral MTases suggests that NS5 requires distinct amino acids for its N-7 and 2′-O MTase activities. The entire K
61
-D
146
-K
182
-E
218
motif is essential for 2′-O MTase activity, whereas N-7 MTase activity requires only D
146
. The other three amino acids facilitate, but are not essential for, guanine N-7 methylation. Amino acid substitutions within the K
61
-D
146
-K
182
-E
218
motif in a WNV luciferase-reporting replicon significantly reduced or abolished viral replication in cells. Additionally, the mutant MTase-mediated replication defect could not be
trans
complemented by a wild-type replicase complex. These findings demonstrate a critical role for the flavivirus MTase in viral reproduction and underscore this domain as a potential target for antiviral therapy.
Marek's Disease Virus Encodes MicroRNAs That Map to
meq
and the Latency-Associated Transcript
Authors: Burnside Joan , Bernberg Erin , Anderson Amy , Lu Cheng , Meyers Blake C. , Green Pamela J. , Jain Neeta , Isaacs Grace , Morgan Robin W. 
Published: August 2006

ABSTRACT

MicroRNAs (miRNAs) are a class of small (∼22-nucleotide) regulatory molecules that block translation or induce degradation of target mRNAs. These have been identified in a wide range of organisms, including viruses. In particular, the oncogenic gammaherpesviruses Kaposi's sarcoma herpesvirus and Epstein-Barr virus encode miRNAs that could potentially regulate either viral or host genes. To determine if Marek's disease virus (MDV), an oncogenic alphaherpesvirus of chickens, encodes miRNAs, we isolated small RNAs from MDV-infected chicken embryo fibroblasts (CEF) and used the 454 Life Sciences sequencing technology to obtain the sequences of 13,679 candidate host and viral small RNAs. Eight miRNAs were found, five of which flank the
meq
oncogene and three that map to the latency-associated transcript (LAT) region of the genome. The
meq
gene is unique to pathogenic serotypes of MDV and is transcriptionally active during latency and transformation, and the LAT region of the MDV genome is antisense to the immediate-early gene ICP4. Secondary structure analysis predicted that the regions flanking the miRNAs could form hairpin precursors. Northern blot analysis confirmed expression of all miRNAs in MDV-infected CEF, MDV-induced tumors, and MDV lymphoblastoid cell lines. We propose that the MDV miRNAs function to enable MDV pathogenesis and contribute to MDV-induced transformation of chicken T cells.
Tertiary Structural and Functional Analyses of a Viroid RNA Motif by Isostericity Matrix and Mutagenesis Reveal Its Essential Role in Replication
Authors: Zhong Xuehua , Leontis Neocles , Qian Shuiming , Itaya Asuka , Qi Yijun , Boris-Lawrie Kathleen , Ding Biao 
Published: August 2006

ABSTRACT

RNA-templated RNA replication is essential for viral or viroid infection, as well as for regulation of cellular gene expression. Specific RNA motifs likely regulate various aspects of this replication. Viroids of the
Pospiviroidae
family, as represented by the
Potato spindle tuber viroid
(PSTVd), replicate in the nucleus by utilizing DNA-dependent RNA polymerase II. We investigated the role of the loop E (sarcin/ricin) motif of the PSTVd genomic RNA in replication. A tertiary-structural model of this motif, inferred by comparative sequence analysis and comparison with nuclear magnetic resonance and X-ray crystal structures of loop E motifs in other RNAs, is presented in which core non-Watson-Crick base pairs are precisely specified. Isostericity matrix analysis of these base pairs showed that the model accounts for the reported natural sequence variations and viable experimental mutations in loop E motifs of PSTVd and other viroids. Furthermore, isostericity matrix analysis allowed us to design disruptive, as well as compensatory, mutations of PSTVd loop E. Functional analyses of such mutants by in vitro and in vivo experiments demonstrated that loop E structural integrity is crucial for replication, specifically during transcription. Our results suggest that the PSTVd loop E motif exists and functions in vivo and provide loss-of-function genetic evidence for the essential role of a viroid RNA three-dimensional motif in rolling-circle replication. The use of isostericity matrix analysis of non-Watson-Crick base pairing to rationalize mutagenesis of tertiary motifs and systematic in vitro and in vivo functional assays of mutants offers a novel, comprehensive approach to elucidate the tertiary-structure-function relationships for RNA motifs of general biological significance.
Antiviral Potency of APOBEC Proteins Does Not Correlate with Cytidine Deamination
Authors: Bishop Kate N. , Holmes Rebecca K. , Malim Michael H. 
Published: August 2006

ABSTRACT
The human cytidine deaminases APOBEC3G (hA3G) and APOBEC3F (hA3F) are intracellular antiretroviral factors that can hypermutate nascent reverse transcripts and inhibit the replication of human immunodeficiency virus type 1 (HIV-1). Both enzymes have two cytidine deaminase motifs, although only the C-terminal motif is catalytic. Current models of APOBEC protein function imply editing is the principal mechanism of antiviral activity. In particular, hA3G is a more potent inhibitor of HIV-1 infectivity than hA3F and also induces a greater frequency of mutations in HIV-1 cDNA. We used hA3G/hA3F chimeric proteins to investigate whether cytidine deaminase potential reflects antiviral potency. We show here that the origin of the C-terminal deaminase motif is sufficient to determine the degree of mutation induced in a bacterial assay that measures mutations in chromosomal DNA. In contrast, this was not the case in the context of HIV-1 infection where the N-terminal deaminase motif also modulated the editing capabilities of the chimeras. Surprisingly, although three of the chimeric proteins induced levels of mutation that approximated those of parental hA3F, they displayed lower levels of antiviral activity. Most importantly, real-time PCR experiments revealed that the quantity of reverse transcripts detected in target cells, rather than the mutational burden carried by such DNAs, corresponded closely with viral infectivity. In other words, the antiviral phenotype of APOBEC proteins correlates with their ability to prevent the accumulation of reverse transcripts and not with the induction of hypermutation.
Role of Bim in Regulating CD8+T-Cell Responses during Chronic Viral Infection
Authors: Grayson Jason M. , Weant Ashley E. , Holbrook Beth C. , Hildeman David 
Published: August 2006

ABSTRACTApoptosis is critical for the development and maintenance of the immune system. The proapoptotic Bcl-2 family member Bim is important for normal immune system homeostasis. Although previous experiments have shown that Bim is critical for the apoptosis of antigen-specific CD8+T cells during acute viral infection, the role of Bim during chronic viral infection is unclear. Using lymphocytic choriomeningitis virus clone 13 infection of mice, we demonstrate a role for Bim in CD8+T-cell apoptosis during chronic viral infection. Enumeration of antigen-specific CD8+T cells by major histocompatibility complex class I tetramer staining revealed that CD8+DbNP396-404+T cells, which undergo extensive deletion in wild-type mice, exhibited almost no decrease in Bim mutant mice. This contrasts with CD8+DbGP33-41+and CD8+DbGP276-286+T cells that underwent similar decreases in numbers in both Bim mutant and wild-type mice. Increased numbers of CD8+DbNP396-404+T cells in Bim mutant mice were due to lack of apoptosis and could not be explained by altered proliferation, differential homing to tissues, or increased help from CD4+T cells. When viral titers were examined, high levels were initially observed in both groups, but in Bim mutant mice, clearance from the spleen and sera was slightly accelerated. These experiments demonstrate the critical role of Bim during chronic viral infection to down-regulate CD8+T-cell responses and have implications for designing strategies for optimizing immunotherapies during situations where antigen persists, such as chronic infection, autoimmune syndromes, and cancer.
Intra- and Intersubtype Alternative Pak2-Activating Structural Motifs of Human Immunodeficiency Virus Type 1 Nef
Authors: O'Neill Eduardo , Baugh Laura L. , Novitsky Vladimir A. , Essex Myron E. , Garcia J. Victor 
Published: August 2006

ABSTRACT
The design of antiviral strategies against human immunodeficiency virus type 1 (HIV-1) has been largely derived from studies of subtype B viruses, although they constitute only 12% of infections worldwide. At 50% of all HIV-1 infections worldwide, subtype C viruses are the most predominant. Here, we present evidence that subtype C Nefs display functional Pak2-activating motifs that differ from those found in subtype B and E Nefs. The identification of multiple Pak2-activating structural motifs that singly affect one Nef activity revealed a functional plasticity that has implications for future drug and vaccine design aimed at HIV-1 Nef and its effects on the deregulation of the immune system.
Poxviral Regulation of the Host NF-κB Response: the Vaccinia Virus M2L Protein Inhibits Induction of NF-κB Activation via an ERK2 Pathway in Virus-Infected Human Embryonic Kidney Cells
Authors: Gedey Roderick , Jin Xiao-Lu , Hinthong Olivia , Shisler Joanna L. 
Published: August 2006

ABSTRACT
Exposure of eukaryotic cells to viruses will activate the host NF-κB transcription factor, resulting in proinflammatory and immune protein production. Vaccinia virus (VV), the prototypic orthopoxvirus, expresses products that inhibit this antiviral event. To identify novel mechanisms responsible for this effect, we made use of a VV deletion mutant (MVA) that stimulates NF-κΒ activation in infected 293T cells. In this virus-host system, the extents of NF-κΒ-regulated gene expression and nuclear translocation were reduced in the presence of either PD 98059 or U0126, two compounds capable of blocking ERK1 and ERK2 phosphorylation. A similar repression was also observed in cells that contained a dominant, nonactive form of ERK2 but not in cells where ERK1 phosphorylation was inhibited via overexpression of a dominant-negative mutant MEK1 protein. Presumably, proteins expressed from a wild-type VV that block ERK2 activity would also inhibit MVA-induced NF-κB activation. Indeed, the expression of one such open reading frame, M2L, supported this prediction. First, ectopic M2L expression hampered ERK2 phosphorylation induced by exposure to phorbol myristate acetate. Second, viral M2L expression via infection of cells with a recombinant MVA construct that stably expressed M2L decreased the phosphorylation of ERK2 compared to that in cells infected with the parental MVA strain. Finally, the recombinant M2L-expressing virus restored the “wild-type” NF-κB-inhibitory phenotype, as indicated by decreased NF-κB migration to infected cell nuclei and interference in transcription. Thus, in 293T cells, VV apparently utilizes its M2L protein to interfere with a step(s) that would otherwise enable ERK2 phosphorylation and the consequential activation of an NF-κΒ response.
Inducible Yeast System for Viral RNA Recombination Reveals Requirement for an RNA Replication Signal on Both Parental RNAs
Authors: Garcia-Ruiz Hernan , Ahlquist Paul 
Published: August 2006

ABSTRACT

To facilitate RNA recombination studies, we tested whether
Saccharomyces cerevisiae
, which supports brome mosaic virus (BMV) replication, also supports BMV RNA recombination. Yeast strains expressing BMV RNA replication proteins 1a and 2a
pol
were engineered to transiently coexpress two independently inducible, overlapping, nonreplicating derivatives of BMV genomic RNA3. B3Δ3′ lacked the coat protein gene and negative-strand RNA promoter. B3Δ5′ lacked the positive-strand RNA promoter and had the coat gene replaced by the selectable
URA3
gene. After 12 to 72 h of induction, B3Δ3′ and B3Δ5′ transcription was repressed and Ura
+
yeast cells were selected. All Ura
+
cells contained recombinant RNA3 replicons expressing
URA3
. Most replicons arose by intermolecular homologous recombination between B3Δ3′ and B3Δ5′. Such recombinants were isolated only when 1a and 2a
pol
were expressed and after transient transcription of both B3Δ3′ and B3Δ5′, showing that recombination occurred at the RNA, not DNA, level. A minority of
URA3
-expressing replicons were derived from B3Δ5′, independently of B3Δ3′, by 5′ truncation and modification, generating novel positive-strand promoters and demonstrating that BMV can give rise to subgenomic RNA replicons. Intermolecular B3Δ3′-B3Δ5′ recombination occurred only when both parental RNAs bore a functional,
cis
-acting template recognition and recruitment element targeting viral RNAs to replication complexes. The results imply that recombination occurred in RNA replication complexes to which parental RNAs were independently recruited. Moreover, the ability to obtain intermolecular recombinants at precisely measurable, reproducible frequencies, to control genetic background and induction conditions, and other features of this system will facilitate further studies of virus and host functions in RNA recombination.
Mobility of Human Immunodeficiency Virus Type 1 Pr55
Gag
in Living Cells
Authors: Gomez Candace Y. , Hope Thomas J. 
Published: August 2006

ABSTRACT
Human immunodeficiency virus type 1 (HIV-1) assembly requires the converging of thousands of structural proteins on cellular membranes to form a tightly packed immature virion. The Gag polyprotein contains all of the determinants important for viral assembly and must move around in the cell in order to form particles. This work has focused on Gag mobility in order to provide more insights into the dynamics of particle assembly. Key to these studies was the use of several fluorescently labeled Gag derivatives. We used fluorescence recovery after photobleaching as well as photoactivation to determine Gag mobility. Upon expression, Gag can be localized diffusely in the cytoplasm, associated with the plasma membrane, or in virus-like particles (VLPs). Here we show that Gag VLPs are primarily localized in the plasma membrane and do not colocalize with CD63. We have shown using full-length Gag as well as truncation mutants fused to green fluorescent protein that Gag is highly mobile in live cells when it is not assembled into VLPs. Results also showed that this mobility is highly dependent upon cholesterol. When cholesterol is depleted from cells expressing Gag, mobility is significantly decreased. Once cholesterol was replenished, Gag mobility returned to wild-type levels. Taken together, results from these mobility studies suggest that Gag is highly mobile and that as the assembly process proceeds, mobility decreases. These studies also suggest that Gag assembly must occur in cholesterol-rich domains in the plasma membrane.