MIMM 324 midterm

what is a virus?

Infectious, obligate intracellular parasite

where did viruses come from?

1. the virus first hypothesis (viruses predate or coevolved w/their current cellular hosts)
2. the regressive/reduction hypothesis (viruses are remnants of cellular organisms)
3. the progressive/escape hypothesis (viruses arose from genetic elements that gained the ability to move between cells)

2 phases of viruses

inanimate phase, the virion; multiplying phase, in the infected cell

virus classification

nature and sequence of nucleic acid virion, symmetry of capsid, presence or absence of envelope, dimensions of virion and capsid

steps of the infectious cycle

1. receptor binding
2. entry and uncoating
3. early gene expression
4. replication of viral genome
5. Late gene expression
6. Assembly of virions
7. Exit

Receptor binding

virus encoded proteins of virion bind to specific proteins/carbs/lipids on cell surface; cell receptors are specific for each virus and host species

Entry and Uncoating

once they've located the right cell, need to pass through cell wall or membrane(s); bacteriophages drill holes/inject genome, plant viruses penetrate as a result of damage to cell wall, animal viruses taken up via membrane fusion or endocytosis; once inside, capsid disintegrates to release genome (Uncoating)

Early gene expression

viral genome must direct expression of early proteins (typically direct genome rep); molecular pathway depends on chem nature and strandedness of viral genome

Replication of viral genome

early proteins promote rep of viral genome, cell becomes factory for expression and replication of viral genomes

Late gene expression

many viruses express late mRNAs from newly replicated genomes, late viral proteins typically structural proteins used to make viral particles

Assembly of virions

structural proteins package viral genomes and assemble the capsid; enveloped viruses encode glycoproteins inserted into lipid membranes and direct formation of envelope upon release

Exit

progeny virions released from the host cell (death/lysis of host cell, extrusion from cell membrane); virions find and infect new host, reinitiate rep cycle

Hershey-Chase Experiment explained

took bacteriophages, grew in radioactive sulfur; sulfur present in AAs not NAs, labels protein part of phage; took phages and mixed w/bacteria, attached to bacteria, injected NA; blended up bacteria/bacteriophages, bacteriophages fall off surface; radioactivity in supernatant fragment not palate; progeny had no radioactivity; labeled phages w/radioactive phosphorous, labeled NA; now cell palate had radioactivity; radioactive DNA detected in progeny

Hershey-Chase Experiment

confirmed that DNA is the genetic material because only radiolabeled DNA could be found in bacteriophage-infected bacteria

Baltimore classification system

Developed by Nobel laureate David Baltimore
Based around mRNA production methods
Separates viruses into seven classes

positive strand

mRNA (ribosome ready) is always the positive strand; DNA equivalent polarity also considered (+) strand

negative strand

RNA and DNA complements of (+) strands are negative strands; not all (+) RNA is mRNA, not all translated

what is encoded in viral genome

gene products/regulatory signals for:
- rep of viral genome
- assembly/packaging of genome
- reg and timing of rep cycle
- modulation of host defenses
- spread to other cells and hosts

info not in viral genomes

no genes encoding complete protein synthesis machinery; no genes encoding proteins involved in energy production or membrane biosynthesis; no classical centromere or telomeres

Baltimore class 1: dsDNA genomes

genomes copied by host DNA pols (eg polyomaviridae, pappilomaviridae); genomes encode DNA pol (eg adenoviridae, poxviridae)

Baltimore class 2: ssDNA genomes

can be circular or linear (eg circoviridae- circular, parvoviridae- linear); must be converted to dsDNA to make mRNA; rarely infect humans (2 can, TT virus, b19 parvovirus)

RNA genomes

mammalian cells don't have RdRps; RNA virus genomes have to encode RdRp; RdRp produce RNA genomes and mRNA from RNA templates

Baltimore class 3: dsRNA genomes

dsRNA genomes can't be translated directly (ribosomes can't access it), must be copied to mRNA; must carry RdRp in viral particle; eg: reoviridae

Baltimore class 4: (+) ssRNA genomes

can be directly translated; don't have to carry RdRp in particle b/c they can be directly translated; eg: Picornaviridae, Flaviviridae, Coronaviridae

Baltimore class 5: (-) ssRNA genomes

complement to mRNA, can't be directly translated; must carry RdRp in viral particle to produce mRNA; eg: Orthomyxoviridae (segmented), Paramyxoviridae and Rhabdoviridae (non segmented)

Baltimore class 6: (+) ssRNA-RT

1 viral fam (retroviridae); 2 human pathogens HIV and HTLV; looks like an mRNA, but not translated immediately upon entry to cell; copied by reverse transcriptase to (-) ssRNA then to dsDNA; dsDNA integrates into host DNA, then host pol makes mRNA to make viral proteins

Baltimore class 7: dsDNA-RT

partially dsDNA, gapped DNA, which can't be copied to mRNA; partially dsDNA converted to fully dsDNA then transcribed mRNA; eg: hepadnaviridae (hep B virus)

the plaque assay

quantitative measure of infectious units (PFU); allows scientists to count the number of infectious viral particles in a suspension w/high degree of precision and reproducibility

Focus forming unit assay

modification of plaque assay; after viral infection cells are permeabilized and stained w/an antibody against a viral protein

Endpoint dilution assay

looking for cytopathic effects; instead of looking for specific plaques, just look for evidence of clearing/cytopathic effects

Multiplicity of Infection (MOI)

# infectious particles added per susceptible cell (not the # infectious particles a cell received)

transfection

transformation-infection; production of infectious virus after transformation of cells by viral DNA/RNA; first done w/bacteriophage lambda

functions of structural proteins

Protection of the genome and delivery of the genome

metastability of virus particles

2 states: must protect genome (stable), must come apart upon infection (unstable); energy put into virus particle during assembly; PE used for disassembly if cell provides right signal

How is metastability achieved?

stable structure: created by symmetrical arrangement of many identical proteins to provide max contact
unstable structure: not usually permanently bonded together, can be taken apart on infection to release/expose genome

rules for self-assembly

1: each subunit has identical bonding contacts w/its neighbors
2: these bonding contacts usually non-covalent

Helical symmetry

coat protein molecule engage in identical equivalent interactions w/one another and w/viral genome; allows you to construct large, stable structure from single protein subunit

icosahedral symmetry

icosahedron: solid w/20 faces, each an equilateral triangle; allows formation of closed shell w/smallest # (60) identical subunits

Quasiequivalence

when capsid contains more than 60 subunits, each occupies quasi equivalent position; non covalent binding properties of subunits in dif structural environments are similar but not identical

triangulation number (T)

number of facets per triangular face of an icosahedron; combining several triangular facets allows assembly of larger face from same structural unit; when you have more than T= 1, also have 6 fold axis of symmetry

tailed bacteriophages

head: icosahedral capsid
contractile head: attached to one 5 fold access of icosahedral capsid
baseplate: for attachment

enveloped virions

capsids can be covered by host membrane (envelope), acquired by budding of nucleocapsid through cellular membrane

unstructured envelopes

can vary in size and shape, pleomorphic

structured envelopes

capsid and envelope proteins interact in 1:1 interaction that imparts structure on envelope; very symmetrical

exceptions to rules

poxvirus: doesn't fit into icosahedral mold
pandoravirus, pithovirus: apical pore, elongated structure

Pathogenesis

process of producing a disease

requirements of a successful infection

sufficient quantity of virus; accessible, susceptible and permissive cells; local antiviral absent or overcome; lots of protection

Susceptible cell

has a functional receptor for a given virus, the cell may or may not be able to support viral replication

resistant cell

has no receptor, may or may not be competent to support viral replication

permissive cell

has the capacity to replicate a virus, may or may not be susceptible

viral spread (ex: mousepox)

initial entry through footpad; immediately have access to lymph system b/c it reps in lymph node after breaching skin; pours into blood, from there can go to other organs; undergoes secondary rep in spleen/liver; spills into blood, can be disseminated again into skin; shed through skin, makes rash on skin

virus shedding

release of infectious viruses from an infected host; resp secretions, nasal secretions, mucosal shedding, skin lesions, blood/blood supply, urine, feces, semen, insect vectors, germline/vertical

airborne transmission

if you can inhale particles, breathing in aerosols; the tiniest suspended particles can remain airborne for hours (need good ventilation); infection control strategies for resp viruses focused on limiting droplet and fomite transmission

geography and seasonality affect viral transmission

- geography may restrict presence of virus due to requirement for specific vector/reservoir
- see dif waves of infection at dif latitudes, could be due to humidity or to dif behaviors during dif seasons

Non-eveloped viruses and viral receptors

gain access to receptors by binding to capsid surface or protrusions; sometimes can directly inject genome

enveloped viruses and viral receptors

bind via transmembrane glycoproteins

penetration and uncoating at plasma membrane

conformational change; hairpinning motion brings two membranes together

viral fusion w/endosomal membrane

low pH in endosome causes conformational change, causes it to put fusion peptide into host cell membrane; hair pinning brings membranes close together until fusion

Class I fusion proteins

perpendicular to membrane (spikes); mostly alpha-helical; form trimers

Class II fusion proteins

parallel to membrane; mostly beta sheets; form dimers; conformational change occurs to flat complex, extends fusion peptide into host cell membrane, hair-pinning, fusion

getting into the nucleus

don't want to release DNA into cytoplasm; wait for cell division and breakdown of nuclear membrane

RNA directed RNA synthesis

RNA genome must be copied from end to end w/no loss of nucleotide sequence; production of viral mRNAs that can be efficiently translated by cellular protein synthesis machinery; performed by RdRp

alphavirus RNA

has 1 piece of genomic RNA; when enters cell has cap so viral proteins synthesized right away; doesn't translate whole genome, only to stop codon; proteins are rep proteins, can go and make full length (-) copy of RNA, can make (+) strand and subgenomic RNA

unimolecular (-) ssRNA genomes

when arrives in cell undergoes mRNA synthesis; makes all viral proteins; problem is all these mRNAs shorter than full length (-) strand genome, so can't serve as template

switch from mRNA synthesis to genomic RNA synthesis

coated in N protein, helps make mRNAs, first makes N mRNA; N protein comes back, binds pol and binds nascent transcripts made from viral genome, forces it to continue along genome, make full length (+) strand, can be used as template for more (-) strands

cap snatching

influenza takes host cap mRNAs, cleaves them, uses them as primer to make mRNA

eukaryotic DNA dependent RNA pols

only DNA viruses that rep in cytoplasm encode DdRp

regulation of transcription by viral proteins

could have positive or negative auto-regulatory loop; product promotes synthesis (positive) or inhibits it (negative); cascade regulation, first gene protein binds to next gene, etc, etc

benefits of splicing

marks mRNA for nuclear export, alternative splicing creates dif mRNAs (increase coding capacity), coding info of small genomes expanded, regulation of genome expression

why do DNA viruses need the host

viral DNA rep requires synthesis of at least one, if not more viral proteins; simple viruses need more host proteins, complex viruses encode many but not all proteins required for rep

the 5' end problem

Each round of DNA replication, the DNA length shortens, resulting in a small loss of DNA.
The RNA primers cannot be replaced with DNA.

ways to solve the 5' end problem

circular genome (no ends), DNA priming, protein priming, rolling circle rep

DNA Priming in Parvoviruses

hairpin at both ends, inverted repeats make hairpins; initiate synthesis from 3' end of hairpin; get elongation of 3' OH to end of genome

protein priming in adenoviruses

origins at both ends; strand displacement synthesis; semi-conservative DNA rep; ends complementary so you get dsDNA at the end which looks like og dsDNA

rolling circle replication

nick in circular DNA, nick displaced by extended 3'OH, keeps going around and around, solves 5' end problem b/c you don't have an end

5' end dependent initiation

40S subunit binds ternary complex, creates 40S pre-initiation complex; binds eiF4G which binds cap/helps recruit cap; 48S subunit scans to find start codon

juxtaposition of mRNA ends

eiF4G and eiF4E interact; in pea enation mosaic virus, have 3' CITE elements

Ribosome shunting

viral protein/viral structure can force ribosome to jump over region of RNA and surpass a structure to go to another ORF; predicted to decrease dependence of mRNA on eiF4F complex during initiation by reducing need for mRNA unwinding

internal ribosome entry site (IRES)

A site within an mRNA sequence where a ribosome can bind and initiate translation.

Methionine-independent initiation

can assemble 80S ribosomes w/out any eIFs of Met-tRNAi; don't have traditional start codon

polyproteins

if you're stuck w/one mRNA and can only make 1 protein, what do you do? make polyproteins and use proteases to cleave it into mature viral proteins

leaky scanning

- Occurs when 40S ribosomal subunit bypasses first AUG to initiate translation farther downstream
- Allows multiple viral proteins to be synthesized from a single mRNA

PKR and cellular antiviral response

PKR present in inactive form in cell, induced and activated by virus infection; leads to inhibition translation and apoptosis; viruses try to stop PKR activation

common set of assembly reactions

- must assemble protein shell/coat
- selectively package viral genome
- all need to be released from cell
- for some, have to require envelope
- some also have maturation step to virus particles

localization of viral proteins to nucleus

DNA virus wouldn't go out of nucleus to package b/c it could get sensed in cytoplasm and there are no export pathways to move DNA from nucleus to cytoplasm; easier to package in nucleus

localization of viral proteins to plasma membrane

often go into ER; if cell surface protein, passage through Golgi, travel on microtubules, fuse to allow to be surface localized

strategies for making subassemblies

- produce individual proteins that have high affinity and form assembly
- proteins made as polyprotein, associate before cleaved
- chaperone assisted assembly, make protein, and protein needs to be folded by chaperone to take on functional form

Packaging of segmented genomes

each segment has distinct packaging signal; serial dependence of packaging

Acquiring an envelope

typically occurs after assembly of internal structures; at many cellular membranes; could be enveloped glycoproteins help drive budding or internal/matrix proteins

ESCT pathway

required for retrovirus budding; occurs after release from cell by protease cleavage event in virion; not infectious until undergo this maturation step

release of non-enveloped viral particle

lysis: apoptosis, necroptosis; viral proteins can induce rupture of membranes, loss of membrane integrity w/inhibition of protein synthesis
non lytic release: vesicular release, exocytosis/exosomes

Viruses of Archaea

unusual morphology; have dsDNA genome (1 has ssDNA); most have internal or external lipid envelopes; many are template viruses that integrate their genome into host cell DNA; many don't have an identifiable DNA pol gene

phage therapy

treatment of bacterial diseases using bacteriophages specific to a particular bacterium

Bacteriophage

A virus that infects bacteria

MS2

belong to levivirus genus; naked icosahedral capsid; linear ssRNA (+) sense; inside it has folded phage RNA; phage first finds sex pilus then injects its RNA into bacterium; once phage in bacteria, translated right away, copies into (-) strand, then copied back into (+) stand; coat protein find and capture phage (+) strand; phage protein = lysis protein, lyses bacteria so phage can leave

MS2 protein synthesis

translation of coat genes allow production of replicase and lysis proteins; replicase binds start codon, blocks coat protein synthesis; copies (+) to (-) to (+); newly synthesized (+) folds to allow synthesis of maturation protein; when coat proteins reach threshold amount they form dimers that shut down replicase production and start assembly of phage particles

MS2 genome replication

replicase associated w/3 host proteins; S1 protein of small ribosomal subunits, translation elongation factors EF-Tu and EF-Ts; S1 directs replicase to start of coat gene; EF-Tu helps replicase to start RNA synthesis; EF-Ts recycles Ef-Tu-GDP to EF-Tu-GTP

φX174

microvirus; naked icosahedral capsid; circular ssDNA; replication has 2 phases: 1- ssDNA --> dsDNA, enzymes from host, 2- synthesize ssphage DNA

Assembly of φX174 capsid

2 scaffolding proteins; coat protein forms 9S pentamer; w/internal scaffolding

T7-podoviridae

naked icosahedral capsid; 60 nm (big); short tail and tail fibers; lytic no lysogeny; linear dsDNA genome