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- Title
Requirement of a functional ion channel for Sindbis virus glycoprotein transport, CPV-II formation, and efficient virus budding.
- Authors
Elmasri, Zeinab; Negi, Vashi; Kuhn, Richard J.; Jose, Joyce
- Abstract
Many viruses encode ion channel proteins that oligomerize to form hydrophilic pores in membranes of virus-infected cells and the viral membrane in some enveloped viruses. Alphavirus 6K, human immunodeficiency virus type 1 Vpu (HIV-Vpu), influenza A virus M2 (IAV-M2), and hepatitis C virus P7 (HCV-P7) are transmembrane ion channel proteins that play essential roles in virus assembly, budding, and entry. While the oligomeric structures and mechanisms of ion channel activity are well-established for M2 and P7, these remain unknown for 6K. Here we investigated the functional role of the ion channel activity of 6K in alphavirus assembly by utilizing a series of Sindbis virus (SINV) ion channel chimeras expressing the ion channel helix from Vpu or M2 or substituting the entire 6K protein with full-length P7, in cis. We demonstrate that the Vpu helix efficiently complements 6K, whereas M2 and P7 are less efficient. Our results indicate that while SINV is primarily insensitive to the M2 ion channel inhibitor amantadine, the Vpu inhibitor 5-N, N-Hexamethylene amiloride (HMA), significantly reduces SINV release, suggesting that the ion channel activity of 6K is similar to Vpu, promotes virus budding. Using live-cell imaging of SINV with a miniSOG-tagged 6K and mCherry-tagged E2, we further demonstrate that 6K and E2 colocalize with the Golgi apparatus in the secretory pathway. To contextualize the localization of 6K in the Golgi, we analyzed cells infected with SINV and SINV-ion channel chimeras using transmission electron microscopy. Our results provide evidence for the first time for the functional role of 6K in type II cytopathic vacuoles (CPV-II) formation. We demonstrate that in the absence of 6K, CPV-II, which originates from the Golgi apparatus, is not detected in infected cells, with a concomitant reduction in the glycoprotein transport to the plasma membrane. Substituting a functional ion channel, M2 or Vpu localizing to Golgi, restores CPV-II production, whereas P7, retained in the ER, is inadequate to induce CPV-II formation. Altogether our results indicate that ion channel activity of 6K is required for the formation of CPV-II from the Golgi apparatus, promoting glycoprotein spike transport to the plasma membrane and efficient virus budding. Author summary: Alphaviruses cause acute and long-term diseases ranging from febrile illness to fatal encephalitis in infected individuals. Identifying new drug targets for antiviral development is crucial to controlling and combating alphavirus infections. Inhibitors for viral ion channels such as Influenza M2, HIV Vpu, and HCV P7 have been proven to be effective antivirals. Alphavirus 6K is a 6 kDa structural protein that forms ion channels on membranes of infected cells. However, no structural information is available to establish its functional role in alphavirus life cycle. Using a miniSOG-tagged 6K SINV, we determined the spatial and temporal organization of 6K for the first time in infected cells. Our results reveal the colocalization of 6K with E2 in the Golgi, where its ion channel activity is essential for CPV-II formation. This study establishes that 6K is required for efficient glycoprotein transport to the plasma membrane, a function that can be partially complemented in cis by other viral ion channels that traffic to the Golgi. We demonstrate that the inhibition of 6K by a channel-blocking drug, 5-N, N-Hexamethylene amiloride impedes alphavirus budding, laying the foundation for developing inhibitors targeting 6K as an attractive antiviral strategy.
- Subjects
ION channels; ALPHAVIRUSES; CYTOSKELETAL proteins; CELL membranes; GOLGI apparatus; LIFE cycles (Biology); HIV
- Publication
PLoS Pathogens, 2022, Vol 18, Issue 10, p1
- ISSN
1553-7366
- Publication type
Article
- DOI
10.1371/journal.ppat.1010892