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

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

Jun 03, 2023 154 likes •279 views

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

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AN INVESTIGATION OF THE EFFECT OF SFV INFECTION ON P32 LEVELS AND DISTRIBUTION IN BHK CELLS

By Liam Rayman Supervisor: Dr Martina Scallan

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SLIDE 2

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

  • f C1q (gC1qR)
  • Functions:
  • The exact function is unknown
  • Inducing apoptosis
  • In oxidative phosphorylation
  • HSV-1:
  • Nuclear Egress
  • Redistribution of snRNA
  • Association with HIV-1 protein Rev
  • Redistribution of mitochondria in

Rubella Infection

  • Associates with Rubella

capsid via arginine clusters

  • Fig. 1: Quaternary Structure of

p32

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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

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SLIDE 4

Objectives

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

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Experimental Process

Seed T25cm3 with BHK cells Infect BHK cells

24hr

REAP Cell Fractionation Whole Cell Lysate Preparation Perinuclear Extraction

24hr

Cytosol, Core Nucleus and Perinuclear Extract Whole Cell, Cytosolic and Nuclear Fraction

Whole Protein Cell Lysate Pierce BCA Protein Assay Western Blot Analysis

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SLIDE 6

Assessing p32 levels in Whole Cell Lysates

Mock Infected SFV Infected

p32

First set of Whole Cell Lysate Samples probed for p32

Mock Infected SFV Infected

p32

Second set of Whole Cell Lysate Samples probed for p32

Mock Infected SFV Infected

p32 α-Tubulin

Third set of Whole Cell Lysates probed for p32 and α-tubulin

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SLIDE 7

Evaluating p32 Levels in Cytosolic Fraction

Mock Infected SFV Infected

p32

First set of Cytosolic Fractions probed for p32

Mock Infected SFV Infected

p32

Second set of Cytosolic Fractions probed for p32

Mock Infected SFV Infected

p32 α-Tubulin

Third set of Cytosolic Fractions probed for p32 and α-tubulin

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SLIDE 8

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

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SLIDE 9

Investigating the Presence of p32 in SFV Virions Lysates

100nm

vA774 vY193F

vY184193F

Virions of A7(74), Y193F and Y183193F as seen under Electron Microscope

Y184193F Y193F +Control A7(74) prA7(74)

p32 SFV Capsid Viral lysates and Positive Control Probed for p32

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SLIDE 10

Bioinformatic analysis of Rubella Capsid and SFV Capsid

ClustalΩ

  • determined

sequences to have 18.66% identity

BLAST2

  • gave the sequences

an identity of 19%

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SLIDE 11

Future Studies

p32

  • Knockout or knockdown studies
  • Levels at different time points post infection with SFV
  • Fluorescence studies

Methods

  • Evaluate the cell fractions

SFV

  • Virion lysates: is p32 present in the virus particle
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SLIDE 12

Questions