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Principle of molcular virology > Lecture 3 - Assembly > Flashcards

Lecture 3 - Assembly Flashcards

1
Q

What are the different sets of mechanisms employed by viruses for assembly?

A
  1. Coat protein can assemble alone (e.g. polio)
  2. Coat protein and RNA can associate in an unspecific way due to the charge distribution of RNA
  3. Coat protein and RNA with packaging signals/stemloops - sophisticated production to ensure only viral RNA is packaged
  4. DNA packaging (active)
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2
Q

What forces drive the assembly of virus particles?

A

Hydrophobic and electrostatic interactions Covelent bonds are rarely used as the process of assembly needs to be reviersibe

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3
Q

How did Fraenkel-conrat and Williams shows that RNA may be necessary for the coat assembly of tobacco mosiac virus?

A

When mixtures of purified TMV RNA and coat protein are incubated together, virus particles form spontaenously Thought to be due to electrostatic and some hydrophobic interactions between the coat protein and RNA

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4
Q

What are the features of TMV coat and RNA?

A
  • 2130 molecules of coat protein which form a helical structure around the RNA
  • 6400 bases of RNA
  • protein monomer consists of 158 amino acids
  • length of the virion: 300nm and diameter, 18nm
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5
Q

What are the features of TMV assembly?

A
  • coat protein and RNA spontaenously assemble into comple TMV virions
  • Protomers come together to form disks of two layers of protomers
  • RNA passes through the hole and the helical capsid grows by the addition of protomers to the end of the rod with the RNA loop leading
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6
Q

What is the detailed sequence of TMV particle assembly?

A
  1. around 30 subunit form a stacked 2 layer disk
  2. the origin of assembly sequence (OAS) on the RNA acts act a packaging signal - ensuring that this is the correct piece of genetic information to be incorperated into the capsid, by making the association specific - specifically intreacts with the disk and nudges it slightly out of shape. This allosteric conformational change converts the disk structure to a ‘locked washer’ structure
  3. This starts the formation of the helix, with the coat protein building blocks added and extended in a spiral manner
  4. Meanwhile the OAS gets fed through the hole due to the specific interaction of RNA and the coat protein
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7
Q

How is the assembly of larger (DNA) icosohedral viruses different to that of the TMV?

A
  • DNA viruses often have large isocohedral heads (herpes simplex T = 16)
  • Assembly does not happen spontaenously
  • Scaffolding proteins form a mold from which the coat protein can extend
  • Mold is then removed leaving either through the portal protein or by being degraded in the head
  • Maturation then occurs to fix the icosohedral shape, stabilising through cross linking
  • then DNA pumped in
  • often forms empty capsids, wouldn’t happen for RNA viruses
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8
Q

What is virus maturation?

A
  • When a virion becomes infectious
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9
Q

When does virus maturation occur?

A

After assembly of the protocapsid

Often concomitant with DNA packaging

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10
Q

How does viral maturation occur?

A

Small proteolytic cleavages by maturation proteins lead to increased stability and subtle structual changes (cross linking)

Sometimes a whole set of modifications

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11
Q

Give an example of the maturation process as a good antiviral target

A

HIV forms a poly protein that gets packaged into the virion, however on arrivial the actual proteins are needed so as the virus moves cell-cell protease cleaves the polyprotein into the reqired protein. The protease could be a good antiviral target so that the inhibition leads to the virus to be ‘dead on arrival’

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12
Q

What are the features of the equilibrium of capsid assembly?

A
  • for most viruses, assembly tends to be reversible (e.g. depending on pH)
    • often have all coat proteins or all capsid but few reaction intermediates
  • stronger binding not favourable, leads to kinetically trapped species
  • reaction kinetics drive viral capsid assembly - reaction driven to produce as many bonds as possible with the addition of new coat proteins
  • when have an almost fully formed capsid only one block is missing and the addition of this releases the most energy, driving the completion of the capsid
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13
Q

Why is it important that capsid assembly is reversible?

A

Aside from the need to uncoat when entering cells, revsible is neccesary because malformed bonds formed during assembly can be undone and fixed.

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14
Q

Why are virus capsids often found in either the coat protein or capsid state, not the intermediates?

A

Formation is driven by environmental conditions e.g. pH

When forming, intermediates come together very quickly, driven by the reaction kinects, in order to form the capsid and release as much energy as possible when bonding

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15
Q

What issues are there in viral assembly?

A
  1. The formation of the capsid from building blocks
  2. Attaining the relevent molecular intereactions
  3. Attaining host machinery if necessary
  4. Comparisons between RNA and DNA
  5. Achieving specificity for the packaging of viral nucleic acid
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16
Q

Why is charge problematic for viral assembly?

A

Nucleotide phosphate backbones are negatively charged leading to electrostatic repulsion

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17
Q

How is the negative charge of phosphate groups on the nucleic acid backbone overcome by viruses for assembly?

A
  • small positively charged ions (Na, Mg, K)
  • Nucleocapsid protein (+ charged protein) buffers electrostatic charge to allow the confinement of genetic material
  • Histone like (e.g. adenovirus polypeptide VII)
  • Histone/chromatin-like using cellular histones (e.g. polyomavirus)
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18
Q

Why is viral nucleic acid specificity a problem in viral capsid assembly?

A
  • Viruses are very small compared to the volume of the cell
  • Need to package specific viral nucleic acids over the cellular nucleic acids
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19
Q

How is viral nucleic acid specificity overcome in viral assembly?

A
  1. Stochasti approach (luck) not all virions are infectious e.g. herpes virus. Large numbers of viral RNA/capsid work in favour of the association of viral nucleic acids with the capsid
  2. Nucleocapsid proteins associate the nuleic acid with coat protein
  3. Packaging signals (e.g. a retroviral psi element, a TMV OAS, TR in MS2) The crucial interaction of the NA secondary structure with coat protein (MS2) leading to an allosteric conformation switching (dimer switching in MS2, disk vs locked washer in TMV)
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20
Q

Where does viral budding occur?

A

Can bud from any membrane, specific to the virus - crucial for cell entry and exit

Often have a matrix protein: to ensure nucleocapsid gets associated with membrane and the correct glycoproteins are enclosed in the envelope when budding

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21
Q

What are the exmaple viruses for each of the baltimore classifications?

A

Group I: dsDNA - HSV, HPV/SV40, tailed phages

Group II: ssDNA - M13, Gemini

Group III: dsRNA - Rotavirus

Group IV: (+)ssRNA - MS2, Picorna

Group V: (-)ssRNA - Ebola, Influenza

Group VI: (+)ssRNA-RT - HIV

Group VII: dsDNA-RT - Hep B

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22
Q

Give three examples of class I viruses?

A

Complex, tailed phages with icosohedral and helical symmetry

Papovaviruses, Herpesvirales, Caudovirales

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23
Q

What are the features of Herpesvirales capsids?

A
  • t = 16
  • Around 100 genes
  • Complex life cycle
  • Uses scaffolding proteins
  • >30 scaffolding proteins
  • scaffolding in the nucleus helps to bud from the nuclear membrane (using the primary tegument protein UL30) then lose envelope on the outer nuclear membrane
  • More tegument proteins (alpha-TIF, vhs) form the final envelope via exocytotic vesicles
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24
Q

What is the series of assembly of the herpesvirales envelope?

A
  1. VP5 (UL19) with associated scaffolding proteins (UL26/UL26.5) transported in from the cytoplasm to form the scaffold
  2. UL18 and UL38 assemble around scaffolding proteins to form the procapsid
  3. Autoprotolytic cleavage of the scaffolding protein to form the mature capsid
  4. Cleavage/packaging proteins encapsidate the DNA into the capsid
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25
Q

How does the caudovirus head assemble?

A

Head is assembled with scaffolding proteins around the core, to form a protocopsid

DNa is internalised through the portal with the assistance of a terminase concomitant with the degradation of the scaffolding proteins

Maturation occurs to form the infectious phage

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26
Q

What is the assemble pathway of the bacteriophage T4?

A

Two assembly pathways

  1. Prohead I forms (core only no DNA)
  2. Prohead II (no DNA)
  3. Prohead III (50% DNA)
  4. Mature head (100% DNA)
  5. Collar added

Second pathway running parallel

  1. Plug and wedge come together to form base plate
  2. The addition of the tube and sheath
  3. Tail forms

Combining pathways

  1. Head and tail come together with tail fibres to form completed T4 plage
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27
Q

What are the features of the Class II ssDNA virus phage M13?

A
  • 900nm long, 9nm diameter
  • around 3000 major coat protein g8p (50 amino acids), 4 minor, around 5 at each end
  • Aplha helix with + charge in the middle associates with the - charge of DNA
  • Hydrophobic interactions between adjacent g8p
  • Initially g5p forms a protective sheath arounf the newly synthesised DNA, whilst g8p accumulates at the cytoplasmic membrane then the two are interchanged upon exit
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28
Q

How can M13 be unsed in recombinant technology?

A

There is no limit to the length of the moleule, can use recombinant DNA techniques to add material to the phage

Phage can encapsidate extra genetic info

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29
Q

What is a bipartite genome?

A

Genome consists of two separate molecules anchored in different icosohedra

30
Q

What is a multipartite genome?

A

when genetic material is packaged in separate plates

Thought to be related to the geometry of the virus

31
Q

What is the structure of the geminivirus?

A
  • ssDNA
  • plant virus
  • Geminivirus particles consist of T=1 twinned icosohedra
  • pack a mono- or bipartite genome (one has 8 segments)
  • 22nm diameter
  • 38 nm length
32
Q

What are the features of the Rotavirus? (class III dsRNA)

A
  • Reoviruses: 10-12 genome segments (of around 1 gene each) of dsRNA
  • Capsid consists of 2(generally rhiovirus) or 3 concenrtic icosohedral shells
  • After RM endocytosis and uncoating the segments remain in their core shells
  • Triple protein coat
  • RNA is replicated and double layer particles assembled in the viroplasm
  • DLPs migrate to the ER to obtain third layer formed by VP7 and VP4
  • Virions are released by lysis
33
Q

Why is mRNA transcription difficult to arrange in the Reovirus (type of/general name fpr rotavirus)?

A

Uses dsRNA which is very stable

34
Q

Folllowing RM endocytosis and uncoating, why do the gene segments of Rotaviruses remain in their core shells?

A

Make it easier to maintain conditions to denature dsRNA to create mRNA

Or to evade cellular RNAi which is triggered by the presenece of dsRNA

35
Q

Why do Rotaviuses has a triplet protein coat?

A

To make the viruses resistant to the pH of the stomach and disgestive enzymes in the guy

36
Q

What is the viroplasm?

A

Viral factories for the rotavirus probably made by NSP5 and NSP2

37
Q

Why does the rotavirus capsid consist of two or three cocentric icosohedral shells?

A

Rotavirus causes severe diarrhea in infants

Needs to pass through the digestive tract and be resistant to digestive enzymes

dsRNA more stable than dsDNA but harder to pull apart to make mRNA

needs strong denaturing conditions to pull apart

Multiple coats used to contain dsRNA and contain strong denaturing conditions inside capsid

38
Q

What are the features of the HIV structure and genome?

A

Class VI, ssRNA-RT

  • envelope potein is in the plasma membrane. Capsids assembled in the cytoplasm around RNA
  • immature viruses bud from the plasma membrane
  • maturation takes place following virion release (by cleavage of pp into enzymes like integrase and reverse transcriptase)
  • Psi element as a packaging signal/gammaretro virus core encapsidation signal
39
Q

What technique can be used to view single molecules inside the cell?

A

Single molecule fluorescence assays by fluoresence labelling

Highly sensititve fluoresence microscopy techniques

Allow single nanoparticles to be tracked during their uptake into living cells with millisecond time and nanometer spatial recognition

Analysis of the tragectories can discriminate between random motion and active transport

Provides important information regarding the uptake pathways and location of viruses and nanoparticles

40
Q

What are the features of the capsid and genome of class VII dsDNA Hep B?

A
  • 4 genes
  • C-mRNA gets incapsidated the -cDNA gets generated by reverse transcription
  • RT RNase H degrades template RNA and partial +cDNA gets synthesised during maturation in the virion
  • after partial uncoating ds is completed by RT (when enter the cell) free ends get ligated in the nucleus to form a mini-chromosome (w histones from the nucleus)
  • 4 mRNAs are synthesised, the longest cmRNA gets pckaged
  • capsid formed of HBc (core) antigen
  • t=3 or t=4
41
Q

What are the features fo mass spec?

A
  • Able to look at a wide range of things: lipids to whole envelopes, peptides to whole proteins, macromolecular complexes, intact viruses
  • Wide range of methodologies, preparations and strategies
  • Complements other approaches e.g. muational analysis, protein and genetic analysis, EM, NMR and X-ray
42
Q

How does mass spec work?

A

Specimen is transformed into the gas phase by ionisation (ESI/MALDI) and ions are separated by mass-to-charge ratio (ToF, ion traps)

. Fragmentation e.g. for protein sequence, fragments can be analysed in ion traps

43
Q

How can mass spec be used to look at the virus life cycle?

A

Prepare smaple from different stages in the viral life cycle and track changes, then compare this to known gene/protein banks etc.

Determine what is expressed when.

Get different characteristic degraded products if test viruses at different points in the life cycle.

Look at: incorperated host protein, post translational modification, metabolome, complexes (stoichiometry, protein-ligan interactions, HBV) virus structure and dynamics

44
Q

How can the averaging be overcome in Cryo -EM?

A

If pick out singular distinguished vertex e.g. tail on herpes simplex, can 5-fold average about that vertex

If the absolute orientation is known can do asymmetric reconstruction

45
Q

How can 5 fold averaging be used (Cryo-EM) on MS2?

A

MS2 infects male escherichia coli via the F pillus via the maturation protein. Therefore it attaches by one specific protein allowing 5 fold averaging around that face. This allows also some structuratal information about the RNA in the capsid to be viewed. However under 5 fold averaging, pathway is not as would be seen if the RNA were to meet each vertex exactly once

46
Q

How was it shown that virus assembly could not be looked at in isolation?

A
  • In the absence of genomic RNA, MS2 capsid assembly is very slow
  • TR sequence initiates assembly by associating with the maturation protein in MS2
  • TR sequence forms a stemloop
  • stemloop changes the conformation of the symmetric C/C dimer to the assymetric A/B dimer (allosteric switch)
  • 60 asymmetric dimers are needed to form the capsid -therefore there must also be 60 stemloops
  • RNA needs a path to visit 12 vertices 5 times to make this allosteric change via 60 independent RNA contacts.
47
Q

What is a hamiltonian path? How does it relate to MS2?

A
  • A hamiltonian path
  • occurs on a polyhedron
  • a connected path along the edges such that every vertex (corner point) of the polyhedron is visited once

The assemble pathways of MS2 capsid correspond 1-1 with the hamiltonian paths on its polyhedron

Over 40,500 hamiltonian theorectical paths can be imagined (without information from biochemistry of assembly kinetics.)

48
Q

How was the exact hamiltonian path for RNA in MS2 elucidated?

A
  1. Fiure out total number of hamiltonian paths possible to meet 12 vertices of theMS2 polyhedron 5 times, once (40,500 paths)
  2. Use biohemical information - maturation protein associates with one special vertex (c.f. F-pillus receptor) and both ends of the RNA. RNA circularises, therefore we need a Hamiltonian pseudo-cycle (narrows down to 66 paths being compatible)
  3. Use insights from assembly kinetics. The hamiltonian path encodes the order of dimer additions in assembly - look for paths which favour the continuous complete of closely spaced hexamers and decamers. (3 paths have good thermodynamic properties favoured by the reaction kinetics - illustrate the formation of complete hexamers or decamers in succession with a high number of contacts between them. Reaction proceeds where most new bonds are made with each addition of coat protein, some paths more energetically favourable than others)
49
Q

What is the MS2 dimer switching model?

A

Multiple stem loops from within the RNA genome aid allosteric dimer switching at the 60 positions of the asymmetric dimer (A/B)

50
Q

How was an asymmetric reconstruction of MS2 acheived?

A

Cryo EM

Knew the absolute orientation - MS2 attaches to the F-pillus at an angle, but this is tricky to measure

In cyro-em tomogram, MS2 bound to E.coli F pillus for asymmetric reconstruction and 1500 virions averaged

Observed asymmetric RNA, implying conserved RNA configuration

51
Q

How was the path of RNA elucidated in MS2?

A

Used cyro-em with absolute orientation to identify asymmetric RNA within the capsid.

Formulated constraints based on the density information: 6/30 edges identified as occupied, 4/30 idetified as not occupied, 20/30 not classified. Look at hamiltonian paths that are posistent with these constraints. 5 paths conststent with these constraints, further narrowed down by 5 fold averaging - only one available, same as one that was derived theoretically.

52
Q

How was the outer RNA shell density explained as seen in MS2 through cryo-EM?

A

The confinement of the 5’ and 3’ ends of the genome to a five fold axis by maturation proteni and the requirement that the capsid builds through low energy intermediates explains the outer RNA shell density as seen in ryo EM.

53
Q

Is it theoretically possible for viruses to evolve new paths of RNA paths within the capsid?

A

Unlikely. Not much leway for change and the most efficient paths are significantly different enough that single mutations and unlikely to result in them.

Paths appear to be conserved, GA virus is related to MS2, has the same RNA hamiltonian path, therefore viral RNA seems to be a very specific process.

54
Q

How are viruses said to behave like self packing suitcases?

A

RNA ensures that all the bits that need to bond are in the right proximity to each other and with the correct conformation. Highly specific assembly.

55
Q

Why are hamiltonian paths though to be evolutionarily conserved?

A

Only a small number of hamiltonian paths that make sense in a biological context. Those that do are sufficiently different that a mutation would no necccessarily take one path into the others. GA v. MS2

56
Q

By what methods was the RNA configuration of MS2 elucidated?

A
  • Kinetic modelling
  • Bioinformatics analysis
  • Analysis of cryo-EM tomogram
57
Q

What in RNA is absolutely esssential for packaging?

A

Packaging signals to allow building blocks to come together orretly during assembly

58
Q

What are the features of the bonds between specific regions of RNA and their capsid component interactions?

A
  • Not all bonds are very stong (allows process to be reversible)
  • Stong interaction however between maturation protein to direct RNA down the correct path
59
Q

How was it shown that increasing coat protein concentration and packaging signals are sufficient for selective RNA pakcaging?

A

Using competition experiements. Compared viral RNA with cellular RNA. If start with a complete mix, and the viral coat protein is ‘dumped’ into the mix, what follows is a mix of viral and cellular RNA packaged.

In a realistic situation, virus makes these slowly resulting in a build up of protein/RNA. If the same experiemtn is repeated but the coat protein added slowly, it attached to nucleic acid it binds to best through competitive binding, results in integration of almost only viral RNA.

60
Q

How can insights gained into RNA packaging into capsids provide a solution to drug resistance in viruses?

A
  • Escape mutants can occur when viruses are challenged by a drug, small changes in the capsid structure make drugs less likely to bind
  • As hamiltonian path of RNA forming the capsid is an evolutionarily stable feature, dominated by geometry not biochemistry, the virus is less likely to be able to mutant away.
  • Looking into in: Hep B and C, HIV, SARS, Norovirus, foot-and-mouth disease virus, human parechovirus
61
Q

What are the multiple purposes of RNA stem loops and the maturation protein?>

A

RNA stem loops

conformer switching, initiation of assembly (TR), recruitment of dimers and assembly pathway selection

Maturation protein

circularisation of genomic DNA, pilus binding for infection

62
Q

What variety of experimental and theoretical approaches were used to determine geometry of RNA hamiltonian paths in viral capsid formation?

A

Theory: group theory, graph theory, kinetic modelling, bioinformatics, biomolecular simulations

Experiment: Cryo-EM, Mass spec, SELEX and other biochem experiments

63
Q

What is the MS2 TR?

A

Translational repression

An RNA hairpin which hides the AUG start codon of the replicase gene

64
Q

What is the role of the MS2 TR?

A
  1. TR binding to coat protein blocks the translation of the RdDp gene leading to an increase in the coat protein translation, and the beginning of mif life cycle gene expression
  2. TR binding to the coat protein also creates and assembly competent RNA/CP complex. Signals that packaging of the viral RNA into capsids will start shortly.
65
Q

What is the basic life cycle of a +ssRNA virus?

A
  1. Attachment
  2. RNA release
  3. Poly-protein production
  4. Processing
  5. RdRP translation blocked, upregulation of coat protein translation and mid life cycle gene expression
  6. Packaging initiated
  7. Escape through pore
66
Q

Why is timing of viral gene expression important? What types of genes are expressed at different stages of viral infection?

A
  1. Viruses often have early, middle and late gene expression
  2. in early stages of infection, virus needs to take contorl of host defences and resources. Expressed predominantly proteins that supress host defenses
  3. In middle stage, virus prepares to copy itself. Requires expression of viral genes which copy its genome (RNA dependent RNApolymerase) and produce new viral structural proteins (capsid proteins)
  4. late stage of infection virus needs to assemble new progeny birions and prepare for exit. Genes are expressed to aid in assembly and exit (lysis)
67
Q

How can gene timing expresssion be controlled in DNA/RNA viruses?

A

DNA viruses (+retroviruses): Viral gene expression can be ontrolled in the host nucleus by the viral DNA genome

RNA viruses (specificially +/- sense ssRNA): control must be carried out by the RNA itself as there is no viral DNA within the host nucleus to control mRNA transcription (viral genome must also act as mRNA). Control of gene expression, packaging etc. all controlled by the RNA and its interactions with the host and viral proteins.

68
Q

What are the early, middle and late genes of MS2?

A

Early genes: RNA dependent RNA polymerase (RdRp). Purpose - copy viral genome by specifically recognising a promoter in viral RNA

Middle genes: Coat protein and maturation protein. Purpose: CP serves as a structural protein for viral capsid whilst MP is a special protein required for cell entry by the progeny as it binds to the F pillus

Late genes: Lysis protein. Purpose - degrades the peptidoglycan matrix of the bacterial cell wall. Needed for viral exit from the host.

69
Q

What is the definition of allostery?

A

Allosteric regulation is the reggulation of an enzyme or other protein by binding an effector molecule at the proteins allosteric site, physically distinct from the proteins active site

70
Q

How does the allosteric switch work?

A
  • change in the displacement pattern of the protein dimers, especially in the motion of the GF loops
  • flip of the FG loops rom a symmetric to an asymmetric conformation
71
Q

What does the modelling approach imply about the allosteric switch?

A

The allosteric effect is non-sequence specific and therefore can occur at many places in the genome. Resulted in the simer switching model of capsid assembly.

Principle of molcular virology (7 decks)

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