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?.
How can viruses be purified and analyzed?
How can viral proteins and nucleic acid be detected in infected cells?
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.
Centrifugation to equilibrium
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
Analysis of viral proteins by denaturing SDS gel electrophoresis
RNA or DNA isolated from purified virions by phenol extraction
Measure length of known and unknown DNAs. Calculate MWs based on known MW of known
FX174 DNA = ~5 kb
HSV DNA =
When the DNA is heated above its melting temperature or Tmit is denatured into two single strands
At a temperature 10-25 degrees below the Tm the DNA will reanneal to reform the duplex structure. This process is called hybridization.
This is a powerful way to detect viral RNA or DNA in the infected cell
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
Synthesize new DNA (use Taq polymerase)
Denature; add primers
Denature; make new DNA
Repeat denature; synthesize DNA n times
Small product increases exponentially
Example: PCR and RT PCR used to detect Herpes simplex virus DNA and mRNA in latently infected rabbit ganglia
RNA isolated from latently infectedrabbits
Latently infected ganglion
RNA isolated from reactivated rabbits
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