Publications by authors named "Nicholas F Parrish"

29 Publications

  • Page 1 of 1

Virus-derived variation in diverse human genomes.

PLoS Genet 2021 Apr 26;17(4):e1009324. Epub 2021 Apr 26.

Genome Immunobiology RIKEN Hakubi Research Team, RIKEN Center for Integrative Medical Sciences and RIKEN Cluster for Pioneering Research, Yokohama, Japan.

Acquisition of genetic material from viruses by their hosts can generate inter-host structural genome variation. We developed computational tools enabling us to study virus-derived structural variants (SVs) in population-scale whole genome sequencing (WGS) datasets and applied them to 3,332 humans. Although SVs had already been cataloged in these subjects, we found previously-overlooked virus-derived SVs. We detected non-germline SVs derived from squirrel monkey retrovirus (SMRV), human immunodeficiency virus 1 (HIV-1), and human T lymphotropic virus (HTLV-1); these variants are attributable to infection of the sequenced lymphoblastoid cell lines (LCLs) or their progenitor cells and may impact gene expression results and the biosafety of experiments using these cells. In addition, we detected new heritable SVs derived from human herpesvirus 6 (HHV-6) and human endogenous retrovirus-K (HERV-K). We report the first solo-direct repeat (DR) HHV-6 likely to reflect DR rearrangement of a known full-length endogenous HHV-6. We used linkage disequilibrium between single nucleotide variants (SNVs) and variants in reads that align to HERV-K, which often cannot be mapped uniquely using conventional short-read sequencing analysis methods, to locate previously-unknown polymorphic HERV-K loci. Some of these loci are tightly linked to trait-associated SNVs, some are in complex genome regions inaccessible by prior methods, and some contain novel HERV-K haplotypes likely derived from gene conversion from an unknown source or introgression. These tools and results broaden our perspective on the coevolution between viruses and humans, including ongoing virus-to-human gene transfer contributing to genetic variation between humans.
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http://dx.doi.org/10.1371/journal.pgen.1009324DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8101998PMC
April 2021

Virus-like insertions with sequence signatures similar to those of endogenous nonretroviral RNA viruses in the human genome.

Proc Natl Acad Sci U S A 2021 02;118(5)

Laboratory of RNA Viruses, Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto 606-8507, Japan;

Understanding the genetics and taxonomy of ancient viruses will give us great insights into not only the origin and evolution of viruses but also how viral infections played roles in our evolution. Endogenous viruses are remnants of ancient viral infections and are thought to retain the genetic characteristics of viruses from ancient times. In this study, we used machine learning of endogenous RNA virus sequence signatures to identify viruses in the human genome that have not been detected or are already extinct. Here, we show that the -mer occurrence of ancient RNA viral sequences remains similar to that of extant RNA viral sequences and can be differentiated from that of other human genome sequences. Furthermore, using this characteristic, we screened RNA viral insertions in the human reference genome and found virus-like insertions with phylogenetic and evolutionary features indicative of an exogenous origin but lacking homology to previously identified sequences. Our analysis indicates that animal genomes still contain unknown virus-derived sequences and provides a glimpse into the diversity of the ancient virosphere.
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http://dx.doi.org/10.1073/pnas.2010758118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7865133PMC
February 2021

Chromosomally-integrated human herpesvirus 6 and autoimmune connective tissue diseases.

J Clin Virol 2021 01 5;134:104714. Epub 2020 Dec 5.

Laboratory for Statistical and Translational Genetics, RIKEN Center for Integrative Medical Sciences, Yokohama, Japan.

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http://dx.doi.org/10.1016/j.jcv.2020.104714DOI Listing
January 2021

Endogenous retroviruses drive species-specific germline transcriptomes in mammals.

Nat Struct Mol Biol 2020 10 7;27(10):967-977. Epub 2020 Sep 7.

Division of Reproductive Sciences, Division of Developmental Biology, Perinatal Institute, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA.

Gene regulation in the germline ensures the production of high-quality gametes, long-term maintenance of the species and speciation. Male germline transcriptomes undergo dynamic changes after the mitosis-to-meiosis transition and have been subject to evolutionary divergence among mammals. However, the mechanisms underlying germline regulatory divergence remain undetermined. Here, we show that endogenous retroviruses (ERVs) influence species-specific germline transcriptomes. After the mitosis-to-meiosis transition in male mice, specific ERVs function as active enhancers to drive germline genes, including a mouse-specific gene set, and bear binding motifs for critical regulators of spermatogenesis, such as A-MYB. This raises the possibility that a genome-wide transposition of ERVs rewired germline gene expression in a species-specific manner. Of note, independently evolved ERVs are associated with the expression of human-specific germline genes, demonstrating the prevalence of ERV-driven mechanisms in mammals. Together, we propose that ERVs fine-tune species-specific transcriptomes in the mammalian germline.
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http://dx.doi.org/10.1038/s41594-020-0487-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8246630PMC
October 2020

Endogenization and excision of human herpesvirus 6 in human genomes.

PLoS Genet 2020 08 10;16(8):e1008915. Epub 2020 Aug 10.

Laboratory for Statistical and Translational Genetics, RIKEN Center for Integrative Medical Sciences, Yokohama, Japan.

Sequences homologous to human herpesvirus 6 (HHV-6) are integrated within the nuclear genome of about 1% of humans, but it is not clear how this came about. It is also uncertain whether integrated HHV-6 can reactivate into an infectious virus. HHV-6 integrates into telomeres, and this has recently been associated with polymorphisms affecting MOV10L1. MOV10L1 is located on the subtelomere of chromosome 22q (chr22q) and is required to make PIWI-interacting RNAs (piRNAs). As piRNAs block germline integration of transposons, piRNA-mediated repression of HHV-6 integration has been proposed to explain this association. In vitro, recombination of the HHV-6 genome along its terminal direct repeats (DRs) leads to excision from the telomere and viral reactivation, but the expected "solo-DR scar" has not been described in vivo. Here we screened for integrated HHV-6 in 7,485 Japanese subjects using whole-genome sequencing (WGS). Integrated HHV-6 was associated with polymorphisms on chr22q. However, in contrast to prior work, we find that the reported MOV10L1 polymorphism is physically linked to an ancient endogenous HHV-6A variant integrated into the telomere of chr22q in East Asians. Unexpectedly, an HHV-6B variant has also endogenized in chr22q; two endogenous HHV-6 variants at this locus thus account for 72% of all integrated HHV-6 in Japan. We also report human genomes carrying only one portion of the HHV-6B genome, a solo-DR, supporting in vivo excision and possible viral reactivation. Together these results explain the recently-reported association between integrated HHV-6 and MOV10L1/piRNAs, suggest potential exaptation of HHV-6 in its coevolution with human chr22q, and clarify the evolution and risk of reactivation of the only intact (non-retro)viral genome known to be present in human germlines.
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http://dx.doi.org/10.1371/journal.pgen.1008915DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7444522PMC
August 2020

Evolutionary History of Endogenous Human Herpesvirus 6 Reflects Human Migration out of Africa.

Mol Biol Evol 2021 01;38(1):96-107

Institut für Virologie, Freie Universität Berlin, Berlin, Germany.

Human herpesvirus 6A and 6B (HHV-6) can integrate into the germline, and as a result, ∼70 million people harbor the genome of one of these viruses in every cell of their body. Until now, it has been largely unknown if 1) these integrations are ancient, 2) if they still occur, and 3) whether circulating virus strains differ from integrated ones. Here, we used next-generation sequencing and mining of public human genome data sets to generate the largest and most diverse collection of circulating and integrated HHV-6 genomes studied to date. In genomes of geographically dispersed, only distantly related people, we identified clades of integrated viruses that originated from a single ancestral event, confirming this with fluorescent in situ hybridization to directly observe the integration locus. In contrast to HHV-6B, circulating and integrated HHV-6A sequences form distinct clades, arguing against ongoing integration of circulating HHV-6A or "reactivation" of integrated HHV-6A. Taken together, our study provides the first comprehensive picture of the evolution of HHV-6, and reveals that integration of heritable HHV-6 has occurred since the time of, if not before, human migrations out of Africa.
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http://dx.doi.org/10.1093/molbev/msaa190DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7782865PMC
January 2021

piRNA-Guided CRISPR-like Immunity in Eukaryotes.

Trends Immunol 2019 11 31;40(11):998-1010. Epub 2019 Oct 31.

Genome Immunobiology RIKEN Hakubi Research Team, Cluster for Pioneering Research, RIKEN, Yokohama 235-0045, Japan; Center for Integrative Medical Sciences, RIKEN, Yokohama 235-0045, Japan. Electronic address:

Eukaryotic genomes contain virus-derived sequences called endogenous virus elements (EVEs). The majority of EVEs are related to retroviruses, which integrate into the host genome in order to replicate. Some retroviral EVEs encode a function; for example, some produce proteins that block infection by related viruses. EVEs derived from nonretroviral viruses - also recently found in many eukaryotic genomes - are more enigmatic. Here, we summarize the evidence that EVEs can act as templates to generate Piwi-interacting RNAs (piRNAs), whose canonical function is sequence-specific silencing of transposable elements (TEs) to maintain genomic integrity. We argue that EVEs may thus enable heritable, sequence-specific antiviral immune memory in eukaryotes - analogous to CRISPR-Cas immunity in prokaryotes.
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http://dx.doi.org/10.1016/j.it.2019.09.003DOI Listing
November 2019

The Changing Face of Liver Transplantation in the United States: The Effect of HCV Antiviral Eras on Transplantation Trends and Outcomes.

Transplant Direct 2019 Mar 20;5(3):e427. Epub 2019 Feb 20.

Division of Hepatobiliary Surgery and Liver Transplantation, Section of Surgical Sciences, Vanderbilt University Medical Center, Nashville, TN.

Background: Hepatitis C virus (HCV) cirrhosis is the leading indication for liver transplantation in the United States, although nonalcoholic steatohepatitis (NASH) is on the rise. Increasingly effective HCV antivirals are available, but their association with diagnosis-specific liver transplantation rates and early graft survival is not known.

Methods: The Scientific Registry of Transplant Recipients database records were retrospectively stratified by HCV antiviral era: interferon (2003-2010), protease inhibitors (2011-2013), and direct-acting antivirals (2014 to present). Kaplan-Meier, χ, and multivariable Cox proportional hazards regression models evaluated the effects of antiviral era and etiology of liver disease on transplantation rates and graft survival over 3 years.

Results: Liver transplants for HCV decreased (35.3% to 23.6%), whereas those for NASH and alcoholic liver disease increased (5.8% to 16.5% and 15.6% to 24.0%) with each advancing era (all < 0.05). Early graft survival improved with each advancing era for HCV but not for hepatitis B virus, NASH, or alcoholic liver disease (multivariable model era by diagnosis interaction < 0.001). Era-specific multivariable models demonstrated that the risk of early graft loss for NASH was 22% lower than for HCV in the interferon era (hazard ratio, 0.78; 95% confidence interval, 0.64-0.96; = 0.02) but risks associated with these diagnoses did not differ significantly in the protease inhibitor ( = 0.06) or direct-acting antiviral eras ( = 0.08).

Conclusions: Increasing effectiveness of HCV antivirals corresponds with decreased rates of liver transplantation for HCV and improved early graft survival. As the rates of liver transplant for NASH continue to increase, focus will be needed on the prevention and effective therapies for this disease.
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http://dx.doi.org/10.1097/TXD.0000000000000866DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6411219PMC
March 2019

Species-specific host factors rather than virus-intrinsic virulence determine primate lentiviral pathogenicity.

Nat Commun 2018 04 10;9(1):1371. Epub 2018 Apr 10.

Institute of Molecular Virology, Ulm University Medical Center, 89081, Ulm, Germany.

HIV-1 causes chronic inflammation and AIDS in humans, whereas related simian immunodeficiency viruses (SIVs) replicate efficiently in their natural hosts without causing disease. It is currently unknown to what extent virus-specific properties are responsible for these different clinical outcomes. Here, we incorporate two putative HIV-1 virulence determinants, i.e., a Vpu protein that antagonizes tetherin and blocks NF-κB activation and a Nef protein that fails to suppress T cell activation via downmodulation of CD3, into a non-pathogenic SIVagm strain and test their impact on viral replication and pathogenicity in African green monkeys. Despite sustained high-level viremia over more than 4 years, moderately increased immune activation and transcriptional signatures of inflammation, the HIV-1-like SIVagm does not cause immunodeficiency or any other disease. These data indicate that species-specific host factors rather than intrinsic viral virulence factors determine the pathogenicity of primate lentiviruses.
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http://dx.doi.org/10.1038/s41467-018-03762-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5893559PMC
April 2018

A Viral (Arc)hive for Metazoan Memory.

Cell 2018 01;172(1-2):8-10

Department of Virus Research, Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto, Japan; Department of Mammalian Regulatory Network, Graduate School of Biostudies, Kyoto University, Kyoto, Japan. Electronic address:

Arc, a master regulator of synaptic plasticity, contains sequence elements that are evolutionarily related to retrotransposon Gag genes. Two related papers in this issue of Cell show that Arc retains retroviral-like capsid-forming ability and can transmit mRNA between cells in the nervous system, a process that may be important for synaptic function.
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http://dx.doi.org/10.1016/j.cell.2017.12.029DOI Listing
January 2018

Endogenized viral sequences in mammals.

Curr Opin Microbiol 2016 06 26;31:176-183. Epub 2016 Apr 26.

Department of Viral Oncology, Institute for Virus Research, Kyoto University, Kyoto 606-8507, Japan; Department of Tumor Viruses, Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan; Department of Mammalian Regulatory Network, Graduate School of Biostudies, Kyoto University, Kyoto 606-8507, Japan. Electronic address:

Reverse-transcribed RNA molecules compose a significant portion of the human genome. Many of these RNA molecules were retrovirus genomes either infecting germline cells or having done so in a previous generation but retaining transcriptional activity. This mechanism itself accounts for a quarter of the genomic sequence information of mammals for which there is data. We understand relatively little about the causes and consequences of retroviral endogenization. This review highlights functions ascribed to sequences of viral origin endogenized into mammalian genomes and suggests some of the most pressing questions raised by these observations.
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http://dx.doi.org/10.1016/j.mib.2016.03.002DOI Listing
June 2016

An integrated map of structural variation in 2,504 human genomes.

Nature 2015 Oct;526(7571):75-81

University of California San Diego (UCSD), 9500 Gilman Drive, La Jolla, CA 92093, USA.

Structural variants are implicated in numerous diseases and make up the majority of varying nucleotides among human genomes. Here we describe an integrated set of eight structural variant classes comprising both balanced and unbalanced variants, which we constructed using short-read DNA sequencing data and statistically phased onto haplotype blocks in 26 human populations. Analysing this set, we identify numerous gene-intersecting structural variants exhibiting population stratification and describe naturally occurring homozygous gene knockouts that suggest the dispensability of a variety of human genes. We demonstrate that structural variants are enriched on haplotypes identified by genome-wide association studies and exhibit enrichment for expression quantitative trait loci. Additionally, we uncover appreciable levels of structural variant complexity at different scales, including genic loci subject to clusters of repeated rearrangement and complex structural variants with multiple breakpoints likely to have formed through individual mutational events. Our catalogue will enhance future studies into structural variant demography, functional impact and disease association.
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http://dx.doi.org/10.1038/nature15394DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4617611PMC
October 2015

Transcription Profiling Demonstrates Epigenetic Control of Non-retroviral RNA Virus-Derived Elements in the Human Genome.

Cell Rep 2015 Sep 28;12(10):1548-54. Epub 2015 Aug 28.

Department of Viral Oncology, Institute for Virus Research, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan. Electronic address:

Endogenous bornavirus-like nucleoprotein elements (EBLNs) are DNA sequences in vertebrate genomes formed by the retrotransposon-mediated integration of ancient bornavirus sequence. Thus, EBLNs evidence a mechanism of retrotransposon-mediated RNA-to-DNA information flow from environment to animals. Although EBLNs are non-transposable, they share some features with retrotransposons. Here, to test whether hosts control the expression of EBLNs similarly to retrotransposons, we profiled the transcription of all Homo sapiens EBLNs (hsEBLN-1 to hsEBLN-7). We could detect transcription of all hsEBLNs in at least one tissue. Among them, hsEBLN-1 is transcribed almost exclusively in the testis. In most tissues, expression from the hsEBLN-1 locus is silenced epigenetically. Finally, we showed the possibility that hsEBLN-1 integration at this locus affects the expression of a neighboring gene. Our results suggest that hosts regulate the expression of endogenous non-retroviral virus elements similarly to how they regulate the expression of retrotransposons, possibly contributing to new transcripts and regulatory complexity to the human genome.
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http://dx.doi.org/10.1016/j.celrep.2015.08.007DOI Listing
September 2015

piRNAs derived from ancient viral processed pseudogenes as transgenerational sequence-specific immune memory in mammals.

RNA 2015 Oct 17;21(10):1691-703. Epub 2015 Aug 17.

Department of Viral Oncology, Institute for Virus Research, Kyoto University, Kyoto 606-8507, Japan Department of Tumor Viruses, Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan Department of Mammalian Regulatory Network, Graduate School of Biostudies, Kyoto University, Kyoto 606-8507, Japan.

Endogenous bornavirus-like nucleoprotein elements (EBLNs) are sequences within vertebrate genomes derived from reverse transcription and integration of ancient bornaviral nucleoprotein mRNA via the host retrotransposon machinery. While species with EBLNs appear relatively resistant to bornaviral disease, the nature of this association is unclear. We hypothesized that EBLNs could give rise to antiviral interfering RNA in the form of PIWI-interacting RNAs (piRNAs), a class of small RNA known to silence transposons but not exogenous viruses. We found that in both rodents and primates, which acquired their EBLNs independently some 25-40 million years ago, EBLNs are present within piRNA-generating regions of the genome far more often than expected by chance alone (ℙ = 8 × 10(-3)-6 × 10(-8)). Three of the seven human EBLNs fall within annotated piRNA clusters and two marmoset EBLNs give rise to bona fide piRNAs. In both rats and mice, at least two of the five EBLNs give rise to abundant piRNAs in the male gonad. While no EBLNs are syntenic between rodent and primate, some of the piRNA clusters containing EBLNs are; thus we deduce that EBLNs were integrated into existing piRNA clusters. All true piRNAs derived from EBLNs are antisense relative to the proposed ancient bornaviral nucleoprotein mRNA. These observations are consistent with a role for EBLN-derived piRNA-like RNAs in interfering with ancient bornaviral infection. They raise the hypothesis that retrotransposon-dependent virus-to-host gene flow could engender RNA-mediated, sequence-specific antiviral immune memory in metazoans analogous to the CRISPR/Cas system in prokaryotes.
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http://dx.doi.org/10.1261/rna.052092.115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4574747PMC
October 2015

Analysis of deletion breakpoints from 1,092 humans reveals details of mutation mechanisms.

Nat Commun 2015 Jun 1;6:7256. Epub 2015 Jun 1.

1] Program in Computational Biology and Bioinformatics, Yale University, 266 Whitney Avenue, New Haven, Connecticut 06520, USA [2] Department of Molecular Biophysics and Biochemistry, School of Medicine, Yale University, New Haven, Connecticut 06520, USA [3] Department of Computer Science, Yale University, New Haven, Connecticut 06520, USA.

Investigating genomic structural variants at basepair resolution is crucial for understanding their formation mechanisms. We identify and analyse 8,943 deletion breakpoints in 1,092 samples from the 1000 Genomes Project. We find breakpoints have more nearby SNPs and indels than the genomic average, likely a consequence of relaxed selection. By investigating the correlation of breakpoints with DNA methylation, Hi-C interactions, and histone marks and the substitution patterns of nucleotides near them, we find that breakpoints with the signature of non-allelic homologous recombination (NAHR) are associated with open chromatin. We hypothesize that some NAHR deletions occur without DNA replication and cell division, in embryonic and germline cells. In contrast, breakpoints associated with non-homologous (NH) mechanisms often have sequence microinsertions, templated from later replicating genomic sites, spaced at two characteristic distances from the breakpoint. These microinsertions are consistent with template-switching events and suggest a particular spatiotemporal configuration for DNA during the events.
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http://dx.doi.org/10.1038/ncomms8256DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4451611PMC
June 2015

Neutralization properties of simian immunodeficiency viruses infecting chimpanzees and gorillas.

mBio 2015 Apr 21;6(2). Epub 2015 Apr 21.

Departments of Medicine and Microbiology, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

Unlabelled: Broadly cross-reactive neutralizing antibodies (bNabs) represent powerful tools to combat human immunodeficiency virus type 1 (HIV-1) infection. Here, we examined whether HIV-1-specific bNabs are capable of cross-neutralizing distantly related simian immunodeficiency viruses (SIVs) infecting central (Pan troglodytes troglodytes) (SIVcpzPtt) and eastern (Pan troglodytes schweinfurthii) (SIVcpzPts) chimpanzees (n = 11) as well as western gorillas (Gorilla gorilla gorilla) (SIVgor) (n = 1). We found that bNabs directed against the CD4 binding site (n = 10), peptidoglycans at the base of variable loop 3 (V3) (n = 5), and epitopes at the interface of surface (gp120) and membrane-bound (gp41) envelope glycoproteins (n = 5) failed to neutralize SIVcpz and SIVgor strains. In addition, apex V2-directed bNabs (n = 3) as well as llama-derived (heavy chain only) antibodies (n = 6) recognizing both the CD4 binding site and gp41 epitopes were either completely inactive or neutralized only a fraction of SIVcpzPtt strains. In contrast, one antibody targeting the membrane-proximal external region (MPER) of gp41 (10E8), functional CD4 and CCR5 receptor mimetics (eCD4-Ig, eCD4-Ig(mim2), CD4-218.3-E51, and CD4-218.3-E51-mim2), as well as mono- and bispecific anti-human CD4 (iMab and LM52) and CCR5 (PRO140, PRO140-10E8) receptor antibodies neutralized >90% of SIVcpz and SIVgor strains with low-nanomolar (0.13 to 8.4 nM) potency. Importantly, the latter antibodies blocked virus entry not only in TZM-bl cells but also in Cf2Th cells expressing chimpanzee CD4 and CCR5 and neutralized SIVcpz in chimpanzee CD4(+) T cells, with 50% inhibitory concentrations (IC50s) ranging from 3.6 to 40.5 nM. These findings provide new insight into the protective capacity of anti-HIV-1 bNabs and identify candidates for further development to combat SIVcpz infection.

Importance: SIVcpz is widespread in wild-living chimpanzees and can cause AIDS-like immunopathology and clinical disease. HIV-1 infection of humans can be controlled by antiretroviral therapy; however, treatment of wild-living African apes with current drug regimens is not feasible. Nonetheless, it may be possible to curb the spread of SIVcpz in select ape communities using vectored immunoprophylaxis and/or therapy. Here, we show that antibodies and antibody-like inhibitors developed to combat HIV-1 infection in humans are capable of neutralizing genetically diverse SIVcpz and SIVgor strains with considerable breadth and potency, including in primary chimpanzee CD4(+) T cells. These reagents provide an important first step toward translating intervention strategies currently developed to treat and prevent AIDS in humans to SIV-infected apes.
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http://dx.doi.org/10.1128/mBio.00296-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4453581PMC
April 2015

Borna disease virus possesses an NF-ĸB inhibitory sequence in the nucleoprotein gene.

Sci Rep 2015 Mar 3;5:8696. Epub 2015 Mar 3.

1] Department of Viral Oncology, Kyoto University, Kyoto 606-8507, Japan [2] Department of Tumor Viruses, Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan [3] Department of Mammalian Regulatory Network, Graduate School of Biostudies, Kyoto University, Kyoto 606-8507, Japan.

Borna disease virus (BDV) has a non-segmented, negative-stranded RNA genome and causes persistent infection in many animal species. Previous study has shown that the activation of the IκB kinase (IKK)/NF-κB pathway is reduced by BDV infection even in cells expressing constitutively active mutant IKK. This result suggests that BDV directly interferes with the IKK/NF-κB pathway. To elucidate the mechanism for the inhibition of NF-κB activation by BDV infection, we evaluated the cross-talk between BDV infection and the NF-κB pathway. Using Multiple EM for Motif Elicitation analysis, we found that the nucleoproteins of BDV (BDV-N) and NF-κB1 share a common ankyrin-like motif. When THP1-CD14 cells were pre-treated with the identified peptide, NF-κB activation by Toll-like receptor ligands was suppressed. The 20S proteasome assay showed that BDV-N and BDV-N-derived peptide inhibited the processing of NF-κB1 p105 into p50. Furthermore, immunoprecipitation assays showed that BDV-N interacted with NF-κB1 but not with NF-κB2, which shares no common motif with BDV-N. These results suggest BDV-N inhibits NF-κB1 processing by the 20S proteasome through its ankyrin-like peptide sequence, resulting in the suppression of IKK/NF-κB pathway activation. This inhibitory effect of BDV on the induction of the host innate immunity might provide benefits against persistent BDV infection.
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http://dx.doi.org/10.1038/srep08696DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4649702PMC
March 2015

Quantitative phosphoproteomics reveals extensive cellular reprogramming during HIV-1 entry.

Cell Host Microbe 2013 May;13(5):613-623

Department of Pathology and Laboratory Medicine, Children's Hospital of Philadelphia Research Institute, Philadelphia, PA 19104, USA. Electronic address:

Receptor engagement by HIV-1 during host cell entry activates signaling pathways that can reprogram the cell for optimal viral replication. To obtain a global view of the signaling events induced during HIV-1 entry, we conducted a quantitative phosphoproteomics screen of primary human CD4(+) T cells after infection with an HIV-1 strain that engages the receptors CD4 and CXCR4. We quantified 1,757 phosphorylation sites with high stringency. The abundance of 239 phosphorylation sites from 175 genes, including several proteins in pathways known to be impacted by HIV-receptor binding, changed significantly within a minute after HIV-1 exposure. Several previously uncharacterized HIV-1 host factors were also identified and confirmed through RNAi depletion studies. Surprisingly, five serine/arginine-rich (SR) proteins involved in messenger RNA splicing, including the splicing factor SRm300 (SRRM2), were differentially phosophorylated. Mechanistic studies with SRRM2 suggest that HIV-1 modulates host cell alternative splicing machinery during entry in order to facilitate virus replication and release.
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http://dx.doi.org/10.1016/j.chom.2013.04.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4104530PMC
May 2013

Phenotypic properties of transmitted founder HIV-1.

Proc Natl Acad Sci U S A 2013 Apr 29;110(17):6626-33. Epub 2013 Mar 29.

Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.

Defining the virus-host interactions responsible for HIV-1 transmission, including the phenotypic requirements of viruses capable of establishing de novo infections, could be important for AIDS vaccine development. Previous analyses have failed to identify phenotypic properties other than chemokine receptor 5 (CCR5) and CD4+ T-cell tropism that are preferentially associated with viral transmission. However, most of these studies were limited to examining envelope (Env) function in the context of pseudoviruses. Here, we generated infectious molecular clones of transmitted founder (TF; n = 27) and chronic control (CC; n = 14) viruses of subtypes B (n = 18) and C (n = 23) and compared their phenotypic properties in assays specifically designed to probe the earliest stages of HIV-1 infection. We found that TF virions were 1.7-fold more infectious (P = 0.049) and contained 1.9-fold more Env per particle (P = 0.048) compared with CC viruses. TF viruses were also captured by monocyte-derived dendritic cells 1.7-fold more efficiently (P = 0.035) and more readily transferred to CD4+ T cells (P = 0.025). In primary CD4+ T cells, TF and CC viruses replicated with comparable kinetics; however, when propagated in the presence of IFN-α, TF viruses replicated to higher titers than CC viruses. This difference was significant for subtype B (P = 0.000013) but not subtype C (P = 0.53) viruses, possibly reflecting demographic differences of the respective patient cohorts. Together, these data indicate that TF viruses are enriched for higher Env content, enhanced cell-free infectivity, improved dendritic cell interaction, and relative IFN-α resistance. These viral properties, which likely act in concert, should be considered in the development and testing of AIDS vaccines.
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http://dx.doi.org/10.1073/pnas.1304288110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3637789PMC
April 2013

Transmitted/founder and chronic HIV-1 envelope proteins are distinguished by differential utilization of CCR5.

J Virol 2013 Mar 26;87(5):2401-11. Epub 2012 Dec 26.

Department of Microbiology, Perelman School of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA.

Infection by HIV-1 most often results from the successful transmission and propagation of a single virus variant, termed the transmitted/founder (T/F) virus. Here, we compared the attachment and entry properties of envelope (Env) glycoproteins from T/F and chronic control (CC) viruses. Using a panel of 40 T/F and 47 CC Envs, all derived by single genome amplification, we found that 52% of clade C and B CC Envs exhibited partial resistance to the CCR5 antagonist maraviroc (MVC) on cells expressing high levels of CCR5, while only 15% of T/F Envs exhibited this same property. Moreover, subtle differences in the magnitude with which MVC inhibited infection on cells expressing low levels of CCR5, including primary CD4(+) T cells, were highly predictive of MVC resistance when CCR5 expression levels were high. These results are consistent with previous observations showing a greater sensitivity of T/F Envs to MVC inhibition on cells expressing very high levels of CCR5 and indicate that CC Envs are often capable of recognizing MVC-bound CCR5, albeit inefficiently on cells expressing physiologic levels of CCR5. When CCR5 expression levels are high, this phenotype becomes readily detectable. The utilization of drug-bound CCR5 conformations by many CC Envs was seen with other CCR5 antagonists, with replication-competent viruses, and did not obviously correlate with other phenotypic traits. The striking ability of clade C and B CC Envs to use MVC-bound CCR5 relative to T/F Envs argues that the more promiscuous use of CCR5 by these Env proteins is selected against at the level of virus transmission and is selected for during chronic infection.
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http://dx.doi.org/10.1128/JVI.02964-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3571396PMC
March 2013

Molecular identification, cloning and characterization of transmitted/founder HIV-1 subtype A, D and A/D infectious molecular clones.

Virology 2013 Feb 1;436(1):33-48. Epub 2012 Nov 1.

Department of Pathology, University of Alabama at Birmingham, Birmingham, AL 35294, USA.

We report the molecular identification, cloning and initial biological characterization of 12 full-length HIV-1 subtype A, D and A/D recombinant transmitted/founder (T/F) genomes. T/F genomes contained intact canonical open reading frames and all T/F viruses were replication competent in primary human T-cells, although subtype D virus replication was more efficient (p<0.05). All 12 viruses utilized CCR5 but not CXCR4 as a co-receptor for entry and exhibited a neutralization profile typical of tier 2 primary virus strains, with significant differences observed between subtype A and D viruses with respect to sensitivity to monoclonal antibodies VRC01, PG9 and PG16 and polyclonal subtype C anti-HIV IgG (p<0.05 for each). The present report doubles the number of T/F HIV-1 clones available for pathogenesis and vaccine research and extends their representation to include subtypes A, B, C and D.
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http://dx.doi.org/10.1016/j.virol.2012.10.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3545109PMC
February 2013

Transmitted/founder and chronic subtype C HIV-1 use CD4 and CCR5 receptors with equal efficiency and are not inhibited by blocking the integrin α4β7.

PLoS Pathog 2012 31;8(5):e1002686. Epub 2012 May 31.

Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.

Sexual transmission of human immunodeficiency virus type 1 (HIV-1) most often results from productive infection by a single transmitted/founder (T/F) virus, indicating a stringent mucosal bottleneck. Understanding the viral traits that overcome this bottleneck could have important implications for HIV-1 vaccine design and other prevention strategies. Most T/F viruses use CCR5 to infect target cells and some encode envelope glycoproteins (Envs) that contain fewer potential N-linked glycosylation sites and shorter V1/V2 variable loops than Envs from chronic viruses. Moreover, it has been reported that the gp120 subunits of certain transmitted Envs bind to the gut-homing integrin α4β7, possibly enhancing virus entry and cell-to-cell spread. Here we sought to determine whether subtype C T/F viruses, which are responsible for the majority of new HIV-1 infections worldwide, share biological properties that increase their transmission fitness, including preferential α4β7 engagement. Using single genome amplification, we generated panels of both T/F (n = 20) and chronic (n = 20) Env constructs as well as full-length T/F (n = 6) and chronic (n = 4) infectious molecular clones (IMCs). We found that T/F and chronic control Envs were indistinguishable in the efficiency with which they used CD4 and CCR5. Both groups of Envs also exhibited the same CD4+ T cell subset tropism and showed similar sensitivity to neutralization by CD4 binding site (CD4bs) antibodies. Finally, saturating concentrations of anti-α4β7 antibodies failed to inhibit infection and replication of T/F as well as chronic control viruses, although the growth of the tissue culture-adapted strain SF162 was modestly impaired. These results indicate that the population bottleneck associated with mucosal HIV-1 acquisition is not due to the selection of T/F viruses that use α4β7, CD4 or CCR5 more efficiently.
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http://dx.doi.org/10.1371/journal.ppat.1002686DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3364951PMC
October 2012

Mucosal simian immunodeficiency virus transmission in African green monkeys: susceptibility to infection is proportional to target cell availability at mucosal sites.

J Virol 2012 Apr 8;86(8):4158-68. Epub 2012 Feb 8.

Division of Microbiology, Tulane National Primate Research Center, Covington, Louisiana, USA.

African green monkeys (AGMs) are naturally infected with a simian immunodeficiency virus (SIVagm) that is nonpathogenic in its host. Although SIVagm is common and widespread, little is known about the mechanisms that govern its transmission. Since the earliest virus-host interactions may provide key insights into the nonpathogenic phenotype of SIVagm, we developed a mucosal transmission model for this virus. Using plasma from an acutely infected AGM as the virus inoculum, we exposed adult and juvenile AGMs, as well as pigtailed macaques (PTMs) as a nonnatural host control, by mucosal routes to increasing titers of virus and compared the doses needed to establish a productive infection. Four juvenile and four adult AGMs as well as two PTMs were intrarectally (IR) exposed, while two additional adult female AGMs were intravaginally (IVAG) exposed. No animal became infected following exposure to 10(5) RNA copies. Both PTMs but none of the AGMs became infected following exposure to 10(6) RNA copies. Finally, all adult AGMs and two of the four juvenile AGMs became infected following exposure to 10(7) RNA copies, acquiring either one (2 IR infected juveniles, 1 IR infected adult, 2 IVAG infected adults) or two (3 IR infected adults) transmitted founder viruses. These results were consistent with immunophenotypic data, which revealed a significant correlation between the percentage of CD4(+) T cells expressing CCR5 in the mucosa and the susceptibility to infection, in terms of both the viral dose and the numbers of transmitted founder viruses. Moreover, studies of uninfected AGMs showed that the fraction of CCR5-expressing CD4(+) T cells increased significantly with age. These results indicate that (i) AGMs are readily infected with SIVagm by both intrarectal and intravaginal routes, (ii) susceptibility to infection is proportional to the number of available CCR5(+) CD4(+) target cells in the mucosa, and (iii) the paucity of CCR5(+) CD4(+) target cells in infant and juvenile AGMs may explain the near absence of vertical transmission.
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http://dx.doi.org/10.1128/JVI.07141-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3318646PMC
April 2012

Primary infection by a human immunodeficiency virus with atypical coreceptor tropism.

J Virol 2011 Oct 10;85(20):10669-81. Epub 2011 Aug 10.

Duke Human Vaccine Institute, Duke University Medical Center, Durham, North Carolina 27710, USA.

The great majority of human immunodeficiency virus type 1 (HIV-1) strains enter CD4+ target cells by interacting with one of two coreceptors, CCR5 or CXCR4. Here we describe a transmitted/founder (T/F) virus (ZP6248) that was profoundly impaired in its ability to utilize CCR5 and CXCR4 coreceptors on multiple CD4+ cell lines as well as primary human CD4+ T cells and macrophages in vitro yet replicated to very high titers (>80 million RNA copies/ml) in an acutely infected individual. Interestingly, the envelope (Env) glycoprotein of this clade B virus had a rare GPEK sequence in the crown of its third variable loop (V3) rather than the consensus GPGR sequence. Extensive sequencing of sequential plasma samples showed that the GPEK sequence was present in virtually all Envs, including those from the earliest time points after infection. The molecularly cloned (single) T/F virus was able to replicate, albeit poorly, in cells obtained from ccr5Δ32 homozygous donors. The ZP6248 T/F virus could also infect cell lines overexpressing the alternative coreceptors GPR15, APJ, and FPRL-1. A single mutation in the V3 crown sequence (GPEK->GPGK) of ZP6248 restored its infectivity in CCR5+ cells but reduced its ability to replicate in GPR15+ cells, indicating that the V3 crown motif played an important role in usage of this alternative coreceptor. These results suggest that the ZP6248 T/F virus established an acute in vivo infection by using coreceptor(s) other than CCR5 or CXCR4 or that the CCR5 coreceptor existed in an unusual conformation in this individual.
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http://dx.doi.org/10.1128/JVI.05249-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3187499PMC
October 2011

Phenotypic and immunologic comparison of clade B transmitted/founder and chronic HIV-1 envelope glycoproteins.

J Virol 2011 Sep 29;85(17):8514-27. Epub 2011 Jun 29.

Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.

Sexual transmission of human immunodeficiency virus type 1 (HIV-1) across mucosal barriers is responsible for the vast majority of new infections. This relatively inefficient process results in the transmission of a single transmitted/founder (T/F) virus, from a diverse viral swarm in the donor, in approximately 80% of cases. Here we compared the biological activities of 24 clade B T/F envelopes (Envs) with those from 17 chronic controls to determine whether the genetic bottleneck that occurs during transmission is linked to a particular Env phenotype. To maximize the likelihood of an intact mucosal barrier in the recipients and to enhance the sensitivity of detecting phenotypic differences, only T/F Envs from individuals infected with a single T/F variant were selected. Using pseudotyping to assess Env function in single-round infectivity assays, we compared coreceptor tropism, CCR5 utilization efficiencies, primary CD4(+) T cell subset tropism, dendritic cell trans-infections, fusion kinetics, and neutralization sensitivities. T/F and chronic Envs were phenotypically equivalent in most assays; however, T/F Envs were modestly more sensitive to CD4 binding site antibodies b12 and VRC01, as well as pooled human HIV Ig. This finding was independently validated with a panel of 14 additional chronic HIV-1 Env controls. Moreover, the enhanced neutralization sensitivity was associated with more efficient binding of b12 and VRC01 to T/F Env trimers. These data suggest that there are subtle but significant structural differences between T/F and chronic clade B Envs that may have implications for HIV-1 transmission and the design of effective vaccines.
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http://dx.doi.org/10.1128/JVI.00736-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3165820PMC
September 2011

Genetic identity and biological phenotype of a transmitted/founder virus representative of nonpathogenic simian immunodeficiency virus infection in African green monkeys.

J Virol 2010 Dec 29;84(23):12245-54. Epub 2010 Sep 29.

Institute of Molecular Virology, University Clinic of Ulm, Ulm, Germany.

Understanding the lack of disease progression in nonpathogenic simian immunodeficiency virus (SIV) infections is essential for deciphering the immunopathogenesis of human AIDS. Yet, in vivo studies have been hampered by a paucity of infectious molecular clones (IMCs) of SIV suitable to dissect the viral and host factors responsible for the nonpathogenic phenotype. Here, we describe the identification, cloning, and biological analysis of the first transmitted/founder (T/F) virus representing a nonpathogenic SIV infection. Blood was collected at peak viremia from an acutely infected sabaeus monkey (Chlorocebus sabaeus) inoculated intravenously with an African green monkey SIV (SIVagm) strain (Sab92018) that had never been propagated in vitro. To generate IMCs, we first used conventional (bulk) PCR to amplify full-length viral genomes from peripheral blood mononuclear cell (PBMC) DNA. Although this yielded two intact SIVagmSab genomes, biological characterization revealed that both were replication defective. We then performed single-genome amplification (SGA) to generate partially overlapping 5' (n = 10) and 3' (n = 13) half genomes from plasma viral RNA. Analysis of these amplicons revealed clusters of nearly identical viral sequences representing the progeny of T/F viruses. Synthesis of the consensus sequence of one of these generated an IMC (Sab92018ivTF) that produced infectious CCR5-tropic virions and replicated to high titers in Molt-4 clone 8 cells and African green monkey PBMCs. Sab92018ivTF also initiated productive infection in sabaeus monkeys and faithfully recapitulated the replication kinetics and nonpathogenic phenotype of the parental Sab92018 strain. These results thus extend the T/F virus concept to nonpathogenic SIV infections and provide an important new tool to define viral determinants of disease nonprogression.
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http://dx.doi.org/10.1128/JVI.01603-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2976391PMC
December 2010

A rev1-vpu polymorphism unique to HIV-1 subtype A and C strains impairs envelope glycoprotein expression from rev-vpu-env cassettes and reduces virion infectivity in pseudotyping assays.

Virology 2010 Feb 8;397(2):346-57. Epub 2009 Dec 8.

Department of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294, USA.

Functional studies of HIV-1 envelope glycoproteins (Envs) commonly include the generation of pseudoviruses, which are produced by co-transfection of rev-vpu-env cassettes with an env-deficient provirus. Here, we describe six Env constructs from transmitted/founder HIV-1 that were defective in the pseudotyping assay, although two produced infectious virions when expressed from their cognate proviruses. All of these constructs exhibited an unusual gene arrangement in which the first exon of rev (rev1) and vpu were in the same reading frame without an intervening stop codon. Disruption of the rev1-vpu fusion gene by frameshift mutation, stop codon, or abrogation of the rev initiation codon restored pseudovirion infectivity. Introduction of the fusion gene into wildtype Env cassettes severely compromised their function. The defect was not due to altered env and rev transcription or a dominant negative effect of the expressed fusion protein, but seemed to be caused by inefficient translation at the env initiation codon. Although the rev1-vpu polymorphism affects Env expression only in vitro, it can cause problems in studies requiring Env complementation, such as analyses of co-receptor usage and neutralization properties, since 3% of subtype A, 20% of subtype C and 5% of CRF01_A/E viruses encode the fusion gene. A solution is to eliminate the rev initiation codon when amplifying rev-vpu-env cassettes since this increases Env expression irrespective of the presence of the polymorphism.
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http://dx.doi.org/10.1016/j.virol.2009.11.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2822091PMC
February 2010

Genetic identity, biological phenotype, and evolutionary pathways of transmitted/founder viruses in acute and early HIV-1 infection.

J Exp Med 2009 Jun 1;206(6):1273-89. Epub 2009 Jun 1.

University of Alabama at Birmingham, Birmingham, AL 35294, USA.

Identification of full-length transmitted HIV-1 genomes could be instrumental in HIV-1 pathogenesis, microbicide, and vaccine research by enabling the direct analysis of those viruses actually responsible for productive clinical infection. We show in 12 acutely infected subjects (9 clade B and 3 clade C) that complete HIV-1 genomes of transmitted/founder viruses can be inferred by single genome amplification and sequencing of plasma virion RNA. This allowed for the molecular cloning and biological analysis of transmitted/founder viruses and a comprehensive genome-wide assessment of the genetic imprint left on the evolving virus quasispecies by a composite of host selection pressures. Transmitted viruses encoded intact canonical genes (gag-pol-vif-vpr-tat-rev-vpu-env-nef) and replicated efficiently in primary human CD4(+) T lymphocytes but much less so in monocyte-derived macrophages. Transmitted viruses were CD4 and CCR5 tropic and demonstrated concealment of coreceptor binding surfaces of the envelope bridging sheet and variable loop 3. 2 mo after infection, transmitted/founder viruses in three subjects were nearly completely replaced by viruses differing at two to five highly selected genomic loci; by 12-20 mo, viruses exhibited concentrated mutations at 17-34 discrete locations. These findings reveal viral properties associated with mucosal HIV-1 transmission and a limited set of rapidly evolving adaptive mutations driven primarily, but not exclusively, by early cytotoxic T cell responses.
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http://dx.doi.org/10.1084/jem.20090378DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2715054PMC
June 2009
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