The Molecular Biology & The Molecular Biology & General Background of the General Background of the

Lassa VirusLassa VirusPresented by Ryan CordellPresented by Ryan Cordell

Presentation OverviewPresentation Overview

General Virus BackgroundGeneral Virus Background General Morphology of the VirusGeneral Morphology of the Virus Ambisense RNA GenomeAmbisense RNA Genome Viral ProteinsViral Proteins Virus Life CycleVirus Life Cycle TreatmentTreatment WeaponizationWeaponization

Lassa Fever BackgroundLassa Fever Background

Found in West AfricaFound in West Africa 100K – 300k 100K – 300k

Cases/yearCases/year 1% of infections result 1% of infections result

in deathin death Early symptoms Early symptoms

resemble flue or resemble flue or enteritis enteritis

Three-week incubation Three-week incubation periodperiod

General Characteristics VirusGeneral Characteristics Virus

Member of the Arena Member of the Arena Virus FamilyVirus Family

Spherical viral Spherical viral particlesparticles

Particle diameter is Particle diameter is 110-130 nm110-130 nm

Lipid membrane coatLipid membrane coat Virion consists of two Virion consists of two

nucleocapsidsnucleocapsids

General Characteristics Cont’dGeneral Characteristics Cont’d

Club-shaped Club-shaped projections on virion projections on virion surfacesurface

Virion Composition:Virion Composition:– 70% Protein weight70% Protein weight

– 2% Genome weight2% Genome weight

– 8% Carbohydrates8% Carbohydrates

– 20% Lipids20% Lipids

40-45% Guanine & 40-45% Guanine & Cytosine contentCytosine content

Thermal inactivation Thermal inactivation point is 56 point is 56 ooC C

Inactivated at Inactivated at << pH 5.5 pH 5.5 and and >> pH 8.5 pH 8.5

RNA Genome:RNA Genome:Coding RegionCoding Region

Composed of 2 strands: S-RNA & L-RNAComposed of 2 strands: S-RNA & L-RNA L:S ratio is 2:5 in virion & infected cellL:S ratio is 2:5 in virion & infected cell Both strands are ambisenseBoth strands are ambisense S-RNA is 3.4 kb and codes for 2 proteinsS-RNA is 3.4 kb and codes for 2 proteins

• Nucleoprotein (NP)Nucleoprotein (NP)

• Glycoprotein (GPC)Glycoprotein (GPC)

L-RNA is 7 kb and codes for 2 proteinsL-RNA is 7 kb and codes for 2 proteins• Viral RNA-dependent RNA polymerase (L)Viral RNA-dependent RNA polymerase (L)

• Zinc RING-finger motif protein (Z)Zinc RING-finger motif protein (Z)

RNA Genome:RNA Genome:Coding RegionCoding Region

RNA Genome:RNA Genome:Noncoding RegionNoncoding Region

No overlap between (+) & (-) ambisense No overlap between (+) & (-) ambisense genesgenes

Noncoding intergenic region found between Noncoding intergenic region found between genesgenes

Intergenic region forms stable hairpin loop Intergenic region forms stable hairpin loop in both L & S strandsin both L & S strands

17 of 19 nt on the 3’ end are conserved 17 of 19 nt on the 3’ end are conserved between L & Sbetween L & S

Viral ProteinsViral Proteins

Viral Proteins:Viral Proteins:Nucleocapsid Protein (NP)Nucleocapsid Protein (NP)

MW 60-68 kDaMW 60-68 kDa 11stst protein expressed in infected cell protein expressed in infected cell Most abundant structural proteinMost abundant structural protein Major protein component of nucleocapsidsMajor protein component of nucleocapsids NP can be cross-linked to carboxy-terminus NP can be cross-linked to carboxy-terminus

of GP2 (essential to virion assembly)of GP2 (essential to virion assembly)

Viral Proteins:Viral Proteins:Viral RNA-Dependent RNA Viral RNA-Dependent RNA

Polymerase (L)Polymerase (L) MW 180-250 kDaMW 180-250 kDa Actual L-protein can be detected in virionsActual L-protein can be detected in virions 22ndnd protein produced in infected cell protein produced in infected cell

Viral Proteins:Viral Proteins:Envelope Glycoprotein (GPC)Envelope Glycoprotein (GPC)

MW 79-80 kDaMW 79-80 kDa Post-translationally cleaved to release GP1 Post-translationally cleaved to release GP1

& GP3& GP3

Viral Proteins:Viral Proteins:Glycoprotein - 1Glycoprotein - 1

MW 40-46 kDaMW 40-46 kDa 4-11 N-linked glycosylation sites4-11 N-linked glycosylation sites Assemble into homotetrameric complexesAssemble into homotetrameric complexes Complexes held together with disulfide Complexes held together with disulfide

bondsbonds

Viral Proteins:Viral Proteins:Glycoprotein - 2Glycoprotein - 2

MW 35 kDaMW 35 kDa 1-4 potential N-linked glycosylation sites1-4 potential N-linked glycosylation sites Membrane spanning domainMembrane spanning domain Interacts with NP in order to assemble virionInteracts with NP in order to assemble virion Assemble into homotetrameric complexesAssemble into homotetrameric complexes Stalk of glycoprotein spikeStalk of glycoprotein spike Acts as viral fusion protein under acidic Acts as viral fusion protein under acidic

conditionsconditions

Viral Proteins:Viral Proteins:Zinc RING-finger Protein (Z)Zinc RING-finger Protein (Z)

MW 11 kDaMW 11 kDa Contains a RING-finger motif and binds to Contains a RING-finger motif and binds to

zinczinc Relatively large amount of Z-mRNA can be Relatively large amount of Z-mRNA can be

found in the virionfound in the virion Might serve as a cofactor in replication Might serve as a cofactor in replication

and/or transcriptionand/or transcription Actual function is not knownActual function is not known

Virus Life CycleVirus Life Cycle

Virus Life CycleVirus Life Cycle

1.1. Virion comes into contact with cellVirion comes into contact with cell

2.2. GP1 bind to cellular receptor (Dystroglycan)GP1 bind to cellular receptor (Dystroglycan)

3.3. Virion enters cell via smooth-walled clathrin-Virion enters cell via smooth-walled clathrin-free vesiclefree vesicle

4.4. Acidification of endosome leads to nucleocapsid Acidification of endosome leads to nucleocapsid deliverydelivery

a.a. GP1 dissociates from GP2GP1 dissociates from GP2

b.b. GP2 fusion peptide is exposedGP2 fusion peptide is exposed

c.c. Core unit is delivered into cytosolCore unit is delivered into cytosol

Virus Life Cycle Cont’dVirus Life Cycle Cont’d

5.5. NP is expressed in large quantitiesNP is expressed in large quantities

6.6. Replication of L & S strands ensuesReplication of L & S strands ensues

7.7. GPC is expressed leading to GP1 & GP2GPC is expressed leading to GP1 & GP2

8.8. GP1 & GP2 homotetrameric complexes migrate GP1 & GP2 homotetrameric complexes migrate to cell surfaceto cell surface

9.9. NPs combine with genomic RNA to from a NPs combine with genomic RNA to from a string of bead like structuresstring of bead like structures

10.10. NP cross-links to carboxy terminus of GP2NP cross-links to carboxy terminus of GP2

11.11. Complete virus forms and buds off from cellComplete virus forms and buds off from cell

Lassa VirusLassa Virus

TreatmentTreatment

Ribavirin for infected Ribavirin for infected individuals (mech not individuals (mech not known)known)

Vaccines have been Vaccines have been successfully tested in successfully tested in animals but not in animals but not in humanshumans

WeaponizationWeaponization

No current human vaccineNo current human vaccine Poorly understood by Poorly understood by

medical communitymedical community Rarity = unexpectedRarity = unexpected Can’t survive in open for Can’t survive in open for

longlong Only 1 known animal Only 1 known animal

reservoirreservoir Simplistic genomeSimplistic genome Stable replicationStable replication

The EndThe End