Intracellular functions and mechanisms of alphavirus ion channel 6K

Mosquito-borne alphaviruses such as chikungunya, Mayaro, and Eastern equine encephalitis viruses cause high morbidity and mortality in their mammalian hosts. Alphaviruses are globally distributed arthropod-borne viruses that are enzootic in nature with the potential to disseminate to new geographical regions due to vector adaptations causing new outbreaks. A 2019 outbreak of 38 human cases of Eastern equine encephalitis virus occurred in the United States, raising concerns about its reemergence. In 2005–2006, a chikungunya outbreak started in the Indian Ocean Island of Réunion had spread around the world, infecting millions of people. Chikungunya fever is characterized by debilitating joint pain that can last up to 2–3 years, causing arthritis-like conditions. No effective antiviral strategies or vaccines are available against any of these pathogens. Studying these viruses to gain a molecular understanding of their lifecycle is essential to discovering novel targets for therapeutic intervention. Specifically, the poorly understood intracellular mechanisms that drive alphavirus assembly and budding represent promising antiviral targets. We reported for the first time that the alphavirus- encoded ion channel protein 6K plays an essential part in virus budding by enabling the formation of cytopathic vacuoles-II and envelope spike protein transport to the plasma membrane. The defects due to the deletion of 6K can be restored to varying levels by the expression of a functional HIV-1 Vpu and influenza A virus M2 ion channel. We also demonstrated that ion channel inhibitors could be utilized as antivirals. Building on these observations, in Aim 1, we will characterize chikungunya and Sindbis virus 6K ion channels and their activity by reverse genetics, transport assays in proteoliposomes and fractionated intracellular membrane vesicles, and live-confocal imaging of virus-infected cells with ion-specific fluorescent probes. In Aim 2, by expressing virus- encoded ion channels, including the SARS-CoV-2 E protein in cells and from an alphavirus, we will determine if ion channel-based Golgi remodeling is a standard mechanism used by enveloped RNA viruses for membrane modification and efficient virus budding. With new reverse genetics tools and CRISPR-Cas9 methods, we will investigate the functional involvement of host-encoded ion channels in alphavirus budding. By completing these aims, we will define a novel mechanism by which ion channel proteins modify the secretory pathway for virus budding and how this process can be exploited as an antiviral target. We will also generate new reverse genetics tools that will be useful to the scientific community. The critical knowledge gaps we will address are 1) what are the ions transported by alphavirus 6K? 2) how does 6K participate in virus budding? and 3) an understanding of a general mechanism involving virus and host ion channels utilized by alphaviruses for efficient budding.