Viral Proteins and Composition
Name 2 viruses that do not contain equimolar numbers of proteins.
Adenovirus and HSV
What are the factors important to fractionate biological molecules and subcellular particles?
Size, density, and charge
What is the sedimentation rate inversely proportional to?
Rate of migration
What is the role of capillary electrophoresis in DNA sequencing?
Separates deoxyribonucleotides using a polymer that provides high enough resolution to separate fragments ranging from 10 to greater than 1000 bases.
What enzyme allows PCR to be carried out in a single tube many times over and why is it effective?
Taq polymerase, it is a heat stable DNA polymerase
Differentiate between structural and nonstructural viral proteins
Nonstructural- nucleic acids and proteins that do not end up in the structure of the virus itself
Structural- Any protein found in purified virions (complete virus particles, i.e., genomes, capsid proteins, and any envelope and membrane‐associated proteins in the virion)
How does fractionation differ for enveloped and nonenveloped viruses?
Non-enveloped viruses are separated by size using rate zonal centrifugation. Enveloped viruses are separated by buoyant density differences.
What is the role of sodium dodecyl sulfate (SDS)?
Mildly denatures the protein and gives it a uniform negative charge
Why can't the same sequencing methods used for RNA molecules be applied to RNA virus genomes, and how is this problem overcome?
Some RNA virus genomes are extremely large and must be converted to DNA using appropriate oligodeoxyribonucleotide primers and retrovirus reverse transcriptase
What does the technique of real-time PCR provide and how is it accomplished?
It provides a more reliable and precise method of quantitatively measuring the products of PCR reactions. It’s accomplished by measuring the formation of the PCR products continually throughout all cycles of annealing and chain elongation
Name the locations of the six gene-encoded proteins in the HSV-1 capsid.
Capsomers, triplexes, inside the capsid, and capsomer tips
How do you isolate viral genomes from purified virions?
Mild disruption of the capsid proteins and extracting the nucleic acid using phenol extraction
What does staining the gels with color reagents provide?
Quantitative measure of the amount of protein of each size. The color reaction is based on reactions with amino acids in the proteins
What does DNA sequence analysis require?
DNA, a method for priming and extending the DNA and incorporating a labeled nucleotide(s), a method for detecting the terminated/labeled nucleotides, and a method either of separating the labeled fragments by size to identify the nucleotide that was labeled, or of identifying the labeled nucleotide addition as it is being extended.
How can PCR be used to detect low amounts of viral RNA?
Retrovirus reverse transcriptase generates a cDNA copy of the RNA, followed by PCR amplification, using a known primer set. If the correct primers are used, PCR can detect vanishingly small numbers of transcripts.
What are the four proteins that make up the poliovirus capsid and how are they distributed?
The four proteins are VP1, VP2, VP3, and VP4, and they are present in equimolar amounts. Each of the 12 vertices of the icosahedral capsid contains five copies of each protein.
What factors determine the sedimentation rate of a macromolecule?
Molecular size and hydrodynamic volume
What is band intensity proportional to and why is this result expected?
Protein size; In an equimolar mixture of proteins of different sizes, a small-protein polypeptide chain will have fewer amino acids than a larger one, resulting in less staining.
How has the Sanger sequencing method improved over time and what major scientific project benefited from this?
the method has been automated so that analysis can be carried out and directly entered into computer databases with little human interfacing; Human Genome Project
Give an example of how PCR can be used as an epidemiological tool and what has it helped to establish?
Used to amplify traces of influenza virus genomes still present in frozen cadavers of victims of the 1918–1920 influenza pandemic. Study of the sequence of such material has allowed scientists to establish some relationships between that virus and modern strains.
What are the important caveats for full stoichiometric analysis of a virus’s protein composition?
The preparation of virions or capsids must be homogeneous. If a preparation is made up of partial capsids, or truncated helical capsids, the analysis is not valid. Second, except for a few small enveloped viruses the number of glycoproteins in the envelope is not stoichiometric.
How do the buoyant force and centrifugal force determine the position of a viral particle in a gradient and what does this position correspond to?
Buoyant force acts to make the particle float and the centrifugal force pushes the particle to sediment lower in the gradient. The particle settles where the forces balance, this position corresponds to the equilibrium buoyant density of the virus particle.
What is the principle of gel electrophoresis?
The migration rate macromolecule in an electrical field depends on the viscosity of the medium. A medium of high viscosity discourages diffusion, such as an acrylamide gel. Macromolecules can separate based on size and shape.
How does high-throughput sequencing differ from Sanger sequencing?
HTS uses small dsDNA linkers ligated on the end of all molecules to create a sequencing library, primers are extended on glass chip (flow cell), uses microfluidics and fluorescent detection system to read bases in real time
Explain what happens when a fluorescent tag is annealed to a non-DNA product vs a DNA product.
When the primer is not annealed to a DNA product, illumination with a laser or other suitable light source will yield no fluorescence, but when the primer is annealed to the target or the amplified strand of DNA, it is able to generate a signal that can be quantitatively measured upon illumination.