Chikungunya virus infectivity, RNA replication and non-structural polyprotein processing depend on the nsP2 protease’s active site cysteine residue

Abstract

Chikungunya virus (CHIKV), genus Alphavirus, family Togaviridae, has a positive-stand RNA genome approximately 12 kb in length. In infected cells, the genome is translated into non-structural polyprotein P1234, an inactive precursor of the viral replicase, which is activated by cleavages carried out by the non-structural protease, nsP2. We have characterized CHIKV nsP2 using both cell-free and cell-based assays. First, we show that Cys478 residue in the active site of CHIKV nsP2 is indispensable for P1234 processing. Second, the substrate requirements of CHIKV nsP2 are quite similar to those of nsP2 of related Semliki Forest virus (SFV). Third, substitution of Ser482 residue, recently reported to contribute to the protease activity of nsP2, with Ala has almost no negative effect on the protease activity of CHIKV nsP2. Fourth, Cys478 to Ala as well as Trp479 to Ala mutations in nsP2 completely abolished RNA replication in CHIKV and SFV trans-replication systems. In contrast, trans-replicases with Ser482 to Ala mutation were similar to wild type counterparts. Fifth, Cys478 to Ala as well as Trp479 to Ala mutations in nsP2 abolished the rescue of infectious virus from CHIKV RNA transcripts while Ser482 to Ala mutation had no effect. Thus, CHIKV nsP2 is a cysteine protease.Chikungunya virus (CHIKV) belongs to genus Alphavirus (family Togaviridae). It is transmitted by Aedes mosquitoes and has caused several massive outbreaks since 2004 1 . CHIKV has a positive-strand RNA genome approximately 12 kb in length. A large 5′ open reading frame (ORF), which covers 2/3 of the viral genome, is translated directly from the viral genomic RNA and encodes for a non-structural (ns) polyprotein designated as P1234. The second ORF encodes for the precursors of viral structural proteins and is translated from a specific subgenomic RNA synthesized in virus-infected cells 2 .All virus-specific enzymatic activities, required for viral RNA synthesis, are present in P1234 and its cleavage products 3 . Allthough uncleaved P1234 possesses several enzymatic activities 4 , it is incapable of performing viral RNA replication at detectable levels 5 . To become active, P1234 must first be processed into the early replicase (P123 polyprotein + nsP4). This complex can, in principle, perform all the essential steps of viral RNA synthesis 6 . However, during alphavirus infection the early replicase is converted into the mature (nsP1 + nsP2 + nsP3 + nsP4) form 7 . All these cleavages are performed by a protease located at the C-terminus of nsP2 [8][9][10] and the processing of P1234 is regulated at multiple levels [11][12][13][14] .The protease part of nsP2 can be easily purified as an active recombinant protein 10,15 . In addition, the protease activity of nsP2 can be studied using in vitro translation and cell culture models 9,16 . Early studies showed that alphavirus nsP2 is similar to papaine-like proteases. The catalytic dyad of Sindbis virus (SINV) nsP2 is represented by Cys481 and His558 residues 8,17,18 ; these correspond to Cys478 and His54...

Structural insights into RNA recognition by the Chikungunya virus nsP2 helicase

“…As shown in the nsP2h structure, the NTD was not directly involved in RNA14 binding and might be involved in the recognition of other viral proteins or protein junctions. Based on this activity, the NTD helps nsP2 regulate ns-polyprotein processing and coordinate the viral RNA replication process (19,40). Domain 1B, which is an accessory domain, was previously described as a human Upf1 protein (27).…”

“…These findings expand our knowledge of CHIKV and related alphaviruses and might also have broad implications for antiviral and vaccine developments against pathogenic alphaviruses. domains (19). Although the structures of the C-terminal protease region of alphavirus nsP2 (nsP2p) (20,21) and uncleaved P23 precursor protein without the N-terminal helicase part of the nsP2 (nsP2h) (22) have been determined, the CHIKV nsP2h structure remains elusive.…”

Ubiquitin‐Conjugating Enzyme E2 L3 is Downregulated by the Chikungunya Virus nsP2 Protease

“…These proteins must not only mediate the entry of the virus and direct the replication of the genome and the production of progeny virions, they must additionally dampen the host cell innate immune responses and re‐engineer the host cell to promote an environment suitable for virion production . While the enzymatic activities of the nsP2‐pro are well described, the involvement of nsP2 (and specifically the nsP2‐pro domain) in other aspects of the virus replication cycle, and particularly how these mediate modulation of the host cell responses remains to be fully understood.…”

Mono-ADP-ribosylation by ARTD10 restricts Chikungunya virus replication by interfering with the proteolytic activity of nsP2

“…( a ) Schematic representation of the synthetic nsP2 protease substrate with the nsP3/nsP4 cleavage site (long peptide described in [51]). ( b ) Bacterially expressed His 6 -tagged nsP2 protease domain (459-798) or the corresponding catalytically inactive CASA mutant were subjected to an in vitro protease assay with bacterially expressed and purified synthetic substrate at 30°C for the indicated times.…”

“…The artificial protease substrate (pGEX4T1-nsP3/nsP4-site-polylinker-EGFP) was created based on the long nsP3/nsP4 site described in Rausalu et al [51]. This sequence was ordered as oligos containing EcoR I (5’-end) and BamH I (3’-end) restriction sites mimicking overhangs (5’-aattcGACGAGTTAAGACTAGACAGGGCAGGTGGGTATATATTCTCGTCGgag-3’, 3’-gatcctcCGACGAGAATATATACCCACCTGCCCTGTCTAGTCTTAACTCGTCg-5’) that were annealed in vitro .…”