An RS Motif within the Epstein-Barr Virus BLRF2 Tegument Protein Is Phosphorylated by SRPK2 and Is Important for Viral Replication

Epstein-Barr virus (EBV) is a gammaherpesvirus that causes infectious mononucleosis, B cell lymphomas, and nasopharyngeal carcinoma. Many of the genes required for EBV virion morphogenesis are found in all herpesviruses, but some are specific to gammaherpesviruses. One of these gamma-specific genes, BLRF2, encodes a tegument protein that has been shown to be essential for replication in other gammaherpesviruses. In this study, we identify BLRF2 interacting proteins using binary and co-complex protein assays. Serine/Arginine-rich Protein Kinase 2 (SRPK2) was identified by both assays and was further shown to phosphorylate an RS motif in the BLRF2 C-terminus. Mutation of this RS motif (S148A+S150A) abrogated the ability of BLRF2 to support replication of a murine gammaherpesvirus 68 genome lacking the BLRF2 homolog (ORF52). We conclude that the BLRF2 RS motif is phosphorylated by SRPK2 and is important for viral replication

Viral Perturbations of Host Networks Reflect Disease Etiology

Many human diseases, arising from mutations of disease susceptibility genes (genetic diseases), are also associated with viral infections (virally implicated diseases), either in a directly causal manner or by indirect associations. Here we examine whether viral perturbations of host interactome may underlie such virally implicated disease relationships. Using as models two different human viruses, Epstein-Barr virus (EBV) and human papillomavirus (HPV), we find that host targets of viral proteins reside in network proximity to products of disease susceptibility genes. Expression changes in virally implicated disease tissues and comorbidity patterns cluster significantly in the network vicinity of viral targets. The topological proximity found between cellular targets of viral proteins and disease genes was exploited to uncover a novel pathway linking HPV to Fanconi anemia

Interpreting Cancer Genomes Using Systematic Host Perturbations by Tumour Virus Proteins

Genotypic differences greatly influence susceptibility and resistance to disease. Understanding genotype-phenotype relationships requires that phenotypes be viewed as manifestations of network properties, rather than simply as the result of individual genomic variations. Genome sequencing efforts have identified numerous germline mutations associated with cancer predisposition and large numbers of somatic genomic alterations. However, it remains challenging to distinguish between background, or “passenger” and causal, or “driver” cancer mutations in these datasets. Human viruses intrinsically depend on their host cell during the course of infection and can elicit pathological phenotypes similar to those arising from mutations. To test the hypothesis that genomic variations and tumour viruses may cause cancer via related mechanisms, we systematically examined host interactome and transcriptome network perturbations caused by DNA tumour virus proteins. The resulting integrated viral perturbation data reflects rewiring of the host cell networks, and highlights pathways that go awry in cancer, such as Notch signalling and apoptosis. We show that systematic analyses of host targets of viral proteins can identify cancer genes with a success rate on par with their identification through functional genomics and large-scale cataloguing of tumour mutations. Together, these complementary approaches result in increased specificity for cancer gene identification. Combining systems-level studies of pathogen-encoded gene products with genomic approaches will facilitate prioritization of cancer-causing driver genes so as to advance understanding of the genetic basis of human cancer

A course-based research experience: how benefits change with increased investment in instructional time

There is widespread agreement that science, technology, engineering, and mathematics programs should provide undergraduates with research experience. Practical issues and limited resources, however, make this a challenge. We have developed a bioinformatics project that provides a course-based research experience for students at a diverse group of schools and offers the opportunity to tailor this experience to local curriculum and institution-specific student needs. We assessed both attitude and knowledge gains, looking for insights into how students respond given this wide range of curricular and institutional variables. While different approaches all appear to result in learning gains, we find that a significant investment of course time is required to enable students to show gains commensurate to a summer research experience. An alumni survey revealed that time spent on a research project is also a significant factor in the value former students assign to the experience one or more years later. We conclude: 1) implementation of a bioinformatics project within the biology curriculum provides a mechanism for successfully engaging large numbers of students in undergraduate research; 2) benefits to students are achievable at a wide variety of academic institutions; and 3) successful implementation of course-based research experiences requires significant investment of instructional time for students to gain full benefit

BLRF2 interacting proteins.

<p>P Prey; B Bait.</p><p>+ Positive;</p><p>− Negative.</p

BLRF2 C-terminus mediates interaction with host and EBV proteins.

<p>(A) GST pull-down assay to confirm binding of putative BLRF2 interacting proteins identified in a yeast two-hybrid assay. Lysates from 293T cells transfected with GST-BLRF2 and the indicated GFP tagged proteins were captured with GST-agarose, washed, resolved by SDS page and proteins detected by western blotting with anti-GFP (upper panels) and anti-GST antibodies (lower panels). Input lysates (2%) are shown in the left panels. (B) Mapping of BLRF2 binding partners confirmed in (A) to the BLRF2 N-terminus (GST-BLRF2 aa1–130 (i)) or C-terminus (GST-BLRF2 aa42–162 (ii)).</p

BLRF2 forms protein complex with SRPK2 and host RNA splice factors.

<p>(A) Schematic of two fractionation procedures tested to extract BLRF2 complexes. The cytosol (C) was removed by Digitonin extraction and split between two procedures. Procedure 1 was a three step process in which the membrane and organelles (M1) were collected after Triton X-100 (TX-100) lysis, the soluble fraction after NP40 lysis (S1) and the remaining insoluble pellet (P1). Procedure 2 was only two steps in which the soluble fraction (S2) was collected after NP40 lysis and the remaining insoluble pellet (P2). (B) Western blot analysis of the fractions obtained using procedure described in (A). BLRF2 extraction was monitored using rabbit polyclonal anti-BLRF2 antibody. Endogenous BLRF2 is indicted with a filled arrowhead and FLAG-HA-BLRF2 with an open arrowhead. Fraction composition was also assessed by western blotting for control cell proteins BRG1 (nuclear and chromatin bound), lamin B (cytoskeleton) and tubulin (cytoplasmic). (C) Silver stain gel of 10% of the final elutions from a tandem affinity purification of FLAG-HA-GFP and FLAG-HA-BLRF2 stable P3HR1-ZHT cell lines. Molecular weights of size markers are shown (left). (D) Network representation of interacting proteins identified in TAP-MS and Y2H generated by Pathway Palette and the BioGrid database. EBV proteins are shown as stars and host proteins as circles. The bait (BLRF2) is shown in pink. Interacting proteins are colored based on the technology that identified them (TAP – green; Y2H – yellow; Both – blue). Edges are colored based on the type of evidence used to infer the interaction (Co-complex - blue edges; Binary – red). EBV protein interactions are all binary as described in the text and host-host interaction data is derived from the Biogrid database. Only the connected components are shown. The table shows enrichment of KEGG Pathways for proteins identified by TAP-MS.</p

BLRF2 rescues ORF52 null MHV68 replication, but the BLRF2-ARA mutant cannot.

<p>(A) Complementation assay measuring MHV68 virion release by quantitative PCR into supernatants of 293T cells co-transfected with replication defective MVH68 ORF52 null BAC and empty vector, wild-type FLAG-BLRF2, or FLAG-BLRF2-ARA mutant. Viral DNA was quantified four days post-transfection and the results shown are representative of two independent experiments performed in triplicate. Western blot with anti-flag antibody shows BLRF2-WT and –ARA expression levels.</p

Characterization of BLRF2 during EBV replication.

<p>(A) Time course of EBV protein expression using whole cell lysates from P3HR1-ZHT cells (parental) or P3HR1-ZHT cells stably expressing FLAG-HA-BLRF2 induced with 4HT for 0, 24, 48 or 72 hours. Detection of tubulin serves as loading control. Endogenous BLRF2 is indicated with a solid arrowhead and FLAG-HA-BLRF2 with an open arrowhead. (B) Immunofluorescence microscopy to determine BLRF2 localization during EBV replication in P3HR1-ZHT cells (parental, top panel) or P3HR1-ZHT cells stably expressing FLAG-HA-BLRF2 (FLAG-HA-BLRF2, bottom panels), either uninduced (left) or induced with 4-Hydroxytamoxifen (4HT) for 48 hours (middle) or 72 hours (right). Anti-BLRF2 and anti-FLAG staining are shown in green and DNA staining is shown in blue. (C) Subcellular fractionation of EBV proteins and control cell proteins from P3HR1-ZHT cells stably expressing FLAG-HA-BLRF2 induced for replication with 4HT for 0, 24, 48 or 72 hours. Equal relative amounts of the cytosol (C), membrane and organelles (M), nucleus (N), chromatin bound (Ch) and cytoskeletal (Cs) fractions were probed for the indicated proteins. Tubulin served as a control for the cytosol fraction and Lamin B for the cytoskeleton fraction. As for panel A, endogenous and FLAG-HA tagged BLRF2 are indicated with filled and open arrowheads, respectively.</p