Spada, S. J. | bioRxiv Science

bioRxiv2026

Restraint of Powassan virus replication by TRIM5α facilitates viral avoidance of antiviral immunity

TRIpartite Motif (TRIM) protein 5 alpha (TRIM5) is a well characterized cellular inhibitor of lentivirus replication that limits transmission of related viruses between primates. We previously reported that TRIM5 derived from humans and rhesus macaques inhibits replication of orthoflaviviruses belonging to the tick-borne encephalitis virus (TBEV) serocomplex, including TBEV, Kyasanur forest disease virus and Langat virus (LGTV), but interestingly not the tick-borne Powassan virus (POWV). To further characterize the primate TRIM5 and orthoflavivirus interface, we screened TRIM5 variants from representative old- and new-world primates for restriction capacity. TRIM5 from old-world African green monkey, De Brazzas monkey and chimpanzee demonstrated virus-specific restriction of tick-borne orthoflaviviruses. Efforts to determine why TRIM5 fails to inhibit POWV revealed that our lab stock had acquired a non-synonymous mutation in NS3 that, when introduced into a POWV molecular clone, facilitated virus replication in the presence of all inhibitory primate TRIM5 proteins. Infection of human dendritic cells with TRIM5-resistant POWV resulted in high early replication and strong induction of interferon responses that limited replication compared with the wild-type virus. Thus, primate TRIM5 functions as a potent cellular barrier to infection with tick-borne orthoflaviviruses that restrains replication to a level that may help avoid early innate immune recognition.

microbiology↗

bioRxiv2024

Human ESCRT-I and ALIX function as scaffolding helical filaments in vivo

The Endosomal Sorting Complex Required for Transport (ESCRT) is an evolutionarily conserved machinery that performs reverse-topology membrane scission in cells universally required from cytokinesis to budding of enveloped viruses. Upstream acting ESCRT-I and ALIX control these events and link recruitment of viral and cellular partners to late-acting ESCRT-III CHMP4 through incompletely understood mechanisms. Using structure-function analyses combined with super-resolution imaging, we show that ESCRT-I and ALIX function as distinct helical filaments in vivo. Together, they are essential for optimal structural scaffolding of HIV-1 nascent virions, the retention of viral and human genomes through defined functional interfaces, and recruitment of CHMP4 that itself assembles into corkscrew-like filaments intertwined with ESCRT-I or ALIX helices. Disruption of filament assembly or their conformationally clustered RNA binding interfaces in human cells impaired membrane abscission, resulted in major structural instability and leaked nucleic acid from nascent virions and nuclear envelopes. Thus, ESCRT-I and ALIX function as helical filaments in vivo and serve as both nucleic acid-dependent structural scaffolds as well as ESCRT-III assembly templates. Significance statementWhen cellular membranes are dissolved or breached, ESCRT is rapidly deployed to repair membranes to restore the integrity of intracellular compartments. Membrane sealing is ensured by ESCRT-III filaments assembled on the inner face of membrane; a mechanism termed inverse topology membrane scission. This mechanism, initiated by ESCRT-I and ALIX, is universally necessary for cytokinesis, wound repair, budding of enveloped viruses, and more. We show ESCRT-I and ALIX individually oligomerize into helical filaments that cluster newly discovered nucleic acid-binding interfaces and scaffold-in genomes within nascent virions and nuclear envelopes. These oligomers additionally appear to serve as ideal templates for ESCRT-III polymerization, as helical filaments of CHMP4B were found intertwined ESCRT-I or ALIX filaments in vivo. Similarly, corkscrew-like filaments of ALIX are also interwoven with ESCRT-I, supporting a model of inverse topology membrane scission that is synergistically reinforced by inward double filament scaffolding.

cell biology↗