MIDTERM BIOC20

Virion consists of

-Nucleic acid genome

-protective protein coat

-Some contain a lipid envelope

Virion capsid

Protective protein coat

Nature of Viruses

Obligate intracellular parasites (only replicate in living cell)

Viruses lack

Essential functions

-synthesis of basic biological building blocks (nucleotides, amino acids, CHO's, lipids)

-generation of ATP

-protein synthesis (ribosomes, tRNAs)

Uncoating

First step, virus particles break down and release genomes inside the cell

-released from protective protein shell

Once virus uncoats

Genome can be used as a template for mRNA synthesis - produce proteins

Newly made viral proteins

Work together to replicate genome, encapsulate the new genomes to form progeny virions

Virus morphology

Distinct, genome sizes, and particle sizes

-smallest 20 nm in diameter, coding few as 2 proteins

-largest 500nm in diameter, coding > 1200 proteins

Virus genomes can be

-RNA or DNA, not both

-single or double stranded

-circular or linear

Problem with RNA genome

-no RNA polymerase - replication problem

-difficulty making RNA transcript

Viruses can infect

All forms of life, animals, plants, insects, bacteria, algae,, fungi

Most abundant form of life

Viruses, 10 fold excess of virus compared to bacteria in earth oceans

Mass = 1 million blue whales

Length - 200 million light years

Study of viruses led to

Identification of promoters for eukaryotic RNA polymerase

Enzymes involved in cellular DNA replication

RNA splicing from studying viral mRNAs in eukaryotic cells

Isolation of numerous cellular oncogenes and the understanding that cancer is caused by their mutation or unregulated expression

First virus discovered

TMV

First virus discovered by

Distinguished by filtration

Russian scientist Dimitri Ivanovski

Dutch scientist Martinus Beijerinck (first name for virus = contagious virus fluidium)

TMV

Tobacco mosaic disease killing tobacco plants

Found by grounding up infected leaves trying to filter bacteria, passed through porcelain filter was infecting leaves

Discovery about viruses in mid 1930s

Highly purified TMV could form crystals

-challenged conventional notions about genes and nature of living organisms

Are virsuses alive?

Not living entities, biochemical processes - only living period inside host

1915-1917 scientist in England and France discovered bacteria

Could be listed by filterable agents

Study of bacteriophages helped establish the field of molecular biology by

Mapping phage genes

Elucidated phage replication cycles

Developed the plaque assay

Bacteriophage

Viruses that infect bacteria

Felix d'Herelle

Worked on phage therapy

-using phage to treat human bacterial disease

-useful against antibiotic resistant bacteria

-use as antibacterial agents in humans is not accepted

Tumor virus study lead to

Understanding of nature of cancer

-reverse transcriptase

-oncogenes

Now being used to construct vectors to express proteins to specifically destroy tumor cells

Plaque assay allows for

Quantitation of virus

-based on principle that bacteria diffract visible light, dense liquid cultures appear 'cloudy'

-bacteriophages lyes their host cell and this lysis causes a loss of diffraction, leading to a clearing of bacterial culture ('clear lysis' = plaque)

Reported as PFU (plaque forming unit)

Method of virus detection and measurement

Spread bacteria on surface of nutrient agar (Petri dish) nd apply serial dilutions of a phage suspension

Occurs after serial dilutions of phage suspension to detect virus

Phage binds to bacterial cell -> replicates -> releases progeny phage particles -> taken up by neighboring cells -> further replication rounds —-> visible plaque

Hemagglutination

Convenient and rapid assay for many virus

Why hemagglutination is convenient

RBC's easy to isolate and store, have visible colour

Viruses often have many copies of receptor binding proteins on their surface, and RBCs contain many copies of these receptors

Virus particles can be seen and counted by

Election microscopy

-mix virus particles with electron dense stain, virus particles exclude the stain and show up light against dark background (NEGATIVE STAINING)

Measurement of # of infections particles (plaque assay) compared to counts via electron microscopy yields

Ratio of Physical particles to infectious particles

Much greater than 1 (10, 100, 1000)

Defective particles arise spontaneously

Not all particles intact (enveloped are sensitive)

Empty capsid (no genome present)

Defective genomes (mutations, missing part or strand)

Cellular anti-viral defenses

Viral replication cycle

Single

-cells infected with an MOI (multiplicity of infection) of 10-100

-all cells infected simultaneously

-allows for synchronization of infection

Mouse polyomavirus replication cycle

May not be detectable for -20 hours (latent period)

Detailed pathway of virus replication

Binding to receptor -> entry and uncoating -> early gene expression -> replication of viral genome -> late gene expression -> assembly of virions ->exit

Step 1 of virus replication cycle

Virions bind to receptors on cell surface

-surface proteins of viruses bind CHOs of glycolipids and glycoproteins widely distributed -> non specific binding

-some require specific proteins only on certain cell types -> high specific binding, limiting virus tropism

- often a non-specific primary receptor, followed by a secondary receptor that is more specific

Step 2 of virus replication cycle

Virion (or viral genome) enters the cell

-Bacteriophage drills holes in cell membrane

-enveloped viruses fuse lipid envelope with plasma membrane

-endocytosis

Step 3 of virus replication cycle

Early viral genes are expressed: Baltimore classification of viruses

Baltimore classification of viruses

Based on pathway to mRNA synthesis

Draws attention to enzymes needed

-where the genome fits into the central dogma of information flow: DNA-> RNA -> protein

Class 1 type and example

dsDNA, herpes, pox virus

Class 2 type and example

ssDNA, adeno-associated viruses

Class 3 type and example

dsRNA, retrovirus

Class 4 type and example

(+) sense ssRNA, hepatitis A/C, picornaviruses

Class 5 type and example

(-) sense ssRNA, influenza virus

Class 6 type and example

ssRNA reverse transcribing HIV

Class 7 type and example

dsDNA reverse transcribing, Hepatitis B

Step 4 of virus replication cycle

Early viral proteins direct replication of viral genomes

-all RNA viruses must encode RNA dependant RNA polymerase (not retroviruses)

-early proteins help form RNA replication complexes

Step 5 of virus replication cycle

Late messenger RNA made from newly replicated genomes

-mechanisms that control switch from early to late gene expression are extensively studied

Step 6 of virus replication cycle

Late viral proteins package viral genomes and assemble virions

-structural proteins are most abundant viral proteins

Step 7 of virus replication cycle

Progeny virions are released from host cell

-can be via lysis (death) of host cell

-budding (with minimal effect to host cell)

-cell-to-cell via passages

Host enzymes

DNA-dependant DNA polymerase (dsDNA from ssDNA)

DNA-dependant RNA polymerase (mRNA transcripts from DNA)

Viral enzymes (not all viruses)

RNA-dependant RNA polymerase (RNA from RNA)

Reverse transcriptase (DNA from RNA template)

Capsid

Package viral genomes and transmit them to host cell

-rigid symmetrical container composed of viral protein

Nucleocapsid

Capsid with enclosed genome

Virion

Complete infectious virus particle, possibly including an envelope

Envelope virus

Many virus capsid surrounded by lipid bilayer

Virions are studied by these types of visual

Electron microscopy

X ray diffraction

Electron microsopy - negative staining

Reveals structure

Electron microscopy - positive staining

In sections of infected cells

-sub-optimal, staining artifacts

Cyroelectron microscopy (cryoEM)

Flash freeze samples

Improves preservation of structure

Data from many images is merged

Crystallization and X-ray diffraction

Higher resolution

Diffraction patterns captured and analyzed

Virus come in simple symmetrical packages

Can assemble spontaneously (self-assembly) not energy intensive, multiple identical subunits

Capsids composed of

Copies of identical subunits (genetic economy)

-avoid exhausting coding capacity of small viral genome

-requirement for symmetry of subunit interactions

Why capsids have identical subunits

Give capsid symmetry

-shape = geometry of its outline

-symmetry = rotational and translational operations that describe it (ex. Twofold axis, threefold axis)

Three types of symmetry of capsid

Tetrahedral - polygon with 4 identical triangular face

Cubic - 6 identical square faces

Icosahedral - 20 triangular faces

Icosaedral symmetry, rotational axes that are

Fivefold (12, pentagon vertices)

Threefold (20 triangular faces)

Twofold (30, oval midpoint of triangular edges)

Example of icosahedral capsid - parvovirus

-5kb ssDNA genome, 20nm diameter

-60 copies of 520aa capsid

-capsid proteins have a jelly-roll beta barrel fold

- series of interacting beta strands and extending loops tie together neighboring subunits

Example of icosahedral capsid - poliovirus

8kb ssRNA genome, 30nm diameter

3 structural subunits from 1 polyprotein: vp1, vp2, vp3

Simple capsids have

Repeating subunits with identical interactions

More complex capsids have

Repeating subunits interacting in a quasi-equivalent manner

Larger viruses come in

More complex packages

-capsids contain two types of structural subunits: herons and pentons

Capsids with helical symmetry are

Organized as helical tubes composed of identical repeating subunits

-rna winds along a groove that follows a helical path of protein subunits

- each protein subunit binds a fixed # of nucleotides

Bacteriophages contain both elements of

Symmetry icosahedral capsid head and helical 'tail'

Advantage of helical symmetry

Longer genome = longer capsid

Differences in genome length dont need to worry about helical capsid

Symmetry of helical capsid is described by

Number of subunits per turn (u) , displacement along the helical axis between one subunit and the next (p)

The pitch of the helix (P) is

The distance along the axis corresponding to one turn

P = u x p

Virus envelopes are derived from

Cellular membranes and are composed of

-lipid bilayers (same composition as the cellular membrane from which they were derived)

-viral glycoproteins (spikes sticking out of envelope)

Viral envelopes are acquired at the cell membrane by a process calledd

Budding

Budding

-viral glycoproteins are inserted to membrane

-nucleocapsids associate with glycoproteins and get wrapped in membrane

Pathway A of budding

Some nucleocapsids interact directly with cytoplasmic tails of envelope proteins (viral spikes) during budding

Pathway B of budding

Some viruses use a matrix (M) protein to interact with both viral spikes (HA and NA) and nucleocapsids (helical RNP)

Togaviruses envelope

Each protein contacts capsid protein underlying membrane -> symmetry of capsid and env proteins

Influenza virus envelope

Helical nucleocapsids and can adopt a variety of shapes/sizes (spherical and elongated) -> no obvious symmetry

How envelope is formed in virus

Viral glycoproteins are inserted into the lipid membrane to form the envelope

-most glycoproteins have a large glycosylated external domain (ECTODOMAIN) a hydrophobic transmembrane anchor domain, and a short internal tail

Multiple modes of capsid assembly exist depending on

Size, shape, and complexity of capsid

Genomic composition

Scaffolding proteins

Assist with formation of pro-capsid

Are not included in final, mature virion

Herpesvirus envelope (dsDNA)

Encapsulates the viral genome into a preassembled capsid or procapsid

Portal for DNA entry

Energy dependent process: ATP-driven motor

Packaging signals

Direct incorporation of viral genome into virions

-packaging sequence on viral genome (eg. stem-loop structure, -ve chargres) interact with capsid protein (+ve charge)

Core proteins

Accompany the viral genome inside the capsid

-condense the viral DNA, nucleoprotein complex

Formation of viral envelopes driven by

Interactions between viral proteins

-capping of viral envelope proteins

-matrix proteins at cytoplasmic face of membrane

Mechanism of viral genome release

Proteolytic cleavage of capsid or fusion proteins (most common)

Unspooling of genome into cell

Interaction of genome with cytoplasmic components

Virus classification based on

Molecular architecture, genetic relatedness and host organism

Virus species

Share a high degree of nucleocapsids acid homology, similar amino acid sequences and antigenic properties

-infect limited organisms, or specific target cells/tissues

-common genetic lineage

Virus genera

Shared characteristics, like genome organization and size, virion structure, and replication strategies

-although related by evolution divergent nt and aa sequences

-infect different organisms or cells/tissues within an organism

Virus families

Share overall/general genome organization, virion structure, and replication mechanisms

-can vary greatly in virion size and genome length

-may have unique genes from other family members

-could have evolved seperately, with limited homology of nt or aa sequences

Criteria used to classify viruses

Type of nucleic acid (DNA or RNA)

Strandedness of nucleic acid (ss or ds)

Topology of nucleic acid (linear, circular, segmented)

Symmetry of capsid (icosahedral,helical, or none)

Presence/absence of envelope

Genetically and evolutionarily viruses infect

Related organisms

-virus taxonomy takes into account viruses infecting different categories of organisms

General knowledge derived from classification groups

Virus with ssDNA genomes tend to be small viruses

Most Plant viruses have + strand RNA genomes

Many fungal viruses have ds RNA genomes

Most bacteriophages have ds DNA genomes

SsDNA genomes

Smallest of all viral genomes

Most are circular or hairpin end (linear)

-protects fragile ____ from nucleases

None are enveloped

Most are icosahedral capsid symmetry

DsDNA genomes

Include largest known viruses

-more stable genome, more complexity/adaptable

Wide range of genome sizes 5-1180kb

Viruses with dsDNA genomes have (compared to ssDNA)

Higher replication = higher error rate

Can hijack dna repair mechanisms to correct deleterious mutation

Larger = enveloped, no known infecting plants