Determination of interactions between tegument proteins of herpes simplex virus type 1

要約のみTohoku University有本　博一課

Identification of structural protein–protein interactions of herpes simplex virus type 1

AbstractIn this study we have defined protein–protein interactions between the structural proteins of herpes simplex virus type 1 (HSV-1) using a LexA yeast two-hybrid system. The majority of the capsid, tegument and envelope proteins of HSV-1 were screened in a matrix approach. A total of 40 binary interactions were detected including 9 out of 10 previously identified tegument–tegument interactions (Vittone, V., Diefenbach, E., Triffett, D., Douglas, M.W., Cunningham, A.L., and Diefenbach, R.J., 2005. Determination of interactions between tegument proteins of herpes simplex virus type 1. J. Virol. 79, 9566–9571). A total of 12 interactions involving the capsid protein pUL35 (VP26) and 11 interactions involving the tegument protein pUL46 (VP11/12) were identified. The most significant novel interactions detected in this study, which are likely to play a role in viral assembly, include pUL35–pUL37 (capsid–tegument), pUL46–pUL37 (tegument–tegument) and pUL49 (VP22)–pUS9 (tegument–envelope). This information will provide further insights into the pathways of HSV-1 assembly and the identified interactions are potential targets for new antiviral drugs

First evidence of overlaps between HIV-Associated Dementia (HAD) and non-viral neurodegenerative diseases: proteomic analysis of the frontal cortex from HIV+ patients with and without dementia

<p>Abstract</p> <p>Background</p> <p>The pathogenesis of HIV-associated dementia (HAD) is poorly understood. To date, detailed proteomic fingerprinting directly from autopsied brain tissues of HAD and HIV non-dementia patients has not been performed.</p> <p>Result</p> <p>Here, we have analyzed total proteins from the frontal cortex of 9 HAD and 5 HIV non-dementia patients. Using 2-Dimensional differential in-gel electrophoresis (2-DIGE) to analyze the brain tissue proteome, 76 differentially expressed proteins (p < 0.05; fold change>1.25) were identified between HAD and HIV non-dementia patients, of which 36 protein spots (based on 3D appearance of spots on the images) were chosen for the mass spectrometry analysis. The large majority of identified proteins were represented in the energy metabolic (mitochondria) and signal transduction pathways. Furthermore, over 90% of the protein candidates are common to both HAD and other non-viral neurodegenerative disease, such as Alzheimer's disease. The data was further validated using specific antibodies to 4 proteins (CA2, GS, CKMT and CRMP2) by western blot (WB) in the same samples used for 2D-DIGE, with additional confirmation by immunohistochemitsry (IHC) using frontal lobe tissue from different HAD and HIV+ non-dementia patients. The validation for all 4 antibodies by WB and IHC was in concordance with the DIGE results, lending further credence to the current findings.</p> <p>Conclusion</p> <p>These results suggest not only convergent pathogenetic pathways for the two diseases but also the possibility of increased Alzheimer's disease (AD) susceptibility in HAD patients whose life expectancy has been significantly increased by highly active antiretroviral therapy.</p

Novel Approaches to Detect Serum Biomarkers for Clinical Response to Interferon-β Treatment in Multiple Sclerosis

Interferon beta (IFNβ) is the most common immunomodulatory treatment for relapsing-remitting multiple sclerosis (RRMS). However, some patients fail to respond to treatment. In this study, we identified putative clinical response markers in the serum and plasma of people with multiple sclerosis (MS) treated with IFNβ. In a discovery-driven approach, we use 2D-difference gel electrophoresis (DIGE) to identify putative clinical response markers and apply power calculations to identify the sample size required to further validate those markers. In the process we have optimized a DIGE protocol for plasma to obtain cost effective and high resolution gels for effective spot comparison. APOA1, A2M, and FIBB were identified as putative clinical response markers. Power calculations showed that the current DIGE experiment requires a minimum of 10 samples from each group to be confident of 1.5 fold difference at the p<0.05 significance level. In a complementary targeted approach, Cytometric Beadarray (CBA) analysis showed no significant difference in the serum concentration of IL-6, IL-8, MIG, Eotaxin, IP-10, MCP-1, and MIP-1α, between clinical responders and non-responders, despite the association of these proteins with IFNβ treatment in MS

Determination of Interactions between Tegument Proteins of Herpes Simplex Virus Type 1

The aim of this study was to elucidate protein-protein interactions between tegument proteins of herpes simplex virus type 1 (HSV-1). To do so, we have cloned and expressed in the LexA yeast (Saccharomyces cerevisiae) two-hybrid system, 13 of the 21 currently known tegument proteins of HSV-1. These included the tegument proteins essential for replication in cell lines, UL17, UL36, UL37, UL48, and UL49, and the nonessential tegument proteins US11, UL11, UL14, UL16, UL21, UL41, UL46, and UL47. A total of 104 combinations were screened in the yeast two-hybrid assay, with 9 interactions identified. These included: UL11-UL16, UL36-UL37, UL36-UL48, UL46-UL48, UL47-UL48, and UL48-UL49. The remaining interactions consisted of self-associations that were observed for US11, UL37, and UL49. The interactions UL36-UL37, UL36-UL48, UL37-UL37, UL46-UL48, and UL47-UL48 have not been previously reported for HSV-1. The interaction of UL46-UL48 was verified using an in vitro pull-down assay. The interactions of UL36-UL37 and UL37-UL37 were verified with a coimmunoprecipitation assay. Knowledge of HSV-1 tegument protein-protein interactions will provide insights into the pathways of tegument assembly, and the identified interactions are potential targets for new antiviral drugs

Herpes Simplex Virus Tegument Protein US11 Interacts with Conventional Kinesin Heavy Chain

Little is known about the mechanisms of transport of neurotropic herpesviruses, such as herpes simplex virus (HSV), varicella-zoster virus, and pseudorabies virus, within neurons. For these viruses, which replicate in the nucleus, anterograde transport from the cell body of dorsal root ganglion (DRG) neurons to the axon terminus occurs over long distances. In the case of HSV, unenveloped nucleocapsids in human DRG neurons cocultured with autologous skin were observed by immunoelectron microscopy to colocalize with conventional ubiquitous kinesin, a microtubule-dependent motor protein, in the cell body and axon during anterograde axonal transport. Subsequently, four candidate kinesin-binding structural HSV proteins were identified (VP5, VP16, VP22, and US11) using oligohistidine-tagged human ubiquitous kinesin heavy chain (uKHC) as bait. Of these viral proteins, a direct interaction between uKHC and US11 was identified. In vitro studies identified residues 867 to 894 as the US11-binding site in uKHC located within the proposed heptad repeat cargo-binding domain of uKHC. In addition, the uKHC-binding site in US11 maps to the C-terminal RNA-binding domain. US11 is consistently cotransported with kinetics similar to those of the capsid protein VP5 into the axons of dissociated rat neurons, unlike the other tegument proteins VP16 and VP22. These observations suggest a major role for the uKHC-US11 interaction in anterograde transport of unenveloped HSV nucleocapsids in axons