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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?.

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slide1

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.
slide2

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
slide3

Velocity Sedimentation

Sucrose density

1.04 g/cm3

Centrifugation

1.12 g/cm3

Start

t1

t2

t3

slide4

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

slide5

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

slide6

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
slide7

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.
slide8

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
slide9

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

slide10

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

slide11

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

slide12

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

slide13

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

slide14

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

slide15

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
slide16

IMMUNOFLUORESCENCE

Fluorescent microsopy

Direct

Indirect