ADP-ribosyl–binding and hydrolase activities of the alphavirus nsP3 macrodomain are critical for initiation of virus replication

Abstract

Alphaviruses are plus-strand RNA viruses that cause encephalitis, rash, and arthritis. The nonstructural protein (nsP) precursor polyprotein is translated from genomic RNA and processed into four nsPs. nsP3 has a highly conserved macrodomain (MD) that binds ADP-ribose (ADPr), which can be conjugated to protein as a posttranslational modification involving transfer of ADPr from NAD + by poly ADPr polymerases (PARPs). The nsP3 MD also removes ADPr from mono ADP-ribosylated (MARylated) substrates. To determine which aspects of alphavirus replication require nsP3 MD ADPr-binding and/or hydrolysis function, we studied NSC34 neuronal cells infected with chikungunya virus (CHIKV). Infection induced ADP-ribosylation of cellular proteins without increasing PARP expression, and inhibition of MARylation decreased virus replication. CHIKV with a G32S mutation that reduced ADPr-binding and hydrolase activities was less efficient than WT CHIKV in establishing infection and in producing nsPs, dsRNA, viral RNA, and infectious virus. CHIKV with a Y114A mutation that increased ADPr binding but reduced hydrolase activity, established infection like WT CHIKV, rapidly induced nsP translation, and shut off host protein synthesis with reduced amplification of dsRNA. To assess replicase function independent of virus infection, a transreplicase system was used. Mutant nsP3 MD s D10A, G32E, and G112E with no binding or hydrolase activity had no replicase activity, G32S had little, and Y114A was intermediate to WT. Therefore, ADP ribosylation of proteins and nsP3 MD ADPr binding are necessary for initiation of alphavirus replication, while hydrolase activity facilitates amplification of replication complexes. These observations are consistent with observed nsP3 MD conservation and limited tolerance for mutation. alphavirus | macrodomain | ADP ribosylation | replication complexes | PARP

Deciphering the Nucleotide and RNA Binding Selectivity of the Mayaro Virus Macro Domain

“…4). These results are in line with the different roles of MD during the early and late stages of viral replication, in which a multi-functional domain is likely required [37]. The observations presented here enlighten the structural and physicochemical determinants of MD-ligand interactions and provide valuable functional insights into the MD of this neglected virus reinforcing the efforts to design novel structure-based inhibitors of this Alphavirus protein domain.…”

“…Subsequently, several studies proved its significance to viral replication and virulence, while recent studies proposed that the function of MDs is associated with their activity as ADP-ribosylhydrolases, suggesting their implication in counteracting responses to infection [28][29][30][31][32][33][34][35][36]. In general, viral MDs are postulated to be critically involved in replication complex formation, cell-specific viral replication and ADP-ribosylation dependent interactions with host proteins [37]. It remains to be investigated if and how distinct properties of different Alphaviruses MDs such as sequence, structure, dynamics and ligand binding can be correlated to different functions and different viral replication profiles.…”

“…Most of the aforementioned functions require the cross-talk of proteins participating in the ADP-ribosylation and in other post-translational modifications, such as ubiquitination [38][39][40][41][42]. Recently, it was reported that ADPribosylation is induced upon Alphavirus infection in an interferon-independent manner [37].…”

Structural insights into RNA recognition by the Chikungunya virus nsP2 helicase

“…The T4 DNA polymerasetreated amplicon and BsaI-treated vector were incubated for 1 h for annealing before transformation. The CHIKV infectious cDNA (icDNA) clone CMV-ICRES1 and the trans-replicase system vectors CMV-P1234 and pol I-Fluc-Gluc have been previously described (10,38,48). Based on the information of the CHIKV nsP2h ternary complex, point mutations were introduced on CHIKV icDNA, CMV-P1234, pol I-Fluc-Gluc, and nsP2h expression constructs using site-directed PCR-based mutagenesis and subcloning.…”

“…Both P123 and mature individual nsPs are involved in viral RNA replication and transcription. Each nsP has its own distinct enzymatic and nonenzymatic functions as follows: nsP1 displays the methyl-and guanylyltransferase activities required for cap synthesis and plasma membrane-anchoring ability (5,6); nsP2 contains an N-terminal RNA helicase with both nucleotide triphosphatase (NTPase) and RNA triphosphatase activities and a cysteine protease at the C-terminal region that is responsible for polyprotein processing (7,8); nsP3 shows ADP ribosyl-binding and hydrolase activities and is used for the initiation of viral genome replication (9,10); and nsP4 exhibits RNA-dependent RNA polymerase activity (11,12). These nsPs together with host factors form replication complexes (RCs) that catalyze the synthesis of a negative-sense (−) antigenome and subsequently new genomic and subgenomic (SG) RNAs (13).…”