Lecture 3 and 7 part III: Virus life cycle, transcription and translation in eukaryotes

One step growth curves

used to study the replication cycle of a virus infection
Plawue assays for quantification of intracellular and extracellular virus titers

Multiplicity of Infection (MOI)

The ratio of infecting virions to host cells.
Influenced by: infectivity of the virus, environmental conditions (pH, T), duration of exposure to the virus

Bacterial growth vs viral growth

Bacteria grow by binary fission (lag, log, stationary, death);
viruses require a host to replicate, going through attachment, entry, replication, assembly, and release.

Steps of viral replication cycle

1. Attachment (adsorption)
2. Penetration (entry)
3. Uncoating
4. Replication (synthesis)
5. Assembly and maturation
6. Release

Plasma membrane

Phospholipid bilayer
Integral and peripheral proteins

Fluid mosaic model

The currently accepted model of cell membrane structure, which envisions the membrane as a mosaic of individually inserted protein molecules drifting laterally in a fluid bilayer of phospholipids.

endocytic pathways

phagocytosis, pinocytosis, macropinocytosis, receptor-mediated endocytosis

Phagocytosis

Cell eating

Pinocytosis

Cell drinking (cell ingests fluids and dissolved particles)

Macropinocytosis

A process in which large amounts of extracellular fluid are taken up into an intracellular vesicle.
Gf-induced, actin-dependent

receptor-mediated endocytosis

The uptake of specific molecules based on surface protein interactions

Virus entry

To invade different hosts viruses have to mutate their surface receptors, the keys to entering the host cells
Sometimes coreceptors and attachment factors are needed

Attachment factors

Help with the attachement of the viral particle to the cell surface

Cofactors

Needed for the activation process of entry when the viral particle is attached

Naked virus

has no membranous envelope

Naked Virus Entry mechanism

Translocation - particle crosses cell membrane intact
Genome injection - through a pore
Endocytosis - can be clathrin or pH dependent

Genome injection

entry method unique to naked virions, ex bacteriophages
They create a pore in the plasma membrane

endocytosis of naked virus

virus attaches to receptors, endocytosis, uncoating

Enveloped virus entry

membrane fusion or endocytosis
Membrane fusion within the endosome
(Viral particles are needed on cell surface and on the envelope particle to facilitate fusion)

Membrane fusion of enveloped viruses

• Lipid bilayers do not fuse spontaneously and each enveloped virus has a specialized glycoprotein responsible for membrane fusion
• pH-independent fusion, e.g. herpesviruses, HIV
• acid-triggered fusion, e.g. influenza viruses, rhabdoviruses

Endocytosis of enveloped virus

1. The virus attaches to the cell receptor.
   2 Endocytosis is initiated.
2. An endosome forms with the virus
   inside.
3. The low pH of the endosome initiates fusion of the viral envelope with the endosome membrane. The nucleocapsids are released.

Ways to interfere with virus entry

Removal of cell surface receptors (CSRs)
Blockage of CSRs with monoclonal abs
Gene-ranger experiments
Silencing of receptor expression with siRNA (knockdown)
Proteomics approaches

Models of viral entry

Viral surfing
Actin-enhanced clathrin-mediated endocytosis

Viral gene expression: translation

Viruses utilize host translation systems
Viruses have small genomes compared to other microbes and organisms

Eukaryotic DNA replication

• DNA polymerase (primer required)
• 5 > 3
• DNA polymerase's proof reading activity
• needs helicase, primase, and ligase activities
• needs ORI binding protein
• etc

Eukaryotic RNA transcription

• RNA polymerases (I, II, and III)
• synthesis 5 > 3
• transcription factors needed
• enhancers needed

Eukaryotic mRNAs

require processing to produce mature mRNAs, such as:

• capping
• polyadenylation
• RNA splicing
• movement to cytoplasm

Viral mRNA structure

5end capped or non capped,
3end poly(A)-tailed or non poly(A)-tailed

The capping occurs in the cytoplasm or in the nucleus

open reading frame (ORF)

a sequence of DNA or RNA that could be translated to give a polypeptide

Eukaryotic translation

• cap dependent
• three step (initiation, elongation, termination)

Ribosomal Scanning Model

initiation of translation. The ribosome scans for the best context of the Kozak's consensus sequence.

Kozak consensus sequence

occurs on euk mRNA; sequence is (gcc)gccRccAUGG where R is either adenine or guanine;

Protein synthesis in eukaryotes: RNAs involved

MRNA, tRNA, rRNA

Protein synthesis in eukaryotes: mRNA components

Initiation codon = AUG
Terminator = UGA, UAA, UAG
Reading frame
Untranslated region (UTR)

mRNA capping

Addition of a 5' cap consisting of a 7-methylguanosine to the 5' end fo the primary transcript.

Methyltransferase

Enzyme responsible for mRNA capping
Takes methyl group from S-adenosylmethionine (SAM) and adds it to the 5´mRNA end creating a cap

MRNA capping function

Increases RNA stability
Promotes translation
Aids in the transport from nucleus to cytoplasm
Assists splicing of the first intron

Polyadenylation

addition of 200-300 adenine (poly A-tail) nucleotides to the 3' end of an mRNA molecule. happens during RNA spilcing in eukaryotes.
Requires polyadenylation signal

Polyadenylation signal

3' end of noncoding sequence 5′-AATAAA-3′, around 20 nt upstream

poly-A tail functions

MRNA stability in cytoplasm
Translational efficiency
Assist in splicing of last intron

5´cap and poly(A) tail

They cooperate to stimulate translation and make it more efficient

Initiation of translation in eukaryotes

Cap dependent: scanning or shunting
IRES-dependent: IRES mediated or AUG-independent

Scanning Initiation in Eukaryotes

The 40S subunit enters at or near the capped 5′-end of the mRNA and then travels down the mRNA until it reaches the initiation site, which is an AUG triplet in a suitable sequence context.

Shunting initiation in Eukaryotes

The 40S subunit enters near the capped 5′-end and travels down the mRNA until it encounters a sequence that promotes partial dissociation from the mRNA strand. A subsequent signal promotes reassociation, and scanning resumes until the initiator AUG is reached.
SO it jumps

IRES- mediated initiation in eukaryotes

the 40S subunit binds directly to a structured RNA element upstream of the initiation site. This positions the ribosomal subunit at the start site or shortly upstream of it; in the latter case, the 40S reaches the initiation site by scanning.

AUG-independent initiation in eukaryotes

mediated by some insect viral IRESs. The 40S subunit is deposited directly at the initiation site, which need not be AUG in this case. Interactions with the IRES allow the first amino acid to be specified by a codon occupying the ribosomal A site, as in translational elongation.