Nsp12

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Nsp12 is a non-structural protein in the Coronavirus genome. Its gene is part of the ORF1ab reading frame and it is part of the pp1ab polyprotein; it is cleaved by 3CLpro. [1]

Contents

Nsp12 is a multi-domain subunit: it consists of an N-terminal nidovirus-specific extension (NiRAN) domain, an interface domain, and a C-terminal RNA-dependent RNA-polymerase domain. The N-terminal portion of SARS-CoV-2 nsp12 additionally contains a β-hairpin which is sandwiched between the NiRAN and RdRp domain. [2]

A representation of the SARS genome with ORF1A, ORF1AB, and the ribosomal frameshift shown. Coronavirus nsp12 is identified and expanded; RdRp, NiRAN domains as well as the interface domain are identified. SARS Genome and nsp12.png
A representation of the SARS genome with ORF1A, ORF1AB, and the ribosomal frameshift shown. Coronavirus nsp12 is identified and expanded; RdRp, NiRAN domains as well as the interface domain are identified.

Coronavirus nsp12 also plays a role in host immune evasion; research has demonstrated that nsp12 inhibits the nuclear translocation of IRF3. [3]

RdRp Domain

The RNA-dependent RNA polymerase domain of nsp12 is C-terminal. In SARS-CoV-2 the domain spans residues 366 to 920. [4] The structure of the RdRp domain shares common structural features with eukaryotic RNA polymerases: the structure consists of a cupped right hand with subdomains referred to as fingers, palms, and thumbs. [1] RdRp activity is dependent on two key zinc ions and conserved metal binding motifs of a histidine and two cysteines each. [2]

The active site has seven catalytic motifs that are labeled A through G. Motif B serves as a hinge which allows the active site to associate with template RNA and Motif F directly interacts with the phosphate group of incoming free nucleotides. [2]

RdRp has to interact with RNA, which is negatively charged, so multiple subdomains including the primer-template entry site, NTP entry site, and the RNA strand exit routes contain positively charged residues. [2] RdRp is unique from host RNA polymerases in that it has to associate with RNA instead of DNA, many RdRp residues interact with RNA bases via 2’-OH groups on the ribose ring which provides a possibly structural explanation for its specificity for RNA. [4]

Coronavirus nsp12 cannot function independently; it has two essential cofactor proteins, nsp7 and nsp8, that form a Replication and Transcription Complex (RTC). [5] Structural studies of the RTC indicate that nsp7 and nsp8 form an 8:8 hexadecamer which acts as a primase to initiate viral replication. [6]

While nsp12 is relatively well conserved across the Coronavirus viral species, there are biochemical and structural differences between the RdRp domain of SARS-CoV and SARS-CoV-2. SARS-CoV-2 RdRp has lower enzymatic activity and lower thermal stability compared to the RdRp domain in SARS-CoV. [7]

Targeting by Remdesivir

Nsp12 is researched as a target for antiviral drugs as it is highly structurally conserved across related viruses and strains, and there are no human proteins with close structural homology. [2] The emergence of SARS-CoV-2 and associated COVID19 disease led to the investigation of Remdesivir as an antiviral drug for SARS-CoV-2. Remdesivir is a nucleoside analog which can compete with ATP for incorporation into the RNA strand and prematurely terminate RNA synthesis. [5]

NiRAN Domain

Coronavirus nsp12 has an N-terminal nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain which is essential for viral replication. The NiRAN domain is capable of transferring nucleotides as functional groups and it contains three key motifs called A, B, and C with seven invariant residues. [8]

The biological function of the nsp12 NiRAN domain is not as well characterized as RdRp, but recent research has elucidated a possible role for the NiRAN domain in viral RNA capping. An additional non-structural protein, nsp9, was shown to associate with nsp12. [9] The biologically active form of nsp9 was additionally shown to be capable of binding nucleic acids with a preference for single-stranded RNA [10] and could cleave nucleotide triphosphates and transfer the resulting nucleotide monophosphates to protein substrates in a process called NMPylation. [9] Park and colleagues demonstrated that the SARS-CoV-2 NiRAN domain could cleave a pyrophosphate from the end of an uncapped RNA genome and transfer the monophosphorylated RNA to nsp9 to RNAylate it. [11] The domain can then transfer the monophosphorylated RNA from nsp9 to a Guanidine Diphosphate (GDP) to form the initial cap structure for SARS-CoV-2. [11]

Related Research Articles

<span class="mw-page-title-main">Coronavirus</span> Subfamily of viruses in the family Coronaviridae

Coronaviruses are a group of related RNA viruses that cause diseases in mammals and birds. In humans and birds, they cause respiratory tract infections that can range from mild to lethal. Mild illnesses in humans include some cases of the common cold, while more lethal varieties can cause SARS, MERS and COVID-19, which is causing the ongoing pandemic. In cows and pigs they cause diarrhea, while in mice they cause hepatitis and encephalomyelitis.

<span class="mw-page-title-main">SARS-related coronavirus</span> Species of coronavirus causing SARS and COVID-19

Severe acute respiratory syndrome–related coronavirus is a species of virus consisting of many known strains phylogenetically related to severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) that have been shown to possess the capability to infect humans, bats, and certain other mammals. These enveloped, positive-sense single-stranded RNA viruses enter host cells by binding to the angiotensin-converting enzyme 2 (ACE2) receptor. The SARSr-CoV species is a member of the genus Betacoronavirus and of the subgenus Sarbecovirus.

<i>Henipavirus</i> Genus of RNA viruses

Henipavirus is a genus of negative-strand RNA viruses in the family Paramyxoviridae, order Mononegavirales containing six established species, and numerous others still under study. Henipaviruses are naturally harboured by several species of small mammals, notably pteropid fruit bats, microbats of several species, and shrews. Henipaviruses are characterised by long genomes and a wide host range. Their recent emergence as zoonotic pathogens capable of causing illness and death in domestic animals and humans is a cause of concern.

An NTP binding site is a type of binding site found in nucleoside monophosphate (NMP) kinases, N can be adenosine or guanosine. A P-loop is one of the structural motifs common for nucleoside triphosphate (NTP) binding sites, it interacts with the bound nucleotide's phosphoryl groups. For the binding site to be able to bind a nucleotide, the nucleotide must be complex bound to Mg2+ or Mn2+. Nucleotide binding will cause conformational changes in the protein because the P-loop will bend.

<i>Nidovirales</i> Order of positive-sense, single-stranded RNA viruses

Nidovirales is an order of enveloped, positive-strand RNA viruses which infect vertebrates and invertebrates. Host organisms include mammals, birds, reptiles, amphibians, fish, arthropods, molluscs, and helminths. The order includes the families Coronaviridae, Arteriviridae, Roniviridae, and Mesoniviridae.

<span class="mw-page-title-main">RNA-dependent RNA polymerase</span> Enzyme that synthesizes RNA from an RNA template

RNA-dependent RNA polymerase (RdRp) or RNA replicase is an enzyme that catalyzes the replication of RNA from an RNA template. Specifically, it catalyzes synthesis of the RNA strand complementary to a given RNA template. This is in contrast to typical DNA-dependent RNA polymerases, which all organisms use to catalyze the transcription of RNA from a DNA template.

<span class="mw-page-title-main">Coronavirus packaging signal</span> Regulartory element in coronaviruses

The Coronavirus packaging signal is a conserved cis-regulatory element found in Betacoronavirus. It has an important role in regulating the packaging of the viral genome into the capsid. As part of the viral life cycle, within the infected cell, the viral genome becomes associated with viral proteins and assembles into new infective progeny viruses. This process is called packaging and is vital for viral replication.

Putative transmembrane domain more commonly known as Non-structural Protein 6 (NSP6) is one of the two non-structural proteins that gene 11 in rotavirus encodes for alongside NSP5. NSP6 is composed of six transmembrane domains and a C terminal tail. In contrast to the other rotavirus non-structural proteins, NSP6 was found to have a high rate of turnover, being completely degraded within 2 hours of synthesis. NSP6 was found to be a sequence-independent nucleic acid binding protein, with similar affinities for ssRNA and dsRNA

<span class="mw-page-title-main">Hepatitis B virus DNA polymerase</span>

Hepatitis B virus DNA polymerase is a hepatitis B viral protein. It is a DNA polymerase that can use either DNA or RNA templates and a ribonuclease H that cuts RNA in the duplex. Both functions are supplied by the reverse transcriptase (RT) domain.

The term proofreading is used in genetics to refer to the error-correcting processes, first proposed by John Hopfield and Jacques Ninio, involved in DNA replication, immune system specificity, enzyme-substrate recognition among many other processes that require enhanced specificity. The proofreading mechanisms of Hopfield and Ninio are non-equilibrium active processes that consume ATP to enhance specificity of various biochemical reactions.

<span class="mw-page-title-main">Deepak T. Nair</span>

Deepak Thankappan Nair is an Indian Structural Biologist and a scientist at Regional Centre for Biotechnology. He is known for his studies on DNA and RNA polymerases. Deepak was a Ramanujan fellow of the Science and Engineering Research Board (2008–2013) and a recipient of the National BioScience Award for Career Development. The Council of Scientific and Industrial Research, the apex agency of the Government of India for scientific research, awarded him the Shanti Swarup Bhatnagar Prize for Science and Technology, one of the highest Indian science awards, for his contributions to biological sciences in 2017.

<span class="mw-page-title-main">Cap snatching</span>

The first step of transcription for some negative, single-stranded RNA viruses is cap snatching, in which the first 10 to 20 residues of a host cell RNA are removed (snatched) and used as the 5′ cap and primer to initiate the synthesis of the nascent viral mRNA. The viral RNA-dependent RNA polymerase (RdRp) can then proceed to replicate the negative-sense genome from the positive-sense template. Cap-snatching also explains why some viral mRNA have 5’ terminal extensions of 10-20 nucleotides that are not encoded for in the genome. Examples of viruses that engage in cap-snatching include influenza viruses (Orthomyxoviridae), Lassa virus (Arenaviridae), hantaan virus (Hantaviridae) and rift valley fever virus (Phenuiviridae). Most viruses snatch 15-20 nucleotides except for the families Arenaviridae and Nairoviridae and the genus Thogotovirus (Orthomyxoviridae) which use a shorter strand.

<span class="mw-page-title-main">IDX-184</span> Chemical compound

IDX-184 is an antiviral drug which was developed as a treatment for hepatitis C, acting as a NS5B RNA polymerase inhibitor. While it showed reasonable effectiveness in early clinical trials it did not progress past Phase IIb. However research using this drug has continued as it shows potentially useful activity against other emerging viral diseases such as Zika virus, and coronaviruses including MERS, and SARS-CoV-2.

Coronavirus genomes are positive-sense single-stranded RNA molecules with an untranslated region (UTR) at the 5′ end which is called the 5′ UTR. The 5′ UTR is responsible for important biological functions, such as viral replication, transcription and packaging. The 5′ UTR has a conserved RNA secondary structure but different Coronavirus genera have different structural features described below.

<span class="mw-page-title-main">Coronavirus envelope protein</span> Major structure in coronaviruses

The envelope (E) protein is the smallest and least well-characterized of the four major structural proteins found in coronavirus virions. It is an integral membrane protein less than 110 amino acid residues long; in SARS-CoV-2, the causative agent of Covid-19, the E protein is 75 residues long. Although it is not necessarily essential for viral replication, absence of the E protein may produce abnormally assembled viral capsids or reduced replication. E is a multifunctional protein and, in addition to its role as a structural protein in the viral capsid, it is thought to be involved in viral assembly, likely functions as a viroporin, and is involved in viral pathogenesis.

<span class="mw-page-title-main">Coronavirus nucleocapsid protein</span> Most expressed structure in coronaviruses

The nucleocapsid (N) protein is a protein that packages the positive-sense RNA genome of coronaviruses to form ribonucleoprotein structures enclosed within the viral capsid. The N protein is the most highly expressed of the four major coronavirus structural proteins. In addition to its interactions with RNA, N forms protein-protein interactions with the coronavirus membrane protein (M) during the process of viral assembly. N also has additional functions in manipulating the cell cycle of the host cell. The N protein is highly immunogenic and antibodies to N are found in patients recovered from SARS and COVID-19.

ORF7b is a gene found in coronaviruses of the genus Betacoronavirus, which expresses the accessory protein Betacoronavirus NS7b protein. It is a short, highly hydrophobic transmembrane protein of unknown function.

<span class="mw-page-title-main">ORF8</span> Gene that encodes a viral accessory protein

ORF8 is a gene that encodes a viral accessory protein, Betacoronavirus NS8 protein, in coronaviruses of the subgenus Sarbecovirus. It is one of the least well conserved and most variable parts of the genome. In some viruses, a deletion splits the region into two smaller open reading frames, called ORF8a and ORF8b - a feature present in many SARS-CoV viral isolates from later in the SARS epidemic, as well as in some bat coronaviruses. For this reason the full-length gene and its protein are sometimes called ORF8ab. The full-length gene, exemplified in SARS-CoV-2, encodes a protein with an immunoglobulin domain of unknown function, possibly involving interactions with the host immune system. It is similar in structure to the ORF7a protein, suggesting it may have originated through gene duplication.

ORF1ab refers collectively to two open reading frames (ORFs), ORF1a and ORF1b, that are conserved in the genomes of nidoviruses, a group of viruses that includes coronaviruses. The genes express large polyproteins that undergo proteolysis to form several nonstructural proteins with various functions in the viral life cycle, including proteases and the components of the replicase-transcriptase complex (RTC). Together the two ORFs are sometimes referred to as the replicase gene. They are related by a programmed ribosomal frameshift that allows the ribosome to continue translating past the stop codon at the end of ORF1a, in a -1 reading frame. The resulting polyproteins are known as pp1a and pp1ab.

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References

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  6. ↑ Zhai, Yujia; Sun, Fei; Li, Xuemei; Pang, Hai; Xu, Xiaoling; Bartlam, Mark; Rao, Zihe (November 2005). "Insights into SARS-CoV transcription and replication from the structure of the nsp7–nsp8 hexadecamer". Nature Structural & Molecular Biology. 12 (11): 980–986. doi:10.1038/nsmb999. ISSN   1545-9993. PMC   7096913 . PMID   16228002.
  7. ↑ Peng, Qi; Peng, Ruchao; Yuan, Bin; Zhao, Jingru; Wang, Min; Wang, Xixi; Wang, Qian; Sun, Yan; Fan, Zheng; Qi, Jianxun; Gao, George F.; Shi, Yi (2020-06-16). "Structural and Biochemical Characterization of the nsp12-nsp7-nsp8 Core Polymerase Complex from SARS-CoV-2". Cell Reports. 31 (11): 107774. doi:10.1016/j.celrep.2020.107774. ISSN   2211-1247. PMC   7260489 . PMID   32531208.
  8. ↑ Gorbalenya, Alexander E.; Enjuanes, Luis; Ziebuhr, John; Snijder, Eric J. (April 2006). "Nidovirales: Evolving the largest RNA virus genome". Virus Research. 117 (1): 17–37. doi:10.1016/j.virusres.2006.01.017. PMC   7114179 . PMID   16503362.
  9. 1 2 Slanina, Heiko; Madhugiri, Ramakanth; Bylapudi, Ganesh; Schultheiß, Karin; Karl, Nadja; Gulyaeva, Anastasia; Gorbalenya, Alexander E.; Linne, Uwe; Ziebuhr, John (2021-02-09). "Coronavirus replication–transcription complex: Vital and selective NMPylation of a conserved site in nsp9 by the NiRAN-RdRp subunit". Proceedings of the National Academy of Sciences. 118 (6): e2022310118. Bibcode:2021PNAS..11822310S. doi: 10.1073/pnas.2022310118 . ISSN   0027-8424. PMC   8017715 . PMID   33472860.
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