(SARS) CoV nsp7 esiintyy hexadekameerina nsp8:n kanssa muodostaen renkaan dsRNA:n ympärille

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 http://english.ibp.cas.cn/rh/ras2/raip/200911/t20091130_47919.html
2005: 
Zihe Rao's group has determined the crystal structure of the hexadecameric super-complex assembly formed by the non-structural proteins nsp7 and nsp8. This research work is the first to reveal interactions between SARS-CoV non-structural proteins, and has provided a first glimpse of the coronavirus replication machinery at the atomic level. This achievement was published in October 2005 as an article in Nature Structural and Molecular Biology.
 

   2010 ( kuva)
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https://www.sciencedirect.com/science/article/pii/S0022283610008193?via%3Dihub

Communication

NMR Structure of the SARS-CoV Nonstructural Protein 7 in Solution at pH 6.5

Author links open overlay panelMargaret A.Johnson¹KristapsJaudzems¹KurtWüthrich¹²³

https://doi.org/10.1016/j.jmb.2010.07.043Get rights and content

Abstract

The NMR structure of the severe acute respiratory syndrome coronavirus nonstructural protein (nsp) 7 in aqueous solution at pH 6.5 was determined and compared with the results of previous structure determinations of nsp7 in solution at pH 7.5 and in the crystals of a hexadecameric nsp7/nsp8 complex obtained from a solution at pH 7.5. All three structures contain four helices as the only regular secondary structures, but there are differences in the lengths and sequence locations of the four helices, as well as between the tertiary folds. The present study includes data on conformational equilibria and intramolecular rate processes in nsp7 in solution at pH 6.5, which provide further insights into the polymorphisms implicated by a comparison of the three presently available nsp7 structures.

Graphical Abstract

 (2)
2012
Tässä kuvataan tarkemmin  nsp7+nsp8 superkompleksin RdRp-toimintaa  toisena  RNA:sta riipuvana RNApolymeraasina.  Ykkönen tässä funktiossa on nsp12 RdPd. Tämän  toissijaisen , nsp8:n katalysoiman RdRP:n   tehtävänä on  ne dovo aloitus ja se operoi primaasin tavoin ja pystyy laajentamaan  primeroituja RNA-templaatteja,  mutta tämä viimeksi mainittu aktiivisuus on 20 kertaa heikompaa  verrattaessa nsp12  RdRp  tehoon  samaa monomeerikonsentraatiota kohden .

Nucleic Acids Res. 2012 Feb;40(4):1737-47. doi: 10.1093/nar/gkr893. Epub 2011 Oct 29.

The SARS-coronavirus nsp7+nsp8 complex is a unique multimeric RNA polymerase capable of both de novo initiation and primer extension.

te Velthuis AJ¹, van den Worm SH, Snijder EJ.

Author information

Abstract

Uniquely among RNA viruses,
 replication of the ~30-kb SARS-coronavirus genome is believed to involve two RNA-dependent RNA polymerase (RdRp) activities.
 The first is primer-dependent and associated with the 106-kDa non-structural protein 12 (nsp12), whereas the second is catalysed by the 22-kDa nsp8. This latter enzyme is capable of de novo initiation and has been proposed to operate as a primase.

Interestingly, this protein has only been crystallized together with the 10-kDa nsp7, forming a hexadecameric, dsRNA-encircling ring structure [i.e. nsp(7+8), consisting of 8 copies of both nsps].

To better understand the implications of these structural characteristics for nsp8-driven RNA synthesis, we studied the prerequisites for the formation of the nsp(7+8) complex and its polymerase activity.

We found that in particular the exposure of nsp8's natural N-terminal residue was paramount for both the protein's ability to associate with nsp7 and for boosting its RdRp activity.

 Moreover, this 'improved' recombinant nsp8 was capable of extending primed RNA templates, a property that had gone unnoticed thus far.
The latter activity is, however, ~20-fold weaker than that of the primer-dependent nsp12-RdRp at equal monomer concentrations.

Finally, site-directed mutagenesis of conserved D/ExD/E motifs was employed to identify residues crucial for nsp(7+8) RdRp activity.

PMID:

22039154

PMCID:

PMC3287201

DOI:

10.1093/nar/gkr893

[Indexed for MEDLINE]

Free PMC Article

• DISCUSSION

  The complex replication and transcription process that coronaviruses initiate upon infection involves up to 16 viral nsps and at least one host factor ( 30–32 ). Both individually as well as in complex with each other, these subunits engage in numerous protein–protein interactions ( 17 , 33 ) and embody various enzymatic activities, including proteolytic ( 19 , 34 ), ATPase ( 35 ), and 5′ cap modifying reactions ( 36 ). Remarkably though, the mechanism and enzymes required to catalyse RNA synthesis in the CoV RTC remain very poorly understood. Moreover, uniquely among RNA viruses which generally employ a single RNA polymerase to drive their RNA synthesis ( 1 , 2 ), the polymerase activity assays and nsp8 mutagenesis documented in this and other studies suggest that, in addition to the presumed nsp12 ‘main RdRp’, other polymerase activities could play a critical role in the synthesis of SARS-CoV RNAs ( 10 , 12 , 37 , 38 ).
  Following up on the description of an nsp8- and nsp7-containing hexadecameric ring structure ( 13 ) and the nsp8-associated polymerase activity ( 12 ), we here demonstrate that the nsp(7+8) hexadecamer is the most probable conformation of the second SARS-CoV polymerase, given the near-complete association of nsp7 and nsp8 when mixed 1:1 in solution ( Figure 2 F). Significant for our understanding of CoV RNA synthesis, we find that this complex is capable of binding dsRNA molecules and extending partially double-stranded RNA templates. This activity is therefore essentially comparable to the activity reported for the nsp12-RdRp ( 10 ).
  A direct comparison with the nsp12 activity is difficult, however. In the course of a one-hour reaction, 0.1 μM monomeric nsp12-RdRp incorporates ∼2 μM NMP into a primed (CU) ₁₀ template ( 10 ). The nsp(7+8) complex, at a 1 μM concentration of nsp7 and nsp8 monomers, incorporates ∼1 μM NMP. Per monomer, the activity difference is therefore 20-fold, but if we assume that most nsp7 and nsp8 monomers assemble into hexadecamers and that each hexadecamer contributes only one functional active site per incorporation event, the difference would be much smaller and only ∼2.5-fold. Presently, however, we do not yet have an estimate for the efficiency and stability of the nsp(7+8) complex, nor do we know the number of active sites in the complex that determine its overall activity.
  Mutagenesis of nsp8 was performed to identify residues that may contribute to the catalytic centre of the nsp(7+8) polymerase, while differently tagged nsp8 recombinant proteins were constructed to explain some striking differences with previous observations. These efforts resulted in two intriguing observations. Firstly, mutation of the conserved N-terminal D/ExD/E motif, comprising D50 and D52 in SARS-CoV, abolished RdRp activity, whereas mutation of the C-terminal motif, including SARS-CoV residues D161 and D163, did not affect polymerase activity ( Figure 5 ). Given the general importance of acidic residues for metal–ion coordination in polymerase active sites ( 3–5 , 22 ) and the D/ExD/E consensus sequence in coronaviruses at positions 50–52, we now postulate that these residues are part of the Mg ²⁺ -binding active site in spite of the more conserved nature of D161 and D163 ( Figure 5 ), and their position in the nsp(7+8) structure (see below for further discussion).
  Secondly, the presence of N-terminal extensions other than nsp7, such as ubiquitin and His ₆ , severely affected the primer extension activity of nsp8 ( Figure 7 ), potentially by changing its oligomeric state ( Figure 2 ). However, the relatively strong activity of nsp7-8 ( Figure 7 ), a potential naturally occurring replicase processing intermediate, implies that nsp8's activity is unlikely to be directly controlled by an N-terminal cleavage event, as was observed for, e.g. the poliovirus polymerase ( 29 ). In addition, these observations suggest that a more diverse array of nsp8-containing RdRps may be involved in CoV replication and transcription.
  Comparing our data against the background of the previously published nsp(7+8) structure ( 13 ), we made four main observations. Firstly, we note that in the published nsp(7+8) crystal structure four of the eight N-terminal D/ExD/E motifs in the complex reside at the border of partially unresolved N-terminal nsp8 domains, where the coordinates of up to 49 nsp8 residues and 5 exogenous amino acids derived from the removed GST fusion partner were not determined. In light of our own finding that unnatural N-terminal extensions severely impair nsp8's RdRp activity ( Figure 5 ), this suggests that the published crystal structure may not represent an active conformation of the nsp(7+8) polymerase. Secondly, we observe that residues D50 and D52, which are both crucial for nsp(7+8) activity, are residing in an α-helix in the nsp(7+8) structure ( Supplementary Figure S5 ), whereas in canonical primases and polymerases, the catalytic centre is preferentially located on β-strands or turns ( 12 , 22 ). Thirdly, we note that Mg ²⁺ was lacking from the published nsp(7+8) crystal structure ( 13 ), even though it is required for nsp(7+8) activity. Fourthly and last, we observe that a 1:1 ratio of nsp7:nsp8 is sufficient to capture all nsp8 in a higher molecular weight complex ( Figure 2 F) whereas previously a 2:1 ratio was required ( 13 ), potentially due to the additional N-terminal residues that altered the dynamics of complex formation.
  The (functional) implications of these observations are not clear at present, but additional structural studies will likely be required to address these issues in detail, and gain insights that may aid in explaining the in vitro results presented here. Likely, such experiments will also offer further information regarding the residues that are involved in nucleotide positioning, Mg ²⁺ coordination and RdRp chemistry.
  In summary, our results provide important novel insights into the functionality of the SARS-CoV hexadecameric nsp(7+8) complex and demonstrate its activity as an RNA polymerase. In addition, our experiments and controls revealed and address a number of disparities between previous claims and hypotheses ( 12 ), and our own observations. The ‘primase hypothesis’ previously formulated by Imbert and co-workers ( 12 ) remains an intriguing model to explain the initiation of SARS-CoV RNA synthesis and is a topic that will be addressed in detail elsewhere. Nevertheless, based on the primer extension activity of nsp(7+8) on non-structured RNA templates, we can no longer exclude the possibility that nsp(7+8) may synthesise substantially longer products than mere oligonucleotide primers in vivo , possibly stimulated by the presence of additional viral protein factors that could e.g. provide RNA-unwinding activity. Consequently, it is now a distinct possibility that CoV RNA synthesis involves structurally different and functionally separable RNA synthesising complexes [e.g. containing nsp12 or nsp(7+8)], each possessing their own dedicated RdRp characteristics and function in viral plus or minus strand RNA synthesis. It will therefore be crucial to study whether these different polymerase activities are part of the same enzyme complex and, if so, whether they can influence each other's activity or are subject to additional control mechanisms.