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

Rachy Abraham, Debra Hauer, Robert Lyle McPherson, Age Utt, Ilsa T. Kirby, Michael S. Cohen, Andres Merits, Anthony Kar Lun Leung, Diane Griffin

Research output: Contribution to journalArticle

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 nsP3MD also removes ADPr from mono ADP-ribosylated (MARylated) substrates. To determine which aspects of alphavirus replication require nsP3MD 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 nsP3MDs 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 nsP3MD ADPr binding are necessary for initiation of alphavirus replication, while hydrolase activity facilitates amplification of replication complexes. These observations are consistent with observed nsP3MD conservation and limited tolerance for mutation.

Original languageEnglish (US)
Pages (from-to)E10457-E10466
JournalProceedings of the National Academy of Sciences of the United States of America
Volume115
Issue number44
DOIs
StatePublished - Oct 30 2018

Fingerprint

Adenosine Diphosphate Ribose
Alphavirus
Hydrolases
Chikungunya virus
Virus Replication
Adenosine Diphosphate
RNA Viruses
Mutation
Proteins
Infection
Polyproteins
Protein Precursors
Poly(ADP-ribose) Polymerases
Viral RNA
Protein Biosynthesis
Virus Diseases
Encephalitis
Post Translational Protein Processing
Exanthema
NAD

Keywords

  • ADP ribosylation
  • Alphavirus
  • Macrodomain
  • PARP
  • Replication complexes

ASJC Scopus subject areas

  • General

Cite this

ADP-ribosyl-binding and hydrolase activities of the alphavirus nsP3 macrodomain are critical for initiation of virus replication. / Abraham, Rachy; Hauer, Debra; McPherson, Robert Lyle; Utt, Age; Kirby, Ilsa T.; Cohen, Michael S.; Merits, Andres; Leung, Anthony Kar Lun; Griffin, Diane.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 115, No. 44, 30.10.2018, p. E10457-E10466.

Research output: Contribution to journalArticle

Abraham, Rachy ; Hauer, Debra ; McPherson, Robert Lyle ; Utt, Age ; Kirby, Ilsa T. ; Cohen, Michael S. ; Merits, Andres ; Leung, Anthony Kar Lun ; Griffin, Diane. / ADP-ribosyl-binding and hydrolase activities of the alphavirus nsP3 macrodomain are critical for initiation of virus replication. In: Proceedings of the National Academy of Sciences of the United States of America. 2018 ; Vol. 115, No. 44. pp. E10457-E10466.
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AU - Hauer, Debra

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AU - Utt, Age

AU - Kirby, Ilsa T.

AU - Cohen, Michael S.

AU - Merits, Andres

AU - Leung, Anthony Kar Lun

AU - Griffin, Diane

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AB - 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 nsP3MD also removes ADPr from mono ADP-ribosylated (MARylated) substrates. To determine which aspects of alphavirus replication require nsP3MD 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 nsP3MDs 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 nsP3MD ADPr binding are necessary for initiation of alphavirus replication, while hydrolase activity facilitates amplification of replication complexes. These observations are consistent with observed nsP3MD conservation and limited tolerance for mutation.

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