Organization of theFlavivirusRNAreplicase complex

Abstract

Flaviviruses, such as Dengue, Japanese encephalitis, West Nile, Yellow Fever, and Zika viruses, are serious human pathogens that cause significant morbidity and mortality globally each year. Flaviviruses are single-stranded, positive-sense RNA viruses, and encode two multidomain proteins, NS3 and NS5, that possess all enzymatic activities required for genome replication and capping. NS3 and NS5 interact within virus-induced replication compartments to form the RNA genome replicase complex. Although the individual enzymatic activities of both proteins have been extensively studied and are well characterized, there are still gaps in our understanding of how they interact to efficiently coordinate their respective activities during positive strand RNA synthesis and capping. Here, we discuss what is known about the structures and functions of the NS3 and NS5 proteins and propose a preliminary NS3:NS5:RNA interaction model based on a large body of literature about how the viral enzymes function, physical restraints between NS3 and NS5, as well as critical steps in the replication process.

Structural disorder in the proteome and interactome of Alkhurma virus (ALKV)

“…All flaviviral NS proteins are multifunctional and play very diverse roles in the control and orchestration of various stages of viral life, including regulation of viral morphogenesis [113] and formation of the replication complex (RC) [114]. Only NS3 and NS5 possess enzymatic activities (which together account for all enzymatic activities required to amplify the RNA genome and to attach a type 1 cap to its 5′ end), whereas biological roles of other NS proteins are not related to the catalysis.…”

“…For example, NS2A plays a role in viral RNA synthesis, viral assembly, inhibition of the interferon α/β response, virus-induced membrane formation, and production of NS1′ through a ribosomal frameshift mechanism [118]. NS2B is known as a regulatory subunit of the flaviviral serine protease NS3, plays a role controlling the localization of NS3 to membranes, possesses membrane-destabilizing activity [114], and may act as a viroporin (a class of small, hydrophobic, viral proteins that have ion-channel activity in vitro, and are involved in enhancing infectivity) [113]. NS3 is a multidomain protein that has three enzymatic activities, such as N-terminal domain with serine protease, and C-terminal domain with RNA helicase and RNA-stimulated nucleoside triphosphate hydrolase and 5′-RNA triphosphatase activities [119].…”

Motif V acts as a Regulator of Energy Transduction Between the Flavivirus NS3 ATPase and RNA Binding Cleft

“…The structural proteins contribute to the formation of mature viral particles, while the nonstructural proteins are responsible for replication of the viral genome and protecting the virus from attacks by the host cell innate immune system (13). Once the viral proteins are post-translationally processed, the positive-sense genomic ssRNA is used as a template to create a negative-sense anti-genomic RNA, forming a double-stranded RNA (dsRNA) intermediate complex (15,16). The nascent negative-sense ssRNA serves as a template strand for producing new positive strand RNAs that can be packaged into viral particles, translated into new proteins, or interfere with the RNA decay pathway (17,18).…”

“…The nascent negative-sense ssRNA serves as a template strand for producing new positive strand RNAs that can be packaged into viral particles, translated into new proteins, or interfere with the RNA decay pathway (17,18). Therefore, the unwinding of dsRNA intermediate to produce a free negative ssRNA for positive strand synthesis is a critical component for the replication of flavivirus RNA genomes (15). This function is achieved by the C-terminal helicase domain of NS3.…”

“…The helical gate and b-wedge are responsible for splitting the dsRNA intermediate into two ssRNA strands: the positive-sense viral ssRNA and the negative-sense template ssRNA (33). Single-molecule and structural biology studies have suggested that the negative-sense ssRNA template enters the RNA binding cleft, where the helicase utilizes the energy produced from the hydrolysis of one ATP molecule to power translocation and unwinding of the dsRNA intermediate one base at a time (15,16,32,(36)(37)(38)(39)(40). A fundamental unanswered question within the SF2 helicase field is how ATP hydrolysis causes structural changes within the helicase to result in translocation and unwinding of dsRNA intermediates.…”