This site is 100% ad supported. Please add an exception to adblock for this site.

Biology 554 Molecular Virology


undefined, object
copy deck
Mechanism causing disease
Severity of disease-causing potential
General Range in Size of Viruses
75 to 200 nm (0.075-0.200 micrometers)
Source of virus
e.g. infected animal
Agent or means by which an infection is spread or transmitted from one individual to another
e.g. aerosol
In a reaction to infection, cellular proteins are made that can interact with neighboring cells and induce those cells to become wholly or partially resistant to virus infection

Interferon production limits spread of virus to uninfected cells.
Incubation Period
The time between initial infection and the observation of clinical symptoms of disease
The infection did something permanent
ex: paralytic polio
Presence of virus in the circulatory system
Innate responses to viral infection:
Early responses: Inflammation, macrophage destruction of infected cells, fever, and interferon production
Adaptive responses to viral infection
requires maturation of both B and T lymphocytes
Effector T cells
Short-lived, kill cells expressing foreign antigens on their surfaces
Helper T cells
drive maturation of B cells into antibody-secreting cells
Functions of Capsid
Viral proteins assemble into capsids that protect viral genomes and allow entry into cells
Destruct infected cells
Individual protein units of a capsule
2 stable shapes of virion structure
Icosahedral and Helical
Baltimore Scheme for classifying viruses
Based on how virus produces mRNAs
Mostly used to distinguish different types of RNA viruses
Positive Sense
virion RNA is like cellular mRNA
Negative Sense
opposite polarity to cellular mRNA, requires a virion-associated enzyme to begin replication cycle
Cis-acting signals
Genetic elements that act in cis work only in the context of the genome in which they are present.

A cis-acting transcription promoter facilitates the transcription of adjacent polypeptide-encoding sequences
Trans-acting signals
Trans-acting elements are just that info expressed to act, more or less freely, at numerous sites within the cell

trans-acting promoters affect the transcription of regions of DNA not in close physical proximity
What is Pol I?
copies ribosomal RNA
What is Pol II?
synthesizes most messenger RNA
What is Pol III?
synthesizes small RNAs like tRNA
Promoter sequences
a DNA sequence that enables a gene to be transcribed. The promoter is recognized by RNA polymerase, which then initiates transcription. In RNA synthesis, promoters are a means to demarcate which genes should be used for messenger RNA creation - and, by extension, control which proteins the cell manufactures.
Enhancer sequences
a short region of DNA that can be bound with proteins (namely, the trans-acting factors, much like a set of transcription factors) to enhance transcription levels of genes (hence the name) in a gene-cluster
encode several genes
there is a particular factor in the cell necessary to allow transcription termination at a specific site

Termination of transcripts at some specific sites requires participation of a protein factor.

A DNA sequence signaling the termination of transcription; termination requires the presence of the rho protein.
In procaryotes, transcription can be terminated at specific sites without the intervention of additional factors
Kozak sequence
Has to have a G or A at -3 position
cellular transmembrane glycoproteins
Virus receptors

Favored by many viruses as receptors to get physically very near to the cell membran

Could act as a signal transducer
cell to cell fusion (syncytia formation)
Some viruses after gaining entry into cells can bypass receptor and infect adjacet cells by cell to cell fusion
Alternate receptor
Used when the normal receptor is not available
A component of the cell surface that is required along with the receptor to get to the next steps in virus entry
How do non-enveloped viruses enter cells?
Receptor-mediated endocytosis is generally employed by non-enveloped viruses.

1. Virion-receptor interaction triggers clathrin coated pit formation
2. Endocytotic vesicle forms and becomes acidified
3. Partial degradation of virion and potential expression of processed antigen
4. Clathrin released virion partially "opened"
5. Viral genome (mRNA) released in cytoplasm
How does acidification differ with non-enveloped virus entry and enveloped virus entry?
Acidification of the enveloped virus causes fusion of the viral and vesicle membrane, therefore releasing its core contents.
Bacteriophage Entry Mechanisms: 3 steps
1. Weak interaction between tail fiber protein and cell surface receptor
2. Strong interaction between tail pins and outer membrane which triggers compression of tail sheath and tail tube penetration through cell wall
3. viral pilot protein enables DNA translocation through inner membrane
Formation of virus envelope occurs in the...
Golgi Complex of the cell.

Glycosyltaion starts in the rough ER, Viral glycoproteins transported to membrane in vesicle.
Enveloped viruses bud from what cellular membranes?
some viruses bud from both nuclear and plasma membranes; others bud only from the plasma membrane
Scaffolding Proteins
A scaffold protein is a protein whose function is to promote other protein-protein interactions.
Scaffold proteins organize chromosome domains within the nucleus.
Two outcomes of virus infection in cells:
1. Abortive or non-productive infection.
2. Productive infection.
Abortive or non-productive infection
e.g. Cell is non-permissive because it lacks some element required fro virus replication or virus has some defective gene product.

Virus got into cell, but lacks something that is required for replication.

Not the same as the virus not being able to find a cellular receptor.
Productive infection
New infectious virus is produced.
Virus latency
Virus latency is the ability of a pathogenic virus to lie dormant within a cell (latent infection).

The entire genome is there, and it can code for an infectious virus, but there is no infectious virus being produced.
What is the fate of the cell following virus infection?
1. The cell survives despite producing infectious virus.
2. Cell is killed by necrosis.
3. Cell is killed by apoptosis.
What is a persistent infection?
The cell survives despite producing infectious virus.
What is necrosis?
The cell contents are released generally leading to immune reaction with inflammation and potential pathology.
What is apoptosis?
Phased or programmed shutdown of cellular functions.

A way for the cell to die in a useful way. Apoptosis normally has a protective function (i.e. eliminates no longer needed highly differentiated cells, aged cells, mutated cells, etc.)

Doesn't cause the immune system to "freak out."
What is equilibrium density centrifugation?
Based on density gradient.

The material is either layered on top of or mixed into some material that can either be preformed into a density gradient or will become a density gradient when it is spun at high speed. The centrifuge is then run until the material finds its place as a band of particular density within the tube.

Typically, a sucrose density gradient is created by gently overlaying lower concentrations of sucrose on higher concentrations in a centrifuge tube. For example, a sucrose gradient may consist of layers extending from 70% sucrose to 20% sucrose in 10% increments (though this is highly variable depending on sample to be purified). The sample containing the particles of interest is placed on top of the gradient and centrifuged at forces in excess of 150,000 x g. The particles travel through the gradient until they reach the point in the gradient at which their density matches that of the surrounding sucrose. This fraction can then be removed and subjected to further analysis.
What is differential centrifugation?
Differential centrifugation is a procedure in which the homogenate is subjected to repeated centrifugations each time increasing the centrifugal force.
Why is SDS-PAGE useful?
Can be used to determine molar ratio of virion proteins, and therefore help determine shape (ex: 12:20:30 ratio)
What is Northern Blotting?
is analogous to Southern Blotting; mRNA transcripts rather than DNA restriction fragments are probed with specific RNA or DNA probes

The gel from the DNA electrophoresis is treated with an alkaline solution (typically containing sodium hydroxide) to cause the double-stranded DNA to denature, separating it into single strands. Denaturation is necessary so that the DNA will stick to the membrane and be hybridized by the probe (see below). Restriction endonucleases are used to break the DNA strands into fragments.
A sheet of nitrocellulose (or, alternatively, nylon) membrane is placed on top of the gel. Pressure is applied evenly to the gel (either using suction, or by placing a stack of paper towels and a weight on top of the membrane and gel). This causes the DNA to move from the gel onto the membrane by capillary action, where it sticks.
The membrane is then baked (in the case of nitrocellulose) or exposed to ultraviolet radiation (nylon) to permanently crosslink the DNA to the membrane.
The membrane is now treated with a hybridization probe - an isolated DNA molecule with a specific sequence that pairs with the appropriate sequence. The probe DNA is labelled so that it can be detected, usually by incorporating radioactivity or tagging the molecule with a fluorescent or chromogenic dye. In some cases, the hybridization probe may be made from RNA, rather than DNA.
After hybridization, excess probe is washed from the membrane, and the pattern of hybridization is visualized on x-ray film by autoradiography in the case of a radioactive or fluorescent probe, or by development of color on the membrane itself if a chromogenic detection is used.
1. Mutant viruses are often recognized because they cannot grow under conditions where wild-type virus grows.
2. If the defects in 2 different mutants involve the same gene, double infection of cells under restrictive conditions produces no virus.
3. If the defects involve different genes, wild-type protein products acting in trans allow virus to grow or complement each other.
4. Almost all the virus resulting from growth by complementation (except for possible recombinants) have the same genotypes as the parents.
Double infection with an HSV temperature sensitive pol mutant and a thymidine kinase deficient virus leads to...
rare recombinant progeny that can be detected under selective conditions
Measuring Recombination frequencies:
Co-infect cells at PERMISSIVE conditions, then score progeny for their genotype.

Recover virus from a double infection under PERMISSIVE conditions and determine PROPORTION of virus showing wild-type genotype.

Double infection of cells with virus mutated in different genes under permissive conditions will allow low frequency recombination between the two different genomes.
Complentation Analysis:
Co-infect cells at NON-PERMISSIVE conditions, then score for what progeny you get.

Yield of infectious virus from double infections vs single infections under RESTRICTIVE conditions.
What are Positive Sense RNA Viruses?
Genome can be translated directly to produce viral protein.

positive-sense genomes which can act as mRNA and be used directly to synthesise proteins without the help of a complementary RNA intermediate. Because of this, these viruses do not need to have an RNA transcriptase packaged into the virion.
What are Negative Senes RNA Viruses?
Negative stranded, has to carry within the virus particle an RNA polymerase (transcriptase)that can copy negative sense genome into mRNA that can then be translated into Viral Protein.

Negative-sense RNA (like DNA) has a nucleotide sequence complementary to the mRNA that it encodes. Like DNA, this RNA cannot be translated into protein directly. Instead, it must first be transcribed into a positive-sense RNA which acts as an mRNA. Some viruses (Influenza, for example) have negative-sense genomes and so must carry an RNA transcriptase inside the virion.
What is the Baltimore Scheme of classification?
The Baltimore Scheme of classification distinguishes between viruses whose genomes can be utilized directly as mRNA (positive stranded RNA viruses) vs. those that require a virion-associated "transcriptase" to produce mRNAs (negative stranded RNA and dsRNA viruses)
What are the major groups of Positive Strand RNA viruses?
Picornaviridae (Poliovirus)
Flaviviridae (Yellow Fever)
Togaviridae (Sindbis Virus)
Coronaviridae (MHV-Mouse Hepatis Virus)
Leviviridae (Qb)
What is a Serotype?
A serovar or serotype is a group of microorganisms or viruses based on the cell surface antigens.

Capsid structure is similar, but amino acid composistion is different.
What are some examples of picornaviruses?
Hepatitus A
Coxsackie A and B
How is polio spread?
Fecal contamination
What are some distinguishing features of the poliovirus genome?
1. About 7700 nucleotides long and encodes a single open reading frame
3. Long 5' untranslated region (~800 nucleotides)contains an Internal Ribosome Entry Site (IRES)
4. Encodes 2 viral proteases (proteins 2A and 3C) that process viral precursor protein. AUTOCLEAVAGE
What is an open reading frame?
An open reading frame or ORF is any sequence of DNA or RNA that can be translated into a protein. In a gene, ORFs are located between the start-code sequence (initiation codon) and the stop-code sequence (termination codon). ORFs are usually encountered when sifting through pieces of DNA while trying to locate a gene.
Poliovirus Replication occurs in what part of the cell?
Exclusively in the cytoplasm
What is VpG?
VpG is a primer for virus RNA replication

It is linked to the 5' end of the poliovirus genome.
How does Poliovirus enter the cell?
receptor mediated endocytosis
Where does viral RNA synthesis take place in the cell?
Viral RNA synthesis takes place in association with virus-modified cellular membranes in the virus-modified endoplasmic reticulum.
How does poliovirus shut off the HOST protein synthesis?
It cleaves the host translation initiation factor (eIF-4)

eIF-4 is what recognizes the mRNA cap structure. Without eIF-4 the cell cannoth begin mRNA translation.
What is an example of Picornaviridae?
What is an example of Flaviviridae?
Yellow Fever
What is an example of Togaviridae?
Sindbis Virus
What is an example of Coronaviridae?
Mouse Hepatitus Virus
What is and example of Leviviridae?
What is Marker Rescue?
Repair of a mutational defect by recombination. For example, when a cell is co-infected with a mutant phage that is unable to replicate and a wild-type phage, recombination between the two phage can repair the replication defect and allow the recombinant derivative of the mutant phage to reproduce.
What are Neurotropic Viruses?
Neurotropic viruses target neurons.
What are some examples of Flaviviridae?
Includes a large group of mosquito-borne agents:

Yellow fever
West Nile
Denque Fever
St. Louis Encephalitis
What are some characteristics of Flaviviridae?
Positive Sense RNA
Have a single ORF
Many are neurotropic
~10000 nucleotides long
5' cap but no poly(A) tail.
What is the purpose of a poly(A) tail on mRNA?
Helps translation by circularizing the RNA.

Poly(A) binding proteins bind to 3' end. These binding proteins bind to initiation factors that contain the cap structure, and circularize the whole thing. This facilitates translation.
Hepatitis A is what type of virus?
Hepatitis C is what type of virus?
What are some characteristics of Togaviridae?
Positive Sense RNA
Encode a subgenomic mRNA
Many are arboviruses and neurotropic.
~11000 nucleotides long
Has 5' cap and a poly(A) tail
Encodes 2 ORFs
What is Rubella?
AKA German Measles

Not an arbovirus, but it is a togavirus; normally causes only mild disease but also associated with arthritis and neurological complications; often leads to miscarriage if acquired during 1st trimester of pregnancy.
What is Sindbis virus?
Prototype of Togaviridae.
The non-structural proteins are encoded at the 5’ end, formed during the first of two characteristic rounds of translation. These proteins are originally translated as a polyprotein, which consequently undergo self cleavage, forming four non-structural proteins responsible for gene expression and replication. The formation of a sub-genomic fragment, encoding the structural proteins and a negative sense fragment, a template for further synthesis of positive sense RNA are the characteristic second phase of translation. Assembly takes place at the cell surface, where the virus buds from the cell, acquiring the envelope. The replication cycle is very fast, taking around 4 hours.
Why does the Sindbis virus encode its own capping enzyme? Why doesn't it use the enzyme from the cell?
Capping occurs in the nucleus of the cell. The virus does not have access to the nucleus, so it has its own capping enzyme.
What is one major difference between Togaviridae and Picornaviridae and Flaviviridae?
Togaviridae have subgenomic mRNA and Picornaviridae and Flaviviridae have only 1 ORF.
What are some examples of Coronaviridae?
SARS, MHV (Mouse Hepatitis Virus)
What are some characteristics of Coronaviridae?
Largest viral RNA genome~30-35 kb
Capped and Poly(A) tail
Positive Sense RNA virus
Helical Nucleocapsid
Five major ORFs translated from 7 3' coterminal subgenomic RNAs
Which ORF on each subgenomic RNA is translated?
Only the first ORF

Deck Info