The Molecular Biology & General Background of the Lassa Virus Presented by Ryan Cordell

Presentation Overview General Virus Background General Virus Background General Morphology of the Virus General Morphology of the Virus Ambisense RNA Genome Ambisense RNA Genome Viral Proteins Viral Proteins Virus Life Cycle Virus Life Cycle Treatment Treatment Weaponization Weaponization

Lassa Fever Background Found in West Africa Found in West Africa 100K – 300k Cases/year 100K – 300k Cases/year 1% of infections result in death 1% of infections result in death Early symptoms resemble flue or enteritis Early symptoms resemble flue or enteritis Three-week incubation period Three-week incubation period

General Characteristics Virus Member of the Arena Virus Family Spherical viral particles Particle diameter is nm Lipid membrane coat Virion consists of two nucleocapsids

General Characteristics Cont’d Club-shaped projections on virion surface Club-shaped projections on virion surface Virion Composition: Virion Composition: –70% Protein weight –2% Genome weight –8% Carbohydrates –20% Lipids 40-45% Guanine & Cytosine content Thermal inactivation point is 56 o C Inactivated at pH 8.5

RNA Genome: Coding Region Composed of 2 strands: S-RNA & L-RNA Composed of 2 strands: S-RNA & L-RNA L:S ratio is 2:5 in virion & infected cell L:S ratio is 2:5 in virion & infected cell Both strands are ambisense Both strands are ambisense S-RNA is 3.4 kb and codes for 2 proteins S-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 proteins L-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: Coding Region

RNA Genome: Noncoding Region No overlap between (+) & (-) ambisense genes No overlap between (+) & (-) ambisense genes Noncoding intergenic region found between genes Noncoding intergenic region found between genes Intergenic region forms stable hairpin loop in both L & S strands Intergenic region forms stable hairpin loop in both L & S strands 17 of 19 nt on the 3’ end are conserved between L & S 17 of 19 nt on the 3’ end are conserved between L & S

Viral Proteins

Viral Proteins: Nucleocapsid Protein (NP) MW kDa MW kDa 1 st protein expressed in infected cell 1 st protein expressed in infected cell Most abundant structural protein Most abundant structural protein Major protein component of nucleocapsids Major protein component of nucleocapsids NP can be cross-linked to carboxy-terminus of GP2 (essential to virion assembly) NP can be cross-linked to carboxy-terminus of GP2 (essential to virion assembly)

Viral Proteins: Viral RNA-Dependent RNA Polymerase (L) MW kDa MW kDa Actual L-protein can be detected in virions Actual L-protein can be detected in virions 2 nd protein produced in infected cell 2 nd protein produced in infected cell

Viral Proteins: Envelope Glycoprotein (GPC) MW kDa MW kDa Post-translationally cleaved to release GP1 & GP3 Post-translationally cleaved to release GP1 & GP3

Viral Proteins: Glycoprotein - 1 MW kDa MW kDa 4-11 N-linked glycosylation sites 4-11 N-linked glycosylation sites Assemble into homotetrameric complexes Assemble into homotetrameric complexes Complexes held together with disulfide bonds Complexes held together with disulfide bonds

Viral Proteins: Glycoprotein - 2 MW 35 kDa MW 35 kDa 1-4 potential N-linked glycosylation sites 1-4 potential N-linked glycosylation sites Membrane spanning domain Membrane spanning domain Interacts with NP in order to assemble virion Interacts with NP in order to assemble virion Assemble into homotetrameric complexes Assemble into homotetrameric complexes Stalk of glycoprotein spike Stalk of glycoprotein spike Acts as viral fusion protein under acidic conditions Acts as viral fusion protein under acidic conditions

Viral Proteins: Zinc RING-finger Protein (Z) MW 11 kDa MW 11 kDa Contains a RING-finger motif and binds to zinc Contains a RING-finger motif and binds to zinc Relatively large amount of Z-mRNA can be found in the virion Relatively large amount of Z-mRNA can be found in the virion Might serve as a cofactor in replication and/or transcription Might serve as a cofactor in replication and/or transcription Actual function is not known Actual function is not known

Virus Life Cycle

1. Virion comes into contact with cell 2. GP1 bind to cellular receptor (Dystroglycan) 3. Virion enters cell via smooth-walled clathrin- free vesicle 4. Acidification of endosome leads to nucleocapsid delivery a.GP1 dissociates from GP2 b.GP2 fusion peptide is exposed c.Core unit is delivered into cytosol

Virus Life Cycle Cont’d 5. NP is expressed in large quantities 6. Replication of L & S strands ensues 7. GPC is expressed leading to GP1 & GP2 8. GP1 & GP2 homotetrameric complexes migrate to cell surface 9. NPs combine with genomic RNA to from a string of bead like structures 10. NP cross-links to carboxy terminus of GP2 11. Complete virus forms and buds off from cell

Lassa Virus

Treatment Ribavirin for infected individuals (mech not known) Ribavirin for infected individuals (mech not known) Vaccines have been successfully tested in animals but not in humans Vaccines have been successfully tested in animals but not in humans

Weaponization No current human vaccine Poorly understood by medical community Rarity = unexpected Can’t survive in open for long Only 1 known animal reservoir Simplistic genome Stable replication

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