Requirement of a functional ion channel for Sindbis virus glycoprotein transport, CPV-II formation, and efficient virus budding

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.

The roles of 6K protein on Getah virus replication and pathogenicity

“…This may be due to that the deletion of the 6K protein leads to occurance of abnormal posttranslational modification on some precursor proteins. The similar case has been shown in Elmasri et al's research 30 …”

“…These results showed that the process of E2 protein transport from the Golgi apparatus to the plasma membrane was inhibited after the deletion of 6K protein, thereby affecting the subsequent processing of viral particles and resulting in a decrease in the release of viral particles (Figure 5). It has recently been found that ion channel activity of SINV 6K protein is necessary for facilitating glycoprotein spike transport to plasma membrane and effective viral budding 30 . Similarly, our experimental results also indicate that the defect in transportation glycoprotein E2 to the cell membrane is caused by the deletion of the 6K protein.…”

“…It has recently been found that ion channel activity of SINV 6K protein is necessary for facilitating glycoprotein spike transport to plasma membrane and effective viral budding. 30 Similarly, our experimental results also indicate that the defect in transportation glycoprotein E2 to the cell membrane is caused by the deletion of the 6K protein. In the research of other alphaviruses, the presence of the 6K protein is crucial for the proper assembly of fully infectious SFV particles, while its absence leads to a significant reduction in viral release.…”

“…The similar case has been shown in Elmasri et al's research. 30 When investigating whether the 6K protein would affect the processing of glycoproteins, we found that both E1 and E2 glycoproteins could be properly processed in cells infected with rGETV-Δ6K. The previous studies have shown that the lack of the entire 6K of SAV3 yielded an E2 protein of larger size.…”

Chikungunya Virus Release is Reduced by TIM-1 Receptors Through Binding of Envelope Phosphatidylserine

“…Assembly of CHIKV is driven by the interaction of capsid protein and the cytoplasmic tail of the E2 protein (4–7). However, recent studies suggest that accessory proteins 6k and TF may also facilitate efficient exit of Sindbis virus, a closely related alphavirus (8).…”