Mammalian orthoreovirus can exit cells in extracellular vesicles

Common Replication Cycle

Attachment

Entry

Genome replication

Translation

Assembly

Exit

Plant Virus Exit

Hijack movement protein and move through plasmodesmata

Release through cell lysis

• Most common release mechanism in bacteriophage

• Mediated by viroporin: Adenoviridae, Picronaviridae

• Phospholipids: Phycodnaviridae

Non-lytic release of naked virus:

Poliovirus and Hep C

ESCRT Dependent Budding

• HIV-1, Ebola, Marburg, Rabies, Newcastle disease

• Viral proteins recruit ESCRT and budding

ESCRT Independent Budding

• Influenza, SARS CoV, RSV, Sindbis Virus

• Viral proteins manipulate cell membrane/other mechanism

Budding through internal membrane and release

Hepatitis B

Evelopment by virus specific mechanism

Vaccinia

Assembly into nucleus

Herpesvirus

Extracellular vessicles (EV)

• Small, membrane bound particles released by cells

• Surrounded by lipid bilayer and contain diverse cargo (Protein, lipid, nucleic acid, etc.)

• Cell-to-cell communication, molecule transport, cell debris removal

EV mediated exit

• Happens on Rotavirus

• Zika virus and Epstein Barr Virus also upregulate EV release

• Does these EVs contain Virus?

• Benefits of release in EV?

Reovirus

• Non-enveloped virus with a segmented, dsRNA

• Human reovirus infects humans, rarely causing disease

Reovirus selectivity to tumor cells

Can selectively infect tumor cells → potential use as oncolytic

Reovirus Exit

• Lytic Cycle: Hela cells and MDCK cells

• Non-lytic cycle: human brain microvascular epithelial cells, primary human airway epithelia

What authors are trying to solve what mystery?

How this virus exits cell?

Figure 2: Do the medium and large EV contain virus

Yes they did

Figure 3:do the small EV contain virus

No they don’t

How did this research group investigate this problem?

Selection of biochemical methods begins with definitions of goals/questions

Is this reovirus physically associated with EV? If so how?

What size EV are associated with the reovirus?

Can we directly see the reovirus on or inside the EV?

Are the viruses contained in the EV still infectious/virulent?

Replication and Membrane Integrity (Trypan Blue) Assay

Used to see if the virus can infect cells and if the virus results in damage to the plasma membrane (suggesting different egress strategies), T1L, T3D, and a MOCK virus used

Differential centrifugation

Separation of large, medium, and small EVs

SDS-PAGE and Estern Blot

Detects viral proteins

Plaque Assay

Determines if there is a complete, infectious virion present in the sample (protein could be anywhere)

Negative Stain Electron Microscopy

An EM method where the sample is NOT sputtered or stained with a heavy metal. Instead, the target grid (or background) is stained. The biological sample then appears as a silhouette

This answers the question, can we directly see the reovirus on or inside the extracellular vesicle?

Antibody Neutralization Assay

Do EVs protect virions from neutralizing antibodies?

High Resolution Melt Assay

Amplification of genetic material and identification of small differences in sequence. Do EVs carry mutliple virions?

Is viral replication ability cell-specific? Does membrane disruption play a role?

No and No

Is viral-EV association correlated with EV size? Does infectivity change with EV size?

EV Egress is not specific to cell type

Are particles packaged within EVs? Does this protect from antibody-mediated neutralization?

EV-mediated protection is virus-strain and cell-type dependent (because it changes between L and Caco-2 cells)

Does reovirus infection affect cell production of proteins?

Yes

Is this association present on the surface of cells?

Yes, on large EV

EV Size Correlates with…

Protection from antibody mediated-neutralization

Key Takeaways

• Reovirus infection increases EV production

• Association regardless of membrane disruption

• Nonspecific Association Does NOT explain EV strategy

• Strain and Cell-Specific Protection from Antibody-Neutralization

• Multiparticle Egress

Is EV egress conserved in other viruses?

Yes! BK, polyomavirus, enterovirus 71, porcine reproductive and respiratory syndrome virus, and Hep A virus

Dual EV Egress

Depending on cell type, either bound externally or packaged internally. Exists in encephalomyocarditis virus. Cannot enrich for only inf-EVs so cannot currently study this further - perhaps cell/virus-types interact differently with EV biosynthesis pathway

Viral apoptotic effect have effect on EV?

Sindbis: viral nucleocapsids and antigens group in/near EV

Chickungunya: forms EVs, neighboring cell infection limited

Perhaps: L vs Caco apoptosis effect Reovirus EV egress differently

Caco does not have fully intact apoptosis pathway