Identification of RACK1 as a novel regulator of non-structural protein 4 of chikungunya virus

Abstract

Chikungunya virus (CHIKV) is a neglected arthropod-borne and anthropogenic alphavirus. Over the past two decades, the CHIKV distribution has undergone significant changes worldwide, from the original tropics and subtropics regions to temperate regions, which has attracted global attention. However, the interactions between CHIKV and its host remain insufficiently understood, which dampens the need for the development of an anti-CHIKV strategy. In this study, on the basis of the optimal overexpression of non-structural protein 4 (nsP4), we explore host interactions of CHIKV nsP4 using mass spectrometry-based protein-protein interaction approaches. The results reveal that some cellular proteins that interact with nsP4 are enriched in the ubiquitin-proteasome pathway. Specifically, the scaffold protein receptor for activated C kinase 1 (RACK1) is identified as a novel host interactor and regulator of CHIKV nsP4. The inhibition of the interaction between RACK1 and nsP4 by harringtonolide results in the reduction of nsP4, which is caused by the promotion of degradation but not the inhibition of nsP4 translation. Furthermore, the decrease in nsP4 triggered by the RACK1 inhibitor can be reversed by the proteasome inhibitor MG132, suggesting that RACK1 can protect nsP4 from degradation through the ubiquitin-proteasome pathway. This study reveals a novel mechanism by which the host factor RACK1 regulates CHIKV nsP4, which could be a potential target for developing drugs against CHIKV.

1. Introduction

Chikungunya virus (CHIKV) is a single positive-sense RNA virus transmitted by mosquitoes and belongs to the Alphavirus genus of the family Togaviridae [1]. The disease caused by CHIKV infection is referred to as chikungunya fever (CHIKF), characterized by sudden fever, rash, myalgia, and other syndromes [2]. Myalgia and arthritis symptoms can persist for months to years [3], which may cause severe spiritual and economic burdens on patients. CHIKV was first reported in Tanzania, a country in Africa, in 1952 when CHIKV was initially limited to local transmission [4,5]. However, since 2004, CHIKV has unexpectedly emerged on the Indian Ocean islands due to adaptive mutations (alanine-to-valine substitution at position 226 of the E1 protein) [6,7]. This mutation significantly increases CHIKV infectivity, leading to the rapid occurrence of large-scale epidemics [8,9]. Global warming, coupled with the development of transportation and tourism, has further accelerated the global spread of CHIKV. To date, over 100 countries have reported outbreaks of CHIKV infection [10]. According to phylogenetic analysis, CHIKV can be divided into four different genotypes, namely, West African (WA), East-Central-South African (ECSA), Asian and Indian Ocean (IOL), corresponding to their respective geographical origins [5].

The CHIKV genome consists of a capped 5′-UTR, two open reading frames (ORFs), and a 3′-UTR poly(A). ORF1 at the 5′ terminus encodes a nonstructural polyprotein that is catalyzed into four individual nsPs, named as nsP1, nsP2, nsP3 and nsP4. The ORF2 at the 3′ terminus encodes a structural polyprotein that is ultimately cleaved into 5‒6 structural proteins, including capsid protein (C), 3 envelope proteins (E1, E2, and E3), 6K and TF [11]. To facilitate CHIKV replication, these viral proteins cooperate and function at various stages of the viral life cycle. In addition, they also interact with host proteins and hijack a significant number of host proteins to participate in cellular pathways for viral replication [12,13]. Non-structural proteins are first translated viral proteins from CHIKV RNA after entry into the cytosol of infected cells and then participate in the synthesis of the viral genome and subgenome. RNA-dependent RNA polymerase (RdRp) is the first essential enzyme for viral RNA synthesis. Therefore, CHIKV nsP4 with RdRp activity is first cleaved from ns-polyprotein by the protease nsP2 and subsequently complexed with P123 to form a replicase for negative-strand RNA synthesis using viral positive RNA as a template. NsP1 is then cleaved by nsP2 and forms the short-lived complex nsP1+P23+nsP4. The mature viral replication complex (vRC) is formed by four fully processed nonstructural proteins and is responsible for the synthesis of the viral genome and subgenome RNA based on the negative RNA template. NsP1 is essential for viral RNA capping through its two enzymatic activities, N7-guanine-methyl-transferase (MTase) and guanylytransferase (GTase) that are critical for viral replication [14]. In addition, nsP1 possesses a membrane-binding peptide (aa 244‒263) that interacts wit