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The SARS-Coronavirus PLnc domain of nsp3 as a replication/transcription scaffolding protein.

ABSTRACT: Many genetic and mechanistic features distinguish the coronavirus replication machinery from that encoded by most other RNA viruses. The coronavirus replication/transcription complex is an assembly of viral and, most probably, cellular proteins that mediate the synthesis of both the unusually large (approximately 30 kb) RNA genome and an extensive set of subgenomic mRNAs. The viral components of the complex are encoded by the giant replicase gene, which is expressed in the form of two polyproteins (pp1a and pp1ab) that are processed into 16 cleavage products (nonstructural proteins 1-16). Using the combination of yeast two-hybrid screening and GST pull-down assays, we have now analyzed all potential interactions between SARS-Coronavirus nonstructural proteins, which may contribute to the structure and/or function of the viral replication/transcription complex. We demonstrate the existence of a complex network of interactions involving all 16 nonstructural proteins. Our results both confirmed previously described associations and identified novel heterodimerizations. The interaction map thus provides a sum of the interactions that may occur at some point during coronavirus RNA synthesis and provides a framework for future research.

SUBMITTER: Imbert I

PROVIDER: S-EPMC7114086 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Multiple roles of the non-structural protein 3 (nsP3) alphavirus unique domain (AUD) during Chikungunya virus genome replication and transcription.

Project description:Chikungunya virus (CHIKV) is a re-emerging Alphavirus causing fever, joint pain, skin rash, arthralgia, and occasionally death. Antiviral therapies and/or effective vaccines are urgently required. CHIKV biology is poorly understood, in particular the functions of the non-structural protein 3 (nsP3). Here we present the results of a mutagenic analysis of the alphavirus unique domain (AUD) of nsP3. Informed by the structure of the Sindbis virus AUD and an alignment of amino acid sequences of multiple alphaviruses, a series of mutations in the AUD were generated in a CHIKV sub-genomic replicon. This analysis revealed an essential role for the AUD in CHIKV RNA replication, with mutants exhibiting species- and cell-type specific phenotypes. To test if the AUD played a role in other stages of the virus lifecycle, the mutants were analysed in the context of infectious CHIKV. This analysis indicated that the AUD was also required for virus assembly. In particular, one mutant (P247A/V248A) exhibited a dramatic reduction in production of infectious virus. This phenotype was shown to be due to a block in transcription of the subgenomic RNA leading to reduced synthesis of the structural proteins and a concomitant reduction in virus production. This phenotype could be further explained by both a reduction in the binding of the P247A/V248A mutant nsP3 to viral genomic RNA in vivo, and the reduced affinity of the mutant AUD for the subgenomic promoter RNA in vitro. We propose that the AUD is a pleiotropic protein domain, with multiple functions during CHIKV RNA synthesis.

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A G-quadruplex-binding macrodomain within the "SARS-unique domain" is essential for the activity of the SARS-coronavirus replication-transcription complex.

Project description:The multi-domain non-structural protein 3 of SARS-coronavirus is a component of the viral replication/transcription complex (RTC). Among other domains, it contains three sequentially arranged macrodomains: the X domain and subdomains SUD-N as well as SUD-M within the "SARS-unique domain". The X domain was proposed to be an ADP-ribose-1"-phosphatase or a poly(ADP-ribose)-binding protein, whereas SUD-NM binds oligo(G)-nucleotides capable of forming G-quadruplexes. Here, we describe the application of a reverse genetic approach to assess the importance of these macrodomains for the activity of the SARS-CoV RTC. To this end, Renilla luciferase-encoding SARS-CoV replicons with selectively deleted macrodomains were constructed and their ability to modulate the RTC activity was examined. While the SUD-N and the X domains were found to be dispensable, the SUD-M domain was crucial for viral genome replication/transcription. Moreover, alanine replacement of charged amino-acid residues of the SUD-M domain, which are likely involved in G-quadruplex-binding, caused abrogation of RTC activity.

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A single inactivating amino acid change in the SARS-CoV-2 NSP3 Mac1 domain attenuates viral replication in vivo.

Project description:Despite unprecedented efforts, our therapeutic arsenal against SARS-CoV-2 remains limited. The conserved macrodomain 1 (Mac1) in NSP3 is an enzyme exhibiting ADP-ribosylhydrolase activity and a possible drug target. To determine the role of Mac1 catalytic activity in viral replication, we generated recombinant viruses and replicons encoding a catalytically inactive NSP3 Mac1 domain by mutating a critical asparagine in the active site. While substitution to alanine (N40A) reduced catalytic activity by ~10-fold, mutations to aspartic acid (N40D) reduced activity by ~100-fold relative to wild-type. Importantly, the N40A mutation rendered Mac1 unstable in vitro and lowered expression levels in bacterial and mammalian cells. When incorporated into SARS-CoV-2 molecular clones, the N40D mutant only modestly affected viral fitness in immortalized cell lines, but reduced viral replication in human airway organoids by 10-fold. In mice, the N40D mutant replicated at >1000-fold lower levels compared to the wild-type virus while inducing a robust interferon response; all animals infected with the mutant virus survived infection. Our data validate the critical role of SARS-CoV-2 NSP3 Mac1 catalytic activity in viral replication and as a promising therapeutic target to develop antivirals.

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SARS coronavirus series

Project description:Set of microarray experiments used to identify an unknown coronavirus in a viral culture derived from a patient with SARS. March 2003. Keywords = SARS Keywords = coronavirus Keywords = viral discovery Keywords = viruses Keywords = respiratory infection

2010-06-05 | E-GEOD-546 | biostudies-arrayexpress

The REEP5/TRAM1 complex binds SARS-CoV-2 NSP3 and promotes virus replication.

Project description:ImportanceGeneration of virus-host protein-protein interactions (PPIs) maps may provide clues to uncover SARS-CoV-2-hijacked cellular processes. However, these PPIs maps were created by expressing each viral protein singularly, which does not reflect the life situation in which certain viral proteins synergistically interact with host proteins. Our results reveal the host-viral protein-protein interactome of SARS-CoV-2 NSP3, NSP4, and NSP6 expressed individually or in combination. Furthermore, REEP5/TRAM1 complex interacts with NSP3 at ROs and promotes viral replication. The significance of our research is identifying virus-host interactions that may be targeted for therapeutic intervention.

| S-EPMC10617467 | biostudies-literature

Dynamics of coronavirus replication-transcription complexes.

Project description:Coronaviruses induce in infected cells the formation of double-membrane vesicles (DMVs) in which the replication-transcription complexes (RTCs) are anchored. To study the dynamics of these coronavirus replicative structures, we generated recombinant murine hepatitis coronaviruses that express tagged versions of the nonstructural protein nsp2. We demonstrated by using immunofluorescence assays and electron microscopy that this protein is recruited to the DMV-anchored RTCs, for which its C terminus is essential. Live-cell imaging of infected cells demonstrated that small nsp2-positive structures move through the cytoplasm in a microtubule-dependent manner. In contrast, large fluorescent structures are rather immobile. Microtubule-mediated transport of DMVs, however, is not required for efficient replication. Biochemical analyses indicated that the nsp2 protein is associated with the cytoplasmic side of the DMVs. Yet, no recovery of fluorescence was observed when (part of) the nsp2-positive foci were bleached. This result was confirmed by the observation that preexisting RTCs did not exchange fluorescence after fusion of cells expressing either a green or a red fluorescent nsp2. Apparently, nsp2, once recruited to the RTCs, is not exchanged with nsp2 present in the cytoplasm or at other DMVs. Our data show a remarkable resemblance to results obtained recently by others with hepatitis C virus. The observations point to intriguing and as yet unrecognized similarities between the RTC dynamics of different plus-strand RNA viruses.

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The coronavirus nucleocapsid protein is dynamically associated with the replication-transcription complexes.

Project description:The coronavirus nucleocapsid (N) protein is a virion structural protein. It also functions, however, in an unknown way in viral replication and localizes to the viral replication-transcription complexes (RTCs). Here we investigated, using recombinant murine coronaviruses expressing green fluorescent protein (GFP)-tagged versions of the N protein, the dynamics of its interactions with the RTCs and the domain(s) involved. Using fluorescent recovery after photobleaching, we showed that the N protein, unlike the nonstructural protein 2, is dynamically associated with the RTCs. Recruitment of the N protein to the RTCs requires the C-terminal N2b domain, which interacts with other N proteins in an RNA-independent manner.

| S-EPMC2953146 | biostudies-literature

A single inactivating amino acid change in the SARS-CoV-2 NSP3 Mac1 domain attenuates viral replication and pathogenesis <i>in vivo</i>.

Project description:Despite unprecedented efforts, our therapeutic arsenal against SARS-CoV-2 remains limited. The conserved macrodomain 1 (Mac1) in NSP3 is an enzyme exhibiting ADP-ribosylhydrolase activity and a possible drug target. To determine the therapeutic potential of Mac1 inhibition, we generated recombinant viruses and replicons encoding a catalytically inactive NSP3 Mac1 domain by mutating a critical asparagine in the active site. While substitution to alanine (N40A) reduced catalytic activity by ~10-fold, mutations to aspartic acid (N40D) reduced activity by ~100-fold relative to wildtype. Importantly, the N40A mutation rendered Mac1 unstable in vitro and lowered expression levels in bacterial and mammalian cells. When incorporated into SARS-CoV-2 molecular clones, the N40D mutant only modestly affected viral fitness in immortalized cell lines, but reduced viral replication in human airway organoids by 10-fold. In mice, N40D replicated at >1000-fold lower levels compared to the wildtype virus while inducing a robust interferon response; all animals infected with the mutant virus survived infection and showed no signs of lung pathology. Our data validate the SARS-CoV-2 NSP3 Mac1 domain as a critical viral pathogenesis factor and a promising target to develop antivirals.

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The SARS Coronavirus 3a protein binds calcium in its cytoplasmic domain.

Project description:The Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) is a positive stranded RNA virus with ?30kb genome. Among all open reading frames (orfs) of this virus, the orf3a is the largest, and encodes a protein of 274 amino acids, named as 3a protein. Sequence analysis suggests that the orf3a aligned to one calcium pump present in Plasmodium falciparum and the enzyme glutamine synthetase found in Leptospira interrogans. This sequence similarity was found to be limited only to amino acid residues 209-264 which form the cytoplasmic domain of the orf3a. Furthermore, this region was predicted to be involved in the calcium binding. Owing to this hypothesis, we were driven to establish its calcium binding property in vitro. Here, we expressed and purified the cytoplasmic domain of the 3a protein, called Cyto3a, as a recombinant His-tagged protein in the E. coli. The calcium binding nature was established by performing various staining methods such as ruthenium red and stains-all. (45)Ca overlay method was also done to further support the data. Since the 3a protein forms ion channels, we were interested to see any conformational changes occurring in the Cyot3a upon calcium binding, using fluorescence spectroscopy and circular dichroism. These studies clearly indicate a significant change in the conformation of the Cyto3a protein after binding with calcium. Our results strongly suggest that the cytoplasmic domain of the 3a protein of SARS-CoV binds calcium in vitro, causing a change in protein conformation.

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The "SARS-unique domain" (SUD) of SARS coronavirus is an oligo(G)-binding protein.

Project description:Caused by a new coronavirus, severe acute respiratory syndrome (SARS) is a highly contagious disease associated with significant fatality that emerged in 2003. The molecular cause of the unusually high human pathogenicity of the SARS coronavirus (SARS-CoV) is still unknown. In an effort to characterize molecular components of the virus that are absent in other coronaviruses, all of which are considerably less pathogenic for humans, we recombinantly produced the SARS-unique domain (SUD) within non-structural protein 3 (Nsp3) of SARS-CoV and characterized its nucleic-acid binding properties. Zone-interference gel electrophoresis and electrophoretic mobility shift assays revealed a specific affinity of SUD for oligo(G)-strings. A few such segments are present in the SARS-CoV genome, but also in mRNAs of host proteins involved in the regulation of signaling pathways. A putative role of SUD in virus-induced apoptosis or survival of host cells is discussed.

| S-EPMC7092865 | biostudies-literature

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