Activity, Template Preference, and Compatibility of Components of RNA Replicase of Eastern Equine Encephalitis Virus.

Eastern equine encephalitis virus (EEEV) usually cycles between Culiseta melanura mosquitoes and birds; however, it can also infect humans. EEEV has a positive-sense RNA genome that, in infected cells, serves as an mRNA for the P1234 polyprotein. P1234 undergoes a series of precise cleavage events producing four nonstructural proteins (nsP1-4) representing subunits of the RNA replicase. Here, we report the construction and properties of a trans-replicase for EEEV. The template RNA of EEEV was shown to be replicated by replicases of diverse alphaviruses. The EEEV replicase, on the other hand, demonstrated limited ability in replicating template RNAs originating from alphaviruses of the Semliki Forest virus complex. The replicase of EEEV was also successfully reconstructed from P123 and nsP4 components. The ability of EEEV P123 to form functional RNA replicases with heterologous nsP4s was more efficient using EEEV template RNA than heterologous alphavirus template RNA. This finding indicates that unlike with previously studied Semliki Forest complex alphaviruses, P123 and/or its processing products have a leading role in EEEV template RNA recognition. Infection of HEK293T cells harboring the EEEV template RNA with EEEV or Western equine encephalitis virus prominently activated expression of a reporter encoded in the template RNA; the effect was much smaller for infection with other alphaviruses and not detectable upon flavivirus infection. At the same time, EEEV infection resulted only in a limited activation of the template RNA of chikungunya virus. Thus, cells harboring reporter-carrying template RNAs can be used as sensitive and selective biosensors for different alphaviruses. IMPORTANCE Infection of EEEV in humans can cause serious neurologic disease with an approximately 30% fatality rate. Although human infections are rare, a record-breaking number was documented in 2019. The replication of EEEV has a unique requirement for host factors but is poorly studied, partly because the virus requires biosafety level 3 facilities which can limit the scope of experiments; at the same time, these studies are crucial for developing antiviral approaches. The EEEV trans-replicase developed here contributes significantly to research on EEEV, providing a safe and versatile tool for studying the virus RNA replication. Using this system, the compatibility of EEEV replicase components with counterparts from other alphaviruses was analyzed. The obtained data can be used to develop unique biosensors that provide alternative methods for detection, identification, quantitation, and neutralization of viable alphaviruses that are compatible with high throughput, semiautomated approaches.

Semliki Forest Virus Chimeras with Functional Replicase Modules from Related Alphaviruses Survive by Adaptive Mutations in Functionally Important Hot Spots.

Alphaviruses (family Togaviridae) include both human pathogens such as chikungunya virus (CHIKV) and Sindbis virus (SINV) and model viruses such as Semliki Forest virus (SFV). The alphavirus positive-strand RNA genome is translated into nonstructural (ns) polyprotein(s) that are precursors for four nonstructural proteins (nsPs). The three-dimensional structures of nsP2 and the N-terminal 2/3 of nsP3 reveal that these proteins consist of several domains. Cleavage of the ns-polyprotein is performed by the strictly regulated protease activity of the nsP2 region. Processing results in the formation of a replicase complex that can be considered a network of functional modules. These modules work cooperatively and should perform the same task for each alphavirus. To investigate functional interactions between replicase components, we generated chimeras using the SFV genome as a backbone. The functional modules corresponding to different parts of nsP2 and nsP3 were swapped with their counterparts from CHIKV and SINV. Although some chimeras were nonfunctional, viruses harboring the CHIKV N-terminal domain of nsP2 or any domain of nsP3 were viable. Viruses harboring the protease part of nsP2, the full-length nsP2 of CHIKV, or the nsP3 macrodomain of SINV required adaptive mutations for functionality. Seven mutations that considerably improved the infectivity of the corresponding chimeric genomes affected functionally important hot spots recurrently highlighted in previous alphavirus studies. These data indicate that alphaviruses utilize a rather limited set of strategies to survive and adapt. Furthermore, functional analysis revealed that the disturbance of processing was the main defect resulting from chimeric alterations within the ns-polyprotein. IMPORTANCE Alphaviruses cause debilitating symptoms and have caused massive outbreaks. There are currently no approved antivirals or vaccines for treating these infections. Understanding the functions of alphavirus replicase proteins (nsPs) provides valuable information for both antiviral drug and vaccine development. The nsPs of all alphaviruses consist of similar functional modules; however, to what extent these are independent in functionality and thus interchangeable among homologous viruses is largely unknown. Homologous domain swapping was used to study the functioning of modules from nsP2 and nsP3 of other alphaviruses in the context of Semliki Forest virus. Most of the introduced substitutions resulted in defects in the processing of replicase precursors that were typically compensated by adaptive mutations that mapped to determinants of polyprotein processing. Understanding the principles of virus survival strategies and identifying hot spot mutations that permit virus adaptation highlight a route to the rapid development of attenuated viruses as potential live vaccine candidates.