Research Article: Multiple roles of the non-structural protein 3 (nsP3) alphavirus unique domain (AUD) during Chikungunya virus genome replication and transcription

Date Published: January 22, 2019

Publisher: Public Library of Science

Author(s): Yanni Gao, Niluka Goonawardane, Joseph Ward, Andrew Tuplin, Mark Harris, Richard J. Kuhn.


Chikungunya virus (CHIKV) is a re-emerging Alphavirus causing fever, joint pain, skin rash, arthralgia, and occasionally death. Antiviral therapies and/or effective vaccines are urgently required. CHIKV biology is poorly understood, in particular the functions of the non-structural protein 3 (nsP3). Here we present the results of a mutagenic analysis of the alphavirus unique domain (AUD) of nsP3. Informed by the structure of the Sindbis virus AUD and an alignment of amino acid sequences of multiple alphaviruses, a series of mutations in the AUD were generated in a CHIKV sub-genomic replicon. This analysis revealed an essential role for the AUD in CHIKV RNA replication, with mutants exhibiting species- and cell-type specific phenotypes. To test if the AUD played a role in other stages of the virus lifecycle, the mutants were analysed in the context of infectious CHIKV. This analysis indicated that the AUD was also required for virus assembly. In particular, one mutant (P247A/V248A) exhibited a dramatic reduction in production of infectious virus. This phenotype was shown to be due to a block in transcription of the subgenomic RNA leading to reduced synthesis of the structural proteins and a concomitant reduction in virus production. This phenotype could be further explained by both a reduction in the binding of the P247A/V248A mutant nsP3 to viral genomic RNA in vivo, and the reduced affinity of the mutant AUD for the subgenomic promoter RNA in vitro. We propose that the AUD is a pleiotropic protein domain, with multiple functions during CHIKV RNA synthesis.

Partial Text

Chikungunya virus (CHIKV; family Togaviridae, genus Alphavirus) [1] is an arbovirus that causes fever, rash and arthralgia with an infrequent fatal outcome [2]. It was first isolated in Tanzania in 1952–1953 [3,4]. During the last 50 years, numerous CHIKV re-emergences have been documented across the world, including Africa, Asia, Europe and America [5,6]. CHIKV is transmitted to humans by mosquitoes, mainly Aedes aegypti and Aedes albopictus. The latter can reproduce in more moderate climates meaning that CHIKV has spread from Southern Africa and is now present across the Americas and parts of Southern Europe (including France and Italy). Increasing global temperatures resulting from climate change raise the concern that CHIKV will spread further. In this regard, there are no antiviral therapies or safe, effective vaccines available to treat CHIKV infection.

Of the four alphavirus non-structural proteins, nsP3 remains the least well understood [25]. The protein consists of three domains, the N-terminal of which has been identified as a macrodomain that binds to ADP-ribose and possesses ADP-ribosylhydrolase activity [9]. Recent studies have proposed a role for this enzymatic activity in virus pathogenesis but as yet the underlying mechanisms remain elusive [11]. The C-terminal hypervariable domain differs dramatically in amino acid sequence between different alphaviruses and is intrinsically disordered. It has been shown to interact with a range of cellular proteins, including components of stress granules [26], and is implicated in the assembly of virus genome replication complexes.