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

2018 ◽  
Vol 115 (44) ◽  
pp. E10457-E10466 ◽  
Author(s):  
Rachy Abraham ◽  
Debra Hauer ◽  
Robert Lyle McPherson ◽  
Age Utt ◽  
Ilsa T. Kirby ◽  
...  
Keyword(s):  
Virus Replication ◽  
Posttranslational Modification ◽  
Infectious Virus ◽  
Nonstructural Protein ◽  
Hydrolase Activity ◽  
Host Protein ◽  
Chikv Infection ◽  
Adp Ribosylation ◽  
Host Protein Synthesis ◽  
Established Infection

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 nsP3MDalso removes ADPr from mono ADP-ribosylated (MARylated) substrates. To determine which aspects of alphavirus replication require nsP3MDADPr-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 nsP3MDADPr binding are necessary for initiation of alphavirus replication, while hydrolase activity facilitates amplification of replication complexes. These observations are consistent with observed nsP3MDconservation and limited tolerance for mutation.

scholarly journals Alphavirus nsP3 ADP-ribosylhydrolase Activity Disrupts Stress Granule Formation

2019 ◽  
Author(s):  
Aravinth Kumar Jayabalan ◽  
Diane E. Griffin ◽  
Anthony K. L. Leung
Keyword(s):  
Translation Initiation ◽  
Protein Modification ◽  
Nonstructural Protein ◽  
Host Protein ◽  
Granule Formation ◽  
Adp Ribosylation ◽  
Initiation Factors ◽  
Translation Initiation Factors ◽  
Host Protein Synthesis ◽  
Lateral Sclerosis

ABSTRACTFormation of stress granules (SGs), cytoplasmic condensates of stalled translation initiation complexes, is regulated by post-translational protein modification. Alphaviruses interfere with SG formation in response to inhibition of host protein synthesis through the activities of nonstructural protein 3 (nsP3). nsP3 has a conserved N-terminal macrodomain that binds and can remove ADP-ribose from ADP-ribosylated proteins and a C-terminal hypervariable domain that binds essential SG component G3BP1. We showed that the hydrolase activity of chikungunya virus nsP3 macrodomain removed ADP-ribosylation of G3BP1 and suppressed SG formation. ADP-ribosylhydrolase-deficient nsP3 mutants allowed stress-induced cytoplasmic condensation of translation initiation factors. nsP3 also disassembled SG-like aggregates enriched with translation initiation factors that are induced by the expression of FUS mutant R495X linked to amyotrophic lateral sclerosis. Therefore, our data indicate that regulation of ADP-ribosylation controls the localization of translation initiation factors during virus infection and other pathological conditions.

scholarly journals COPII Vesicle Transport Is Required for Rotavirus NSP4 Interaction with the Autophagy Protein LC3 II and Trafficking to Viroplasms

2019 ◽  
Vol 94 (1) ◽  
Author(s):  
Sue E. Crawford ◽  
Jeanette M. Criglar ◽  
Zheng Liu ◽  
James R. Broughman ◽  
Mary K. Estes
Keyword(s):  
Endoplasmic Reticulum ◽  
Virus Replication ◽  
Nonstructural Protein ◽  
Vesicle Transport ◽  
Host Protein ◽  
Cellular Membranes ◽  
Particle Assembly ◽  
Cellular Autophagy ◽  
Copii Vesicle ◽  
Copii Vesicles

ABSTRACT Many viruses that replicate in the cytoplasm dramatically remodel and stimulate the accumulation of host cell membranes for efficient replication by poorly understood mechanisms. For rotavirus, a critical step in virion assembly requires the accumulation of membranes adjacent to virus replication centers called viroplasms. Early electron microscopy studies describe viroplasm-associated membranes as “swollen” endoplasmic reticulum (ER). We previously demonstrated that rotavirus infection initiates cellular autophagy and that membranes containing the autophagy marker protein LC3 and the rotavirus ER-synthesized transmembrane glycoprotein NSP4 traffic to viroplasms, suggesting that NSP4 must exit the ER. This study aimed to address the mechanism of NSP4 exit from the ER and determine whether the viroplasm-associated membranes are ER derived. We report that (i) NSP4 exits the ER in COPII vesicles, resulting in disrupted COPII vesicle transport and ER exit sites; (ii) COPII vesicles are hijacked by LC3 II, which interacts with NSP4; and (iii) NSP4/LC3 II-containing membranes accumulate adjacent to viroplasms. In addition, the ER transmembrane proteins SERCA and calnexin were not detected in viroplasm-associated membranes, providing evidence that the rotavirus maturation process of “budding” occurs through autophagy-hijacked COPII vesicle membranes. These findings reveal a new mechanism for rotavirus maturation dependent on intracellular host protein transport and autophagy for the accumulation of membranes required for virus replication. IMPORTANCE In a morphogenic step that is exceedingly rare for nonenveloped viruses, immature rotavirus particles assemble in replication centers called viroplasms, and bud through cytoplasmic cellular membranes to acquire the outer capsid proteins for infectious particle assembly. Historically, the intracellular membranes used for particle budding were thought to be endoplasmic reticulum (ER) because the rotavirus nonstructural protein NSP4, which interacts with the immature particles to trigger budding, is synthesized as an ER transmembrane protein. This present study shows that NSP4 exits the ER in COPII vesicles and that the NSP4-containing COPII vesicles are hijacked by the cellular autophagy machinery, which mediates the trafficking of NSP4 to viroplasms. Changing the paradigm for rotavirus maturation, we propose that the cellular membranes required for immature rotavirus particle budding are not an extension of the ER but are COPII-derived autophagy isolation membranes.

scholarly journals Translational shutdown and evasion of the innate immune response by SARS-CoV-2 NSP14 protein

2021 ◽  
Vol 118 (24) ◽  
pp. e2101161118
Author(s):  
Jack Chun-Chieh Hsu ◽  
Maudry Laurent-Rolle ◽  
Joanna B. Pawlak ◽  
Craig B. Wilen ◽  
Peter Cresswell
Keyword(s):  
Protein Synthesis ◽  
Active Sites ◽  
Nonstructural Protein ◽  
Innate Immune ◽  
Host Protein ◽  
Type I ◽  
Inhibition Activity ◽  
Health Crisis ◽  
Translation Inhibition ◽  
Host Protein Synthesis

The ongoing COVID-19 pandemic has caused an unprecedented global health crisis. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of COVID-19. Subversion of host protein synthesis is a common strategy that pathogenic viruses use to replicate and propagate in their host. In this study, we show that SARS-CoV-2 is able to shut down host protein synthesis and that SARS-CoV-2 nonstructural protein NSP14 exerts this activity. We show that the translation inhibition activity of NSP14 is conserved in human coronaviruses. NSP14 is required for virus replication through contribution of its exoribonuclease (ExoN) and N7-methyltransferase (N7-MTase) activities. Mutations in the ExoN or N7-MTase active sites of SARS-CoV-2 NSP14 abolish its translation inhibition activity. In addition, we show that the formation of NSP14−NSP10 complex enhances translation inhibition executed by NSP14. Consequently, the translational shutdown by NSP14 abolishes the type I interferon (IFN-I)-dependent induction of interferon-stimulated genes (ISGs). Together, we find that SARS-CoV-2 shuts down host innate immune responses via a translation inhibitor, providing insights into the pathogenesis of SARS-CoV-2.

scholarly journals Both ADP-Ribosyl-Binding and Hydrolase Activities of the Alphavirus nsP3 Macrodomain Affect Neurovirulence in Mice

mBio ◽  
2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Rachy Abraham ◽  
Robert L. McPherson ◽  
Morgan Dasovich ◽  
Mohsen Badiee ◽  
Anthony K. L. Leung ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Virus Replication ◽  
Nonstructural Protein ◽  
Severe Disease ◽  
Hydrolase Activity ◽  
Neural Cells ◽  
Wild Type ◽  
Fatal Disease

ABSTRACT Macrodomain (MD), a highly conserved protein fold present in a subset of plus-strand RNA viruses, binds to and hydrolyzes ADP-ribose (ADPr) from ADP-ribosylated proteins. ADPr-binding by the alphavirus nonstructural protein 3 (nsP3) MD is necessary for the initiation of virus replication in neural cells, whereas hydrolase activity facilitates replication complex amplification. To determine the importance of these activities for pathogenesis of alphavirus encephalomyelitis, mutations were introduced into the nsP3 MD of Sindbis virus (SINV), and the effects on ADPr binding and hydrolase activities, virus replication, immune responses, and disease were assessed. Elimination of ADPr-binding and hydrolase activities (G32E) severely impaired in vitro replication of SINV in neural cells and in vivo replication in the central nervous systems of 2-week-old mice with reversion to wild type (WT) (G) or selection of a less compromising change (S) during replication. SINVs with decreased binding and hydrolase activities (G32S and G32A) or with hydrolase deficiency combined with better ADPr-binding (Y114A) were less virulent than WT virus. Compared to the WT, the G32S virus replicated less well in both the brain and spinal cord, induced similar innate responses, and caused less severe disease with full recovery of survivors, whereas the Y114A virus replicated well, induced higher expression of interferon-stimulated and NF-κB-induced genes, and was cleared more slowly from the spinal cord with persistent paralysis in survivors. Therefore, MD function was important for neural cell replication both in vitro and in vivo and determined the outcome from alphavirus encephalomyelitis in mice. IMPORTANCE Viral encephalomyelitis is an important cause of long-term disability, as well as acute fatal disease. Identifying viral determinants of outcome helps in assessing disease severity and developing new treatments. Mosquito-borne alphaviruses infect neurons and cause fatal disease in mice. The highly conserved macrodomain of nonstructural protein 3 binds and can remove ADP-ribose (ADPr) from ADP-ribosylated proteins. To determine the importance of these functions for virulence, recombinant mutant viruses were produced. If macrodomain mutations eliminated ADPr-binding or hydrolase activity, viruses did not grow. If the binding and hydrolase activities were impaired, the viruses grew less well than the wild-type virus, induced similar innate responses, and caused less severe disease, and most of the infected mice recovered. If binding was improved, but hydrolase activity was decreased, the virus replicated well and induced greater innate responses than did the WT, but clearance from the nervous system was impaired, and mice remained paralyzed. Therefore, macrodomain function determined the outcome of alphavirus encephalomyelitis.

scholarly journals Targeting Stem-loop 1 of the SARS-CoV-2 5’UTR to suppress viral translation and Nsp1 evasion

2021 ◽  
Author(s):  
Setu M. Vora ◽  
Pietro Fontana ◽  
Valerie Leger ◽  
Ying Zhang ◽  
Tian-Min Fu ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Nonstructural Protein ◽  
Host Protein ◽  
Locked Nucleic Acid ◽  
Viral Protein Synthesis ◽  
Stem Loop ◽  
Viral Translation ◽  
Nonstructural Protein 1 ◽  
Host Protein Synthesis

SARS-CoV-2 is a highly pathogenic virus that evades anti-viral immunity by interfering with host protein synthesis, mRNA stability, and protein trafficking. The SARS-CoV-2 nonstructural protein 1 (Nsp1) uses its C-terminal domain to block the mRNA entry channel of the 40S ribosome to inhibit host protein synthesis. However, how SARS-CoV-2 circumvents Nsp1-mediated suppression for viral protein synthesis and if the mechanism can be targeted therapeutically remain unclear. Here we show that N- and C-terminal domains of Nsp1 coordinate to drive a tuned ratio of viral to host translation, likely to maintain a certain level of host fitness while maximizing replication. We reveal that the SL1 region of the SARS-CoV-2 5’ UTR is necessary and sufficient to evade Nsp1-mediated translational suppression. Targeting SL1 with locked nucleic acid antisense oligonucleotides (ASOs) inhibits viral translation and makes SARS-CoV-2 5’ UTR vulnerable to Nsp1 suppression, hindering viral replication in vitro at a nanomolar concentration. Thus, SL1 allows Nsp1 to switch infected cells from host to SARS-CoV-2 translation, presenting a therapeutic target against COVID-19 that is conserved among immune-evasive variants. This unique strategy of unleashing a virus’ own virulence mechanism against itself could force a critical trade off between drug resistance and pathogenicity.

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