EXPLORATION OF EARLY-STAGE INFECTION IN ALPHAVIRUSES AND FLAVIVIRUSES

Sindbis virus (SINV) is a mosquito-borne alphavirus with a positive-sense single-stranded RNA genome. It can cause febrile illness in humans, marked by rash, arthritis, and muscle pain. Due to its genetic and structural similarities with other medically significant alphaviruses, SINV serves as a model for studying alphavirus biology. One key SINV protein is a small 6 kDa hydrophobic protein called ‘6K’ that functions as a viroporin—a viral ion channel. Despite evidence suggesting that 6K forms ion channels and is involved in virus budding, its precise mechanisms are not well understood. Understanding the function and inhibition potential of 6K can unveil insights into alphavirus biology and open avenues for therapeutic intervention. There are currently no antiviral therapies targeting alphavirus infections.

In our study, we focus on 6K's ion channel function and its importance in viral growth, glycoprotein trafficking, and viral entry. We show that deleting 6K severely impairs viral growth, glycoprotein trafficking and viral budding. Chimeras in which the 6K transmembrane domain is replaced with ion channels from HIV-1 Vpu, IAV M2, or HCV p7, display partially restored function, demonstrating the conserved functions of viroporins from unrelated viruses.

In addition, we developed a novel chimeric SINV system to study the SARS-CoV-2 Envelope (E) protein’s ion channel activity under biosafety level 2 (BSL-2) conditions. E protein is highly conserved across SARS-CoV-2 variants of concern, making it an attractive target for antiviral drug development. Our system demonstrates that the E protein transmembrane domain (ETM) can partially replace the function of the SINV 6K ion channel, allowing us to screen inhibitors of the E protein. As validation, we show that known ion channel inhibitors such as amantadine and amilorides exhibit similar inhibition profiles in this chimeric system as reported in authentic BSL3 SARS-CoV-2 systems.

Overall, these studies advance our understanding of viroporins in viral infections and support the development of broad-spectrum antivirals against alphaviruses and SARS-CoV-2.