What is p32? 32 kDa protein Also known as pre-mRNA splicing - PowerPoint PPT Presentation

A N I NVESTIGATION OF THE E FFECT OF SFV I NFECTION ON P 32 L EVELS AND D ISTRIBUTION IN BHK C ELLS By Liam Rayman Supervisor: Dr Martina Scallan

What is p32? 32 kDa protein • Also known as pre-mRNA splicing • factor SF2-associated protein, Hyaluronan-binding protein 1 (HABP1), Complement 1q-binding protein (C1qbp), receptor for globular heads of C1q (gC1qR) Functions: • The exact function is unknown o Inducing apoptosis o In oxidative phosphorylation o HSV-1: o • Nuclear Egress • Redistribution of snRNA Fig. 1: Quaternary Structure of Association with HIV-1 protein Rev o p32 Redistribution of mitochondria in o Rubella Infection • Associates with Rubella capsid via arginine clusters

Semliki Forest Virus Well characterised member of the Togaviridae from the Alphavirus genus Enveloped positive sense RNA virus SFV genome codes for 4 non-structural proteins, 4 structural proteins and a non-structural peptide SFV capsid protein has been demonstrated (by reciprocal immunoprecipitation experiments in Dr Scallan’s laboratory ) to bind with p32

Objectives p32 overall levels in SFV p32 localisation in SFV infection infection Comparing the Rubella and SFV The presence of p32 in SFV capsids using Bioinformatic virions analysis

Experimental Process Seed T25cm 3 Infect BHK cells with BHK cells 24hr 24hr Whole Cell REAP Cell Perinuclear Lysate Fractionation Extraction Preparation Whole Cell, Cytosol, Core Whole Protein Cytosolic and Nucleus and Cell Lysate Nuclear Fraction Perinuclear Extract Pierce BCA Protein Assay Western Blot Analysis

Assessing p32 levels in Whole Cell Lysates Mock Infected SFV Infected Mock Infected SFV Infected p32 p32 Second set of Whole Cell Lysate Samples First set of Whole Cell Lysate Samples probed for p32 probed for p32 Mock Infected SFV Infected p32 α -Tubulin Third set of Whole Cell Lysates probed for p32 and α -tubulin

Evaluating p32 Levels in Cytosolic Fraction Mock Infected SFV Infected Mock Infected SFV Infected p32 p32 First set of Cytosolic Fractions probed for Second set of Cytosolic Fractions probed p32 for p32 Mock Infected SFV Infected p32 α -Tubulin Third set of Cytosolic Fractions probed for p32 and α -tubulin

Assessing p32 Levels in Nuclear and Perinuclear Fractions Mock Infected SFV Infected p32 Nuclear Fractions probed for p32 Mock Infected SFV Infected + Control p32 Perinuclear Fractions and Positive Control probed for p32

Investigating the Presence of p32 in SFV Virions Lysates vA774 vY193F vY184193F 100nm Virions of A7(74), Y193F and Y183193F as seen under Electron Microscope A7(74) Y193F +Control prA7(74) Y184193F p32 SFV Capsid Viral lysates and Positive Control Probed for p32

Bioinformatic analysis of Rubella Capsid and SFV Capsid Clustal Ω • determined sequences to have 18.66% identity BLAST2 • gave the sequences an identity of 19%

Future Studies • Knockout or knockdown studies • Levels at different time points post infection with SFV p32 • Fluorescence studies • Evaluate the cell fractions Methods • Virion lysates: is p32 present in the virus particle SFV

Questions