Fidelity of Target Site Duplication and Sequence Preference during Integration of Xenotropic Murine Leukemia Virus-Related Virus

Xenotropic murine leukemia virus (MLV)-related virus (XMRV) is a new human retrovirus associated with prostate cancer and chronic fatigue syndrome. The causal relationship of XMRV infection to human disease and the mechanism of pathogenicity have not been established. During retrovirus replication, integration of the cDNA copy of the viral RNA genome into the host cell chromosome is an essential step and involves coordinated joining of the two ends of the linear viral DNA into staggered sites on target DNA. Correct integration produces proviruses that are flanked by a short direct repeat, which varies from 4 to 6 bp among the retroviruses but is invariant for each particular retrovirus. Uncoordinated joining of the two viral DNA ends into target DNA can cause insertions, deletions, or other genomic alterations at the integration site. To determine the fidelity of XMRV integration, cells infected with XMRV were clonally expanded and DNA sequences at the viral-host DNA junctions were determined and analyzed. We found that a majority of the provirus ends were correctly processed and flanked by a 4-bp direct repeat of host DNA. A weak consensus sequence was also detected at the XMRV integration sites. We conclude that integration of XMRV DNA involves a coordinated joining of two viral DNA ends that are spaced 4 bp apart on the target DNA and proceeds with high fidelity

A comprehensive functional map of the hepatitis C virus genome provides a resource for probing viral proteins.

UnlabelledPairing high-throughput sequencing technologies with high-throughput mutagenesis enables genome-wide investigations of pathogenic organisms. Knowledge of the specific functions of protein domains encoded by the genome of the hepatitis C virus (HCV), a major human pathogen that contributes to liver disease worldwide, remains limited to insight from small-scale studies. To enhance the capabilities of HCV researchers, we have obtained a high-resolution functional map of the entire viral genome by combining transposon-based insertional mutagenesis with next-generation sequencing. We generated a library of 8,398 mutagenized HCV clones, each containing one 15-nucleotide sequence inserted at a unique genomic position. We passaged this library in hepatic cells, recovered virus pools, and simultaneously assayed the abundance of mutant viruses in each pool by next-generation sequencing. To illustrate the validity of the functional profile, we compared the genetic footprints of viral proteins with previously solved protein structures. Moreover, we show the utility of these genetic footprints in the identification of candidate regions for epitope tag insertion. In a second application, we screened the genetic footprints for phenotypes that reflected defects in later steps of the viral life cycle. We confirmed that viruses with insertions in a region of the nonstructural protein NS4B had a defect in infectivity while maintaining genome replication. Overall, our genome-wide HCV mutant library and the genetic footprints obtained by high-resolution profiling represent valuable new resources for the research community that can direct the attention of investigators toward unidentified roles of individual protein domains.ImportanceOur insertional mutagenesis library provides a resource that illustrates the effects of relatively small insertions on local protein structure and HCV viability. We have also generated complementary resources, including a website (http://hangfei.bol.ucla.edu) and a panel of epitope-tagged mutant viruses that should enhance the research capabilities of investigators studying HCV. Researchers can now detect epitope-tagged viral proteins by established antibodies, which will allow biochemical studies of HCV proteins for which antibodies are not readily available. Furthermore, researchers can now quickly look up genotype-phenotype relationships and base further mechanistic studies on the residue-by-residue information from the functional profile. More broadly, this approach offers a general strategy for the systematic functional characterization of viruses on the genome scale

Combining high-resolution genetics and imaging for the study of hepatitis C virus proteins critical for HCV assembly in infected host cells

The establishment of a cell culture system for producing infectious hepatitis C virus (HCV) prompted genetic and functional studies of viral proteins and their roles in the assembly process. Since then, all ten viral proteins have been implicated in HCV assembly. Nonetheless, the exact location of the assembly site within an infected host cell remains unknown. Moreover, an understanding of the chronology of events and individual protein contributions at different stages of the assembly process has been difficult to obtain. The two studies comprising this dissertation apply high-resolution genetics and high-resolution imaging to the study of HCV in cell culture. Study 1 employs a high-resolution genetics approach to reveal a functional map of the entire HCV genome. Next-generation sequencing of an insertion mutant library following passage in cell culture revealed genetic footprints that reflected known biological functions of the underlying protein regions. We show how these genetic footprints can serve as a resource to identify flexible regions that tolerate insertion of tags useful for a variety of protein detection methods. Moreover, using the genetic footprints, we identify a region in the NS4B protein that plays a role in post-RNA-replication steps. Study 2 examines HCV assembly through imaging, applying electron microscopy, electron tomography, superresolution light microscopy, multi-color fluorescence microscopy and live-cell imaging to the study of virus assembly. Our results indicate a juxtaposition of LDs, virus-like particles, membrane vesicles, clusters of HCV core protein and areas containing core-E2-NS5A proteins. The high-resolution snapshots underscore the functional compartmentalization of the LD environment, which provides viral proteins with membranous platforms to carry out a complex process such as virion assembly. We also show how our imaging platform can aid in the phenotypic characterization of an assembly-deficient mutant NS5A virus. The two studies presented in this dissertation further our understanding of the contributions of non-structural proteins such as NS4B and NS5A to the HCV assembly process. We suggest that the combination of the high-resolution genetic platform of study 1 and the high-resolution imaging platform of study 2 facilitates the identification and phenotypic characterization of viral protein regions involved in HCV assembly. A streamlined approach that integrates these two methods has the potential to identify additional targets for therapeutic intervention at post-genome-replication steps

Fidelity of target site duplication and sequence preference during integration of xenotropic murine leukemia virus-related virus.

Xenotropic murine leukemia virus (MLV)-related virus (XMRV) is a new human retrovirus associated with prostate cancer and chronic fatigue syndrome. The causal relationship of XMRV infection to human disease and the mechanism of pathogenicity have not been established. During retrovirus replication, integration of the cDNA copy of the viral RNA genome into the host cell chromosome is an essential step and involves coordinated joining of the two ends of the linear viral DNA into staggered sites on target DNA. Correct integration produces proviruses that are flanked by a short direct repeat, which varies from 4 to 6 bp among the retroviruses but is invariant for each particular retrovirus. Uncoordinated joining of the two viral DNA ends into target DNA can cause insertions, deletions, or other genomic alterations at the integration site. To determine the fidelity of XMRV integration, cells infected with XMRV were clonally expanded and DNA sequences at the viral-host DNA junctions were determined and analyzed. We found that a majority of the provirus ends were correctly processed and flanked by a 4-bp direct repeat of host DNA. A weak consensus sequence was also detected at the XMRV integration sites. We conclude that integration of XMRV DNA involves a coordinated joining of two viral DNA ends that are spaced 4 bp apart on the target DNA and proceeds with high fidelity

Comparable Accuracies of Nonstructural Protein 1- and Envelope Protein-Based Enzyme-Linked Immunosorbent Assays in Detecting Anti-Dengue Immunoglobulin G Antibodies

Background: Dengue virus (DENV) infection remains a global public health concern. Enzyme-linked immunosorbent assays (ELISAs), which detect antibodies targeting the envelope (E) protein of DENV, serve as the front-line serological test for presumptive dengue diagnosis. Very few studies have determined the serostatus by detecting antibodies targeting the nonstructural protein 1 (NS1), which can function as diagnostic biomarkers to distinguish natural immunity from vaccine-induced immunity. Methods: We used community-acquired human serum specimens, with the serostatus confirmed by focus reduction microneutralization test (FRμNT), to evaluate the diagnostic performances of two NS1-based ELISA methods, namely, immunoglobulin G antibody-capture ELISA (NS1 GAC–ELISA) and indirect NS1 IgG ELISA, and compared the results with an E-based virus-like particle (VLP) GAC–ELISA. Results: NS1-based methods had comparable accuracies as VLP GAC–ELISA. Although the sensitivity in detecting anti-NS1 IgM was poor, indirect NS1 IgG ELISA showed similar limits of detection (~1–2 ng/mL) as NS1 GAC–ELISA in detecting anti-NS1 IgG. Combining the results from two or more tests as a composite reference standard can determine the DENV serostatus with a specificity reaching 100%. Conclusion: NS1-based ELISAs have comparable accuracies as VLP GAC–ELISA in determining dengue serostatus, which could effectively assist clinicians during assessments of vaccine eligibility

High-resolution functional profiling of hepatitis C virus genome.

Hepatitis C virus is a leading cause of human liver disease worldwide. Recent discovery of the JFH-1 isolate, capable of infecting cell culture, opens new avenues for studying HCV replication. We describe the development of a high-throughput, quantitative, genome-scale, mutational analysis system to study the HCV cis-elements and protein domains that are essential for virus replication. An HCV library with 15-nucleotide random insertions was passaged in cell culture to examine the effect of insertions at each genome location by insertion-specific fluorescent-PCR profiling. Of 2399 insertions identified in 9517 nucleotides of the genome, 374, 111, and 1914 were tolerated, attenuating, and lethal, respectively, for virus replication. Besides identifying novel functional domains, this approach confirmed other functional domains consistent with previous studies. The results were validated by testing several individual mutant viruses. Furthermore, analysis of the 3' non-translated variable region revealed a spacer role in virus replication, demonstrating the utility of this approach for functional discovery. The high-resolution functional profiling of HCV domains lays the foundation for further mechanistic studies and presents new therapeutic targets as well as topological information for designing vaccine candidates

Integration of retroviral DNA and generation of short direct repeats flanking the provirus.

<p>(A) DNA breaking and joining steps during integration. Viral and target DNA strands are represented by thick black and parallel lines, respectively, and the viral long terminal repeats (LTRs) are depicted as grey boxes. Nucleotides at the top and bottom strands are denoted by uppercase and lowercase letters, respectively. During 3′-end processing, IN removes two nucleotides from the 3′ end of each strand of linear viral DNA so that the viral 3′ ends terminate with a conserved CA dinucleotide. Closed arrowheads denote the positions of strand transfer, a concerted cleavage-ligation reaction during which IN makes a staggered break in the target DNA. Host DNA repair enzymes fill in the resulting single-stranded gaps, denoted by D1 to D4 in the upper strand and d1 to d4 in the lower strand of target DNA, and remove the two unpaired nucleotides at the 5′ ends of the viral DNA (open arrowheads), thereby generating the short direct repeats flanking the provirus. (B) A potential pathway for generating a base transversion in the short direct repeat during XMRV integration. A coordinated integration of the two viral ends occurred at the 4-bp staggered positions as depicted by the closed arrowheads. During repair of the single-stranded gap adjacent to the upstream LTR, an adenine nucleotide was introduced at the D4 position either by misincorporation or aberrant processing of the unpaired AA-dinucleotide at the viral 5′ end. Subsequent repair of the mismatch resulted in the observed transversion (denoted by bold types).</p

Positions of XMRV integration sites and lengths of the target site sequence duplication.

<p>*The nucleotide position corresponds to the position of viral DNA insertion at the top strand of the chromosome indicated. Symbols + and – within the parenthesis indicate the orientation of the viral transcription is the same and opposite, respectively, to the polarity of the top strand. GenBank accession numbers for the integration site sequences are GU816075 to GU816104.</p><p>†The left LTR of the provirus contains a 5-bp deletion that includes the conserved CA dinucleotide at the viral end.</p><p>ψThe target DNA contains a T to A transversion immediately adjacent to the left LTR (position 4).</p

Base composition surrounding XMRV integration sites.

<p>Base compositions of the 4-bp target site duplication (positions D1 to D4; demarcated by the thick vertical lines) and 10 bp upstream (positions −1 to −10) and downstream (positions +1 to +10) of the direct repeat were calculated. The datasets include the 13 integration sites with correct 4-bp direct repeat (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010255#pone-0010255-t001" target="_blank">Table 1</a>), 472 integration sites from acutely infected DU145 cells (GenBank accession numbers EU981292 to EU981799) and 14 integration sites from human prostate cancer tissues (GenBank accession numbers EU981800 to EU981813) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010255#pone.0010255-Kim1" target="_blank">[14]</a>. Integration occurs between positions −1 and D1 on the top strand, and between positions D4 and +1 on the bottom strand (blue arrows). Any base in a position that is significantly overrepresented than the random dataset (<i>P</i><0.0001) is highlighted in green, while any base in a position that is significantly underrepresented than the random dataset (<i>P</i><0.0001) is highlighted in red.</p