PHYSICAL AND CHEMICAL ANALYSIS OF VIRUSES

1. PHYSICAL AND CHEMICAL ANALYSIS OF VIRUSES How can viruses be purified and analyzed? How can viral proteins and nucleic acid be detected in infected cells? • For viruses that bud from the cell plasma membrane into the tissue culture medium, purification is much easier than for viruses where a lot of it remains cell-associated. • Budding into the medium results in freeing the virus from most of the contaminating cellular material.

2. CENTRIFUGATION TECHNIQUES USED TO PURIFY VIRUSES Particles sediment in a centrifugal field at a characteristic rate dependent on the size and shape of the particle. This is called the S value. Differential centrifugation is often the first step in virus purification and takes advantage of the large differences in S values of cellular organelles and viruses. • Nuclei can be pelleted by 600x g for 10 min. • Mitochondria, lysosomes can be pelleted by 15,000 x g for 15 min • Plasma membranes can be pelleted by 100,000 x g for 15 min • Under these conditions viruses will remain in the supernatant

3. Velocity Sedimentation Sucrose density 1.04 g/cm3 Centrifugation 1.12 g/cm3 Start t1 t2 t3

4. Equilibrium Sedimentation Sucrose density 1.04 g/cm3 Centrifugation to equilibrium 1.18 g/cm3 1.30 g/cm3 1.5 g/cm3 Estimation of buoyant density of virus Fraction protein x 1.3 g/cm3 + Fraction RNA/DNA x 1.7 g/cm3+ Fraction lipid x 0.9 g/cm3 Poliovirus: (0.7) (1.3) + (0.3) (1.7) = 1.4 g/cm3 Influenza virus: (0.8) (1.3) + (.02) (1.7) + (0.18) (0.9) = ~1.2 g/cm3 Ribosome: (0.5) (1.3) + (0.5) (1.7) = 1.5 g/cm3

5. Analysis of viral proteins by denaturing SDS gel electrophoresis • SDS is a strong denaturant that disrupts virions • Proteins containing bound SDS migrate toward cathode on gel according to molecular weight • Intensity of protein band is directly correlated with protein mass • If proteins are equimolar, smaller bands will have less mass in proportion to their MW Non-equimolar Equimolar

6. SDS gel electophoresis of the capsid proteins from poliovirus • Poliovirus contains 60 identical subunits (12 capsomeres) each with one copy of four viral proteins • Disrupted virus analyzed by SDS polyacrylamide gel electrophoresis (PAGE) indicates that the proteins are present in equimolar amounts

7. SDS gel electrophoresis of capsid proteins from adenovirus • Adenovirus contains a larger number of proteins than poliovirus and they are not present in equimolar amounts.

8. Analysis of viral genomes RNA or DNA isolated from purified virions by phenol extraction • RNA • Native gel electrophoresis (no SDS)-RNAs migrate according to size • DNA • Analysis by gel electrophoresis. • For larger genomes the DNA is cut into specific pieces using restriction endonucleases and these are separated on PAGE gels. Fragments run according to their MWs. • The restriction fragments can be cloned and sequenced and from this the sequence of the DNA can be determined

9. Other methods for determining size of RNA and DNA genomes • Sedimentation velocity in the ultracentrifuge-same principle as for virus purification • Electron microscopy-useful for large DNA molecules; need an internal standard DNA Measure length of known and unknown DNAs. Calculate MWs based on known MW of known FX174 DNA = ~5 kb HSV DNA = ~140 kb

10. DNA is composed of two complementary strands 5’-GCTAGAACTGCATG-3’ 3’-CGATCTTGACGTAC-5’ When the DNA is heated above its melting temperature or Tmit is denatured into two single strands 5’-GCTAGAACTGCATG-3’ 3’-CGATCTTGACGTAC-5’ At a temperature 10-25 degrees below the Tm the DNA will reanneal to reform the duplex structure. This process is called hybridization. 5’-GCTAGAACTGCATG-3’ 3’-CGATCTTGACGTAC-5’ This is a powerful way to detect viral RNA or DNA in the infected cell

11. Southern and Northern blots are used to detect DNA and RNA, respectively, in infected cells Electrophoresis of RNA or DNA in agarose gel After denaturing, transfer to membrane (nitrocellulose or nylon) Hybridize to radioactive probe Expose to X-ray film

12. Polymerase chain reaction (PCR) Original DNA Synthesize new DNA (use Taq polymerase) Denature; add primers Denature; make new DNA Repeat denature; synthesize DNA n times Small product increases exponentially

13. Example: PCR and RT PCR used to detect Herpes simplex virus DNA and mRNA in latently infected rabbit ganglia RT PCR PCR RNA isolated from latently infectedrabbits Uninfected ganglion Latently infected ganglion HSV HSV RT RNA isolated from reactivated rabbits actin cDNA PCR RT cDNA Size control PCR

14. DETECTION OF VIRAL PROTEINS IN INFECTED CELLS • WESTERN BLOTS Proteins from infected cell separated on SDS gel Blot proteins to filter paper Alternative to labeled staph A is to use antibodies against Fc region from a different animal. Ab comjugated with alkaline phosphatase or horseradish peroxidase Incubate with Ab to viral protein Wash and incubate with labeled staph A protein

15. IMMUNOPRECIPITATION • Label proteins in infected cells with radioactive amino acid precursor (e.g., 35S-methionine) • Prepare cell extract • Incubate with Ab to viral protein • Select Ab-Ag complexes (binding to Staph A-agarose beads is a common method) • Wash and elute proteins into SDS buffer • Run labeled proteins on SDS gels and detect by radioactivity

16. IMMUNOFLUORESCENCE Fluorescent microsopy Direct Indirect