MIMM 324 : Fundamental Virology Final

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Definition of Virus

Infectious, obligate intracellular parasite with genetic material and a protein coat

3 Classes of Viral Capsid

Helical, icosahedral, and complex

Viral Classification Nomenclature

Realm - viria

Kingdom - virea

Phylum - viricota

Class - viricetes

Order - virales

Family - viridae

Genus - virus

2 Virus Phases

Virion - inactive viral particle

Infection - multiplying inside of host

Susceptible Cells

Cells that support viral entry

Permissive Cells

Cells that support entire viral cycle

Early Phase Events

Attachment → Penetration → Uncoating → Early Gene Expression → Genome Replication

Late Phase Events

Late Gene Expression → Assembly → Release

How To Generate New Viral Strains

Reassortment of Segmented Genome in Cell Infected w/ 2+ different strains

Polarity of mRNA Strands

Positive Polarity

Polarity of Anitgenome

Opposite of ssRNA Genome, can be +/-

2 Types of RNA That Are Never Translated

-RNA and dsRNA

Baltimore Class 1

dsDNA virus

Baltimore Class 2

ssDNA virus

Baltimore Class 3

dsRNA virus, carries RDRP

Baltimore Class 4

(+)ssRNA virus, encodes RDRP

Baltimore Class 5

(-)ssRNA virus, carries RDRP

Baltimore Class 6

(+)ssRNA-RT virus, carries reverse transcriptase

Baltimore Class 7

dsDNA-RT virus, carries reverse transcriptase, gapped genome that is first repaired by host mechanisms

Most common baltimore classification in bacteria / archaea viruses

Class 1 - dsDNA

Most common baltimore classification in plants

Class 4 - (+)ssRNA

Most common baltimore classification in humans

Infected equally by Class 1 - dsDNA, Class 4 - (+)ssRNA, and Class 5 - (−)ssRNA viruses.

Chicken Eggs for Vaccine Production

Virus injected into embryonated chicken eggs, used historically for poliovirus vaccine production

Primary Cell Culture

Prepared from animal tissue, only lives 15-20 divisions

Continuous Cell Culture

Tumor tissue or immortalized cell line, replicates indefinitely but may not resemble original cell.

ex. HeLa cells

3 Ways of Making Virus in Cell Culture

1. Infection by viral particle

2. Transfection of (Infectious) viral genome, can never be (-)ssRNA

3. Using an infectious DNA clone, dsDNA plasmid version

Cytopathic Effects

Morphological changes in infected cells. 

ex. Lysis, syncytium (fusion), rounding of cells, detachment, and inclusion bodies

How to Find Life Cycle Time

1. prepare pure viral stock

2. determine its viral titre

3. infect cells at appropriate MOI

4. Perform one step growth cruve

Plaque

Area of lysed cells. Each virion is capable of infecting one cell, lysing it, and releasing new virions to infect neighbouring cells.

Virus Titre

(#plaques / volume plated) * dilution factor

Multiplicity of Infection

Average number of virions added per cell during infection

Cryo Electron Microscopy

Freezing viral particles in water (vitrification), imaging, 3-D and reconstruction of images. Can resolve down to near atomic resolution

Capsid

Protein shell surrounding viral genome

Nucleocapsid

Nucleic acid - protein complex within the virion, normally in enveloped viruses.

Envelope

Host-derived lipid bilayer surrounding the capsid, can contain glycoproteins.

Self Assembly

Most capsid proteins are capable of self-assembly. Each capsid must make identical contacts with its neighbours. Bonding contacts are usually low affinity and non-covalent

Capsid Symmetry

1. helical - for rod shaped

2. icosahdral - roughly spherical

3. platonic polyhedral - spherical or platonic solids (cube, dodecahedral, etc)

Helical Capsids

Usually flexible, rod shaped. Package ssRNA

ex. Tobacco Mosaic Virus

Icosahedral Capsids

Rigid circular shells that package ds/ss DNA/RNA. Made of 60 IAUs.

Icosahedral Asymmetric Unit (IAU)

The smallest repeating unit of an icosahedral capsid from which the complete structure can be built with 5:3:2 symmetry.

Triangulation Number

The number of capsid proteins in the IAU. Smallest capsids have 1 protein per capsomere, so T=1

Quasiequavalent Interactions

head-to-head and tail-to-tail interactions all capsid proteins engage in

Complex Capsids

Capsids that combine different types of symmetry

ex. Bacteriophage: icosahedral head and helical tail

Viral Envelope Glycoproteins

Proteins located on the surface of viral envelopes that facilitate attachment and entry into host cells. Have TM domain, ectodomain, and internal domain.

3 Modes of Interaction Between Envelope and Nucleocapsid

1. direct

2. via matrix protein

3. via multiple proteins

General Viral Life Cycle

1. Attachment

2. Penetration

3. Uncoating

4. Early Gene Expression

5. Replication

6. Late Gene Expression

7. Assembly

8. Release

Viral Entry

Steps up to and including transport of viral genome to site of transcription and replication.

Attachment

Virus reversibly binds to attachment factors via non-specific interactions on cell surface then binds irreversibly to specific receptor(s).

Naked vs. Enveloped Virus Receptor Binding

Naked uses capsid surface or protrusions. Enveloped use TM glycoproteins.

Class 1 Glycoproteins

Forms trimeric spikes at the surface of enveloped viruses. Synthesized as precursor that is processed by host proteases into receptor binding subunit and membrane fusion subunit. Largely a-helical.

Clathrin-Mediated Endocytosis

Virus-receptor complex is taken up by a clathrin-coated pit that is pinched off the membrane by dynamin, a GTPase. It then fuses with an early endosome

Caveolin-Mediated Endocytosis

A dynamin- and caveolin-dependent pathway is used by some viruses, like polyomaviruses (SV40). Virions reach the ER via the caveosome, a pH-neutral endosomal compartmen

Macropinocytosis

Virus incorporates phosphatidylserine phospholipids to mimic apoptotic bodies. Allows take-up by macrophages. Used by Dengue and Ebola virus.

Furin

Golgi protease used to cleave glycoprotein subunits, priming them.

Class 1 Mediated Membrane Fusion

Prehairpin Intermediate

Helical coil that tethers viral membrane and host membrane together via TM anchor domain and fusion peptide respectively. Triggered by receptor binding or low pH.

Class 2 Glycoproteins

Oriented parallel to the surface of the virion, rich in B-strands. Low pH triggers insertion of loops into endosome membrane. Requires priming by cleavage of an associated protein, not the fusion protein itself.

Class 2 Mediated Membrane Fusion

Membrane Perturbation

How naked viruses enter the cell. Either disrupt the membrane, use a membrane penetrating peptide, form a pore, or lipases.

Pore Formation

Ex. poliovirus

1. release of pocket factor, lipid bound protein that stabilizes the virus

2. formation of VP4 hexameric pores in endosome membrane

3. insertion of VP1 N-terminal peptide into membrane as anchor

Uncoating

Occurs concomitantly with membrane crossing, capsid is either degraded or removed to release viral genome. Can occur in cytoplasm, at nuclear membrane, or in nucleus.

Viral Nucleus Entry

Can happen when the nuclear membrane is degraded during division, by entering through the nuclear pore complex, or by disruption of nuclear membrane.

2 Modes of RNA depended RNA Synthesis

De novo initiation - without primer

Primer depended initiation

Viral RdRP

Error prone intentionally to promote genetic diversity. 2 Mg2+ ions catalyze formation of phosphodiester bond. Carried in class 3 and 5, encoded in class 4.

(+)ssRNA Protein Priming

ex. poliovirus

Uses 5’ VPg protein to prime synthesis

(-)ssRNA Protein Priming

Viral nucleoprotein (N) promotes full length synthesis by acting as an anti-termination factor. Coats viral segments to form viral ribonucleoprotein complexes.

Cap Snatching

Viral RdRP has nuclease activity, steals 10-20 nucleotides from host mRNA 5’ caps. Cap primers used to make subgenomic viral mRNAs.

Ambisense RNA Virus

Has both (+) and (-) polarity in its genome.

ex. arenavirus, lassa virus

Lassa Virus Transcription

Transcription termination signal in IGR prevents synthesis of long mRNAs that may bind to each other and trigger host antiviral response

dsRNA Transcription

Transcription and Replication occur in viral subparticle to prevent host antiviral response.

U-Stretches

Template RNA may contain “slippery” stretches of U residues in intergenic region that RdRP to slip and produce long segments of A. Functions as a poly A tail.

IRES

Non-canonical, cap dependent initiation. Uses an Internal Ribosome Entry Site in the 5’ UTR to allow internal initiation based on secondary structure.

Ribosome Shunting

Ribosomes bypass parts of the 5’ UTR to reach start codon or bypass stable secondary elements. Favored by shunting elements such as loops or proteins.

Leaky Scanning

Some, but not all, ribosomes fail to recognize the first start codon and keep going until they find a new one.

Cricket Mosaic Virus Initiation

CrPV IRES mimics met-initiator tRNA. Can recruit 80s ribosome without eIFs and Met-tRNA. Turnip yellow mosaic virus works similarly.

Reinitiation

Fraction of ribosomes that translated an upstream ORF will reinitiate translation at a downstream start codon.

Ribosomal Frameshifting

Ribosome slips by 1 nucleotide, misses stop codon, and continues in a different reading frame.

Readthrough

Ribosome reads through a stop codon but doesn’t stop elongating.

Host Translational Repression

Virus inhibits the host's transcription processes, preventing the expression of host genes.

ex. poliovirus 2A protease cleaves eIF4G, stopping cap dependent initiation.

Factories / Inclusions

Site within infected cells where viral replication and assembly occurs, often containing viral components and host chaperones.

Packaging Signals

Specific DNA sequence or RNA secondary structure needed for selective packaging in sequential assembly

Packaging of Large dsDNA Genomes

Sequential packaging. Capsid formed, and scaffolding proteins bind. Terminases open the cancatimer to pump dsDNA in.

Segmented RNA Packaging

Packaged as a complex of 7 vRNP around central vRNP

Retroviral (+) RNA Packaging

ex. HIV

Psi packaging signals bind GAG precursor which signals to stem loops to interact with kissing loop sequence to form dimer initiation site. Packages in capsid.

Envelope Acquisition

Occurs after assembly of internal structures. Virion takes envelope from nuclear envelope, golgi, ER, or plasma membrane.

Retrovirus Budding

ESCRT recruited to GAG via late domain, used to pinch off bud, releases viral particle. Loss of late domain → failure to release infectious particle.

HIV Maturation

Occurs after budding, involves protease activation to cleave GAG and Gag Pol precursor proteins in individual compartments.

Dengue Virus Maturation

Occurs as virion moves from ER through the TGN, pH induces maturation via pH dependent cleavage

Naked Virus Release

1. Lysis from apoptosis, necropoptosis, or pyropoptosis. Viral proteins can induce rupture of cell membranes (lytic enzymes, vipoporins)

2. Non Lytic release from vesicles, exosomes, and autophagic vesicles

Orthomyoxoviridae

(-)ssRNA virus family. Contains influenzavirus.

Influenza Virion

Enveloped, rounded (sometime filamentous) ~100nm particle. Has segmented linear genome encapsulated by nucleoproteins (vRNP)

Influenza Genome

13.5kb linear segmented. Produces 12-14 proteins.

Influenza Receptors

Neuraminic/Sialic Acids (Sugars)

Coronavirus

(+)ssRNA family. Contains Sars-Cov-2

Coronavirus Virion

Roughly spherical, moderately pleomorphic. ~118-136 nm diameter, spike proteins give crown like appearance

Coronavirus Genome

30kb w/ 5’ cap and 3’ polyA tail. 2/3 is nonstructural proteins, other 1/3 is structural and accessory.

Coronavirus Capping

Encodes own RNA capping mechanism, happens in cytoplasm

Coronavirus Entry Pathways

Early - direct membrane fusion, S2’ cuts at cell surface, major

Late - endosomal fusion in late endosome, minor

Coronavirus Replication Organelles

Double Membrane Vesicles (DMVs), prevents host antiviral response. Membrane taken from ER

Coronavirus NSPs

Responsible for DMV formation, capping proteins, and replication/transcription complex core. Derived from polyprotein precursors