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Hallmarks of Alpha- and Betacoronavirus non-structural protein 7+8 complexes.

ABSTRACT: Coronaviruses infect many different species including humans. The last two decades have seen three zoonotic coronaviruses, with SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) causing a pandemic in 2020. Coronaviral non-structural proteins (nsps) form the replication-transcription complex (RTC). Nsp7 and nsp8 interact with and regulate the RNA-dependent RNA-polymerase and other enzymes in the RTC. However, the structural plasticity of nsp7+8 complexes has been under debate. Here, we present the framework of nsp7+8 complex stoichiometry and topology based on native mass spectrometry and complementary biophysical techniques of nsp7+8 complexes from seven coronaviruses in the genera Alpha- and Betacoronavirus including SARS-CoV-2. Their complexes cluster into three groups, which systematically form either heterotrimers or heterotetramers or both, exhibiting distinct topologies. Moreover, even at high protein concentrations, SARS-CoV-2 nsp7+8 consists primarily of heterotetramers. From these results, the different assembly paths can be pinpointed to specific residues and an assembly model proposed.

SUBMITTER: Krichel B

PROVIDER: S-EPMC7929516 | biostudies-literature | 2021 Mar

REPOSITORIES: biostudies-literature

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Hallmarks of <i>Alpha-</i> and <i>Betacoronavirus</i> non-structural protein 7+8 complexes.

Krichel Boris B Bylapudi Ganesh G Schmidt Christina C Blanchet Clement C Schubert Robin R Brings Lea L Koehler Martin M Zenobi Renato R Svergun Dmitri D Lorenzen Kristina K Madhugiri Ramakanth R Ziebuhr John J Uetrecht Charlotte C

Science advances 20210303 10

Coronaviruses infect many different species including humans. The last two decades have seen three zoonotic coronaviruses, with SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) causing a pandemic in 2020. Coronaviral non-structural proteins (nsps) form the replication-transcription complex (RTC). Nsp7 and nsp8 interact with and regulate the RNA-dependent RNA-polymerase and other enzymes in the RTC. However, the structural plasticity of nsp7+8 complexes has been under debate. Here, we ...[more]

PMID: 33658206

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Project description:Coronavirus genome replication and expression are mediated by the viral replication-transcription complex (RTC) which is assembled from multiple nonstructural proteins (nsp). Among these, nsp12 represents the central functional subunit. It harbors the RNA-directed RNA polymerase (RdRp) domain and contains, at its N terminus, an additional domain called NiRAN which is widely conserved in coronaviruses and other nidoviruses. In this study, we produced bacterially expressed coronavirus nsp12s to investigate and compare NiRAN-mediated NMPylation activities from representative alpha- and betacoronaviruses. We found that the four coronavirus NiRAN domains characterized to date have a number of conserved properties, including (i) robust nsp9-specific NMPylation activities that appear to operate largely independently of the C-terminal RdRp domain, (ii) nucleotide substrate preference for UTP followed by ATP and other nucleotides, (iii) dependence on divalent metal ions, with Mn2+ being preferred over Mg2+, and (iv) a key role of N-terminal residues (particularly Asn2) of nsp9 for efficient formation of a covalent phosphoramidate bond between NMP and the N-terminal amino group of nsp9. In this context, a mutational analysis confirmed the conservation and critical role of Asn2 across different subfamilies of the family Coronaviridae, as shown by studies using chimeric coronavirus nsp9 variants in which six N-terminal residues were replaced with those from other corona-, pito- and letovirus nsp9 homologs. The combined data of this and previous studies reveal a remarkable degree of conservation among coronavirus NiRAN-mediated NMPylation activities, supporting a key role of this enzymatic activity in viral RNA synthesis and processing. IMPORTANCE There is strong evidence that coronaviruses and other large nidoviruses evolved a number of unique enzymatic activities, including an additional RdRp-associated NiRAN domain, that are conserved in nidoviruses but not in most other RNA viruses. Previous studies of the NiRAN domain mainly focused on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and suggested different functions for this domain, such as NMPylation/RNAylation of nsp9, RNA guanylyltransferase activities involved in canonical and/or unconventional RNA capping pathways, and other functions. To help resolve partly conflicting information on substrate specificities and metal ion requirements reported previously for the SARS-CoV-2 NiRAN NMPylation activity, we extended these earlier studies by characterizing representative alpha- and betacoronavirus NiRAN domains. The study revealed that key features of NiRAN-mediated NMPylation activities, such as protein and nucleotide specificity and metal ion requirements, are very well conserved among genetically divergent coronaviruses, suggesting potential avenues for future antiviral drug development targeting this essential viral enzyme.

Project description:Multiple system atrophy (MSA) is a rare, but fatal atypical parkinsonian disorder. The prototypical pathological hallmark are oligodendroglial cytoplasmic inclusions (GCIs) containing alpha-synuclein (α-syn). Currently, two MSA phenotypes are classified: the parkinsonian (MSA-P) and the cerebellar subtype (MSA-C), clinically characterized by predominant parkinsonism or cerebellar ataxia, respectively. Previous studies have shown that the transgenic MSA mouse model overexpressing human α-syn controlled by the oligodendroglial myelin basic protein (MBP) promoter (MBP29-hα-syn mice) mirrors crucial characteristics of the MSA-P subtype. However, it remains elusive, whether this model recapitulates important features of the MSA-C-related phenotype. First, we examined MSA-C-associated cerebellar pathology using human post-mortem tissue of MSA-C patients and controls. We observed the prototypical GCI pathology and a preserved number of oligodendrocytes in the cerebellar white matter (cbw) accompanied by severe myelin deficit, microgliosis, and a profound loss of Purkinje cells. Secondly, we phenotypically characterized MBP29-hα-syn mice using a dual approach: structural analysis of the hindbrain and functional assessment of gait. Matching the neuropathological features of MSA-C, GCI pathology within the cbw of MBP29-hα-syn mice was accompanied by a severe myelin deficit despite an increased number of oligodendrocytes and a high number of myeloid cells even at an early disease stage. Intriguingly, MBP29-hα-syn mice developed a significant loss of Purkinje cells at a more advanced disease stage. Catwalk XT gait analysis revealed decreased walking speed, increased stride length and width between hind paws. In addition, less dual diagonal support was observed toward more dual lateral and three paw support. Taken together, this wide-based and unsteady gait reflects cerebellar ataxia presumably linked to the cerebellar pathology in MBP29-hα-syn mice. In conclusion, the present study strongly supports the notion that the MBP29-hα-syn mouse model mimics important characteristics of the MSA-C subtype providing a powerful preclinical tool for evaluating future interventional strategies.

Dataset Information

Hallmarks of Alpha- and Betacoronavirus non-structural protein 7+8 complexes.

Publications

Hallmarks of <i>Alpha-</i> and <i>Betacoronavirus</i> non-structural protein 7+8 complexes.

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