Viruses

Viruses

1. They are parasitic entities, containing nucleic acid molecules with protective protein coats

   • Parasitic because NA (RNA/DNA) is replicated using host enzymatic machinery
   • Limited host range
     • Animals, plants, fungi + bacteria all have their own
   • Technically not alive because need host to replicate. But sex is reliant on another to ?
   • Definitions
     • Virion: intact virus particle
     • Capsid: protein coat encasing NA core, has structural/scaffold proteins too encoded by viral genome
       • Hollow protein shell enclosing genome
       • Large, symmetric oligomers made of many copies of one/few protein subunits
         • Scaffold proteins interact with capsid subunits
     • Envelope: lipid bilayer that surrounds protein coat of some viruses, not all
       • Contains glycoproteins
         • Protrude from envelope
           • Rest of envelope derived from host (lipid + protein) but glycoproteins are viral
             • Help viruses evade immune system and identify and bind host cell receptors
               • GP120 on HIV binds CD4 causing conf change in GP41 that allows fusion
               • HIV GP120 recognises CD4
               • HA on influenza binds sialic acid
       • Derived from host as buds
       • DISADVANTAGES OF ENVELOPE
         • Lipid bilayer most stable only in host
           • Can dry out quickly
           • Susceptible to heating
           • Susceptible to detergents
       • Useful for entry

2. NUCLEIC ACIDS

   • RNA
     • 1. Directly replicated
     • 2. Or acts as template for DNA synthesis via reverse transcription
     • Single stranded RNA
       • +/SENSE strand = mRNA
       • -/ANTI-SENSE = template for mRNA
   • DNA
   • Single or double stranded
   • Could be entire genome or just processed NA stranded that create the necessary protein

3. VIRAL CAPSID FEATURES

   • NA molecule cannot code for protein big enough to enclose it
     • Watson + crick suggested it was built from multiple copies of subunits in symmetrical fashion
   • Capsid forms 2 different types of structures
     • 1. Rod-like: helical or filamentous virus (TMV)
     • 2. Spherical: Icosahedral virus (FMDV)
       • Electron micrograph images show most viruses are spherical
         • Bacteriophage MS2, Bacteriophage T4 + tobacco bushy shunt virus
           • Bacteriophage T4 has capsid head + tube that acts as syringe so virus sends in RNA without entering cell
       • Spherical capsid made of equilateral triangle faces
         • Believed to be spherical but high-res Xray and EM revealed isohedral
         • Built from 1/few subunits arranged in regular shape (e.g. football)
         • Icosahedral viruses have 20 faces + 12 vertices
         • Due to axis of rotational symmetry have 5:3:2 symmetry
           • 5-fold at vertex
           • 3 fold through centre of each face (20)
             • 60 identical subunits = 20x3
           • 2 fold at centre of each edge
           • Icosahdral symmetry allows lowest-E configuration of particles
         • Large icosahedral viruses form from more than one type subunit, or same one in different arrangements
           • Icosadeltrahedron viruses
             • Gain triangulation (T) number
               • Gives idea of size = bigger T = bigger virus
                 • Gives number of structural units per face
                   • Comes from relationship between number of 5-fold symmetry present to pseudo 6-fold symmetry present (pentamers + hexamers)
               • E.g, T = 1 or 3 often
           • Contains 60 identical sets of subunits
       • Common structure
   • Coat protein interactions critical for capsid formation
   • Virus has to be in particular state inside + outside the host
     • Outside: virion intact to protect NA material
     • Inside: many occasions you don't want capsid as virus needs o replicate
       • Virus in pieces and devoid of identity until reforms

4. TOBACCO MOSAIC VIRUS

   • Causes leaf mottling + discolouration
   • First virus to be discovered + isolated
   • Helical rod virus
   • Coat protein: 150 AAs (~17.5 kDa)
     • 2130 copies in long coat
   • MODEL OF TMV
     • Complete virion = 3000A long
     • Diameter = 180A
     • Coat protein forms hollow right handed helix
       • 16 1/3rd subunits per turn - 2/3rd 17th covered by row above
       • Helical pitch = 23A
     • RNA in model
       • RNA is single stranded, long (~6400 nucleotides)
       • Located in 40 diameter central cavity (hollow shell that holds RNA in centre)
         • winds within turns of helix
           • 3 nucleotides bound per protein subunit
   • VIRAL ASSEMBLY OF TMV
     • Needs structural flexibility to be virion sometimes and devoid of structure others
     • TMV coat can aggregate in solution into "virus-like particles" with NO RNA present
       • Aggregation is ionic strength + pH dependent
         • Highly dependent on environment
           • Virus seeks right environment for assembly giving immune system enough time to recognise foreign entities
         • Low ionic strength/high pH = aggregates of only few subunits
           • virus won't form under these conditions
         • High ionic strength OR neutral ph = disks forms with 2 layer
           • Disk 17 subunits each
             • Each subunit 4 a-helices (17x4) = 68 helices
             • RNA binds flexible loop in disks
               • 3 nucleotides/bases per subunit
                 • Binds arginines (+): Arg41 - Arg90 - Arg92
                   • Same interaction as chromatin - + Arg and - phosphate backbone
               • If you remove protein from capsid left with small spiral of RNA specifically bound in centre
             • Salt bridges between disks in helix
         • Medium ionic strength AND neutral pH = disks aggregate to form protohelices
           • short at first =2 turns (39 subunits)
         • pH5 = protohelices assemble into stacked long helices
           • happy to associate
             • long helices slowly anneal (hours) to form virus like particles
           • First form nicked helix, then slot into place (few hours) = continuous helix
     • Overall: Few proteins aggregate - disk formation - protohelices - nicked helix - continuous helix long helices - long helices anneal into virus-like particles
     • VIRUS ASSMEBLY IN 2 STAGES
       • 1. TMV Nucleation
         • A) Subunits come together - slow process as subunits art necessarily close
           • Rate limiting step
         • B) Results in quasi-stable nucleation complex
         • C) Insertion of imitation RNA sequence - specific hairpin look into central cavity of protohelix
           • Subunit have very high affinity for loop
             • Recognises that it has guanine in every 3rd position
               • Only time base recognised not backbone
           • Experiment looked at I-RNA sequence
             • Mix RNA + small amount of coat protein so only nucleation (not growth) occurs
               • RNAase used to digest RNA - only loop remains
                 • Protected by coat protein - not broken down like rest
           • Essentially a motif that allows virus to recognise its own RNA not host
       • 2. TMV Growth
         • Faster process through cooperative addition of subunits to nucleation complex
           • Addition of protohelices to stack helices forms nicked then continuous helix
         • Overall assembly takes 6 hours from RNA + coat protein mix
           • Takes 10 minutes if RNA mixed with protohelix
             • RNA interacts with coat proteins to form single stranded help structure
               • 3" end help in place via initiation loop at bottom of stack of protohelices
                 • rest of RNA pulled through middle, coiled as it goes
                   • 5" end shortens as spirals
                   • 3" end stays static
                   • Confirmed by EM studies

5. VIRAL DISSAEMBLY

   • General overview
     • Virus only virulent if it can function - so NA material is available for replication
       • Allows it to bud + infect other cells
       • Ergo, host cell environment must destabilise virus
         • Virus wants to retain disassembly to allow time for replication before reassembly
           • Virus assembly entirely dependent on environment
             • Increasing pH inside host plant cell causes deprotonation (basic pH) and would destabilise the virion particle
             • Calcium ions can stabilise TMV coat protein structures
               • Calcium concentrations in lower in plant cells, likely to assist in disassembly

6. 5 KEY POINTS IN VIRUSES

   • 1. Binding to receptors on host cell surface to gain entry
     • HA
   • 2. Cleavage of host cell membrane to gain exit
     • NA
   • 3. Handling the viral NA material
     • HIV uses RT to make double strands
   • 4. Handling the viral protein product
     • Proteolytic cleavage into individual proteins
   • 5. Recruiting host proteins as part of infectious cycle
     • TMV and other plant viruses only have 4-10 proteins

7. WEAK LINKS IN VIRUSES

   • When any of the key features are suppressed
   • Any action that must be carried out by virus
   • Possible to exploit these weak links
     • INFLUENZA VIRUS
       • Can inhibit neuraminidase
         • Drugs fit active site better than the SA it cleaves
           • Zanamivir
           • Prevent budding + spread
     • EBOLA VIRUS
       • Deadly haemorrhagic disease - usually fatal in humans
       • Virus exploits cholesterol transported - Niemann-Pick C1 (NPC1)
         • Transports cholesterol from lysozyme to the ER
           • Riemann-Pick disease sufferers have mutation in NPC1 +2
             • Cholesterol builds up in lysozyme
               • Cells from NPD patients unable to be infected by ebola virus !
     • HIV
       • Uses RT, which we have none of in out genome
         • Good drug target
           • Doravirine belongs to non-nucleoside RT inhibitors
       • Can also target its aspartate proteases for maturation
     • PLANT POTYVIRUS
       • Potyviruses are largest family of plant virus
         • Single stranded RNA virus
       • VPg protein binds to translation initiation factor Elf4E
         • Naturally occurring mutant plants of either protein can't be invade
           • GM plants? to remove protein
     • What link not always enough due to high mutation rate
       • Vaccines induce antibodies
         • Antigen that antibodies recognises has mutated

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