New nsp8 isoform suggests mechanism for tuning viral RNA synthesis

Protein & Cell, Feb 2010

During severe acute respiratory syndrome coronavirus (SARS-CoV) infection, the activity of the replication/transcription complexes (RTC) quickly peaks at 6 hours post infection (h.p.i) and then diminishes significantly in the late post-infection stages. This “down-up-down” regulation of RNA synthesis distinguishes different viral stages: primary translation, genome replication, and finally viron assembly. Regarding the nsp8 as the primase in RNA synthesis, we confirmed that the proteolysis product of the primase (nsp8) contains the globular domain (nsp8C), and indentified the resectioning site that is notably conserved in all the three groups of coronavirus. We subsequently crystallized the complex of SARS-CoV nsp8C and nsp7, and the 3-D structure of this domain revealed its capability to interfuse into the hexadecamer super-complex. This specific proteolysis may indicate one possible mechanism by which coronaviruses to switch from viral infection to genome replication and viral assembly stages.

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New nsp8 isoform suggests mechanism for tuning viral RNA synthesis

Protein Cell New nsp8 isoform suggests mechanism for tuning viral RNA synthesis Shuang Li 2 Qi Zhao 1 Yinjie Zhang 0 Yang Zhang 1 Mark Bartlam 0 Xuemei Li 2 Zihe Rao 0 1 2 0 Tianjin Key Laboratory of Protein Science, College of Life Sciences, Nankai University , Tianjin 300071 , China 1 Structural Biology Laboratory, Tsinghua University , Beijing 100084 , China 2 National Laboratory of Macromolecules, Institute of Biophysics, Chinese Academy of Sciences , Beijing 100101 , China - During severe acute respiratory syndrome coronavirus (SARS-CoV) infection, the activity of the replication/ transcription complexes (RTC) quickly peaks at 6 hours post infection (h.p.i) and then diminishes significantly in the late post-infection stages. This “down-up-down” regulation of RNA synthesis distinguishes different viral stages: primary translation, genome replication, and finally viron assembly. Regarding the nsp8 as the primase in RNA synthesis, we confirmed that the proteolysis product of the primase (nsp8) contains the globular domain (nsp8C), and indentified the resectioning site that is notably conserved in all the three groups of coronavirus. We subsequently crystallized the complex of SARS-CoV nsp8C and nsp7, and the 3-D structure of this domain revealed its capability to interfuse into the hexadecamer super-complex. This specific proteolysis may indicate one possible mechanism by which coronaviruses to switch from viral infection to genome replication and viral assembly stages. nsp8, SARS-CoV, RNA primase, viral life KEYWORDS cycle INTRODUCTION Coronaviruses are positive-strand RNA viruses whose replication machinery is primarily translated by the host *These authors contributed equally to the work ribosome. For positive-strand RNA viruses, the replication of the viral genome requires an RNA-dependent RNA polymerase (RdRp), together with a varied priming strategy. Also, functional non-structural proteins (nsp) utilized as replication machinery are generated via post translational processing. Determining the specific function of these nsp proteins remains one of the most difficult challenges in virology. However, in the light of severe acute respiratory syndrome coronavirus (SARS-CoV) genome sequencing, bioinformatical studies have predicted the function of nsp12 (RNAdependent RNA polymerase), nsp5 (main protease), nsp13 (endo-ribonuclease), nsp14 (helicase), nsp15 (exoribonuclease) and nsp16 (methyltransferase), and domains of nsp3 (ADRP and papain-like protease) (Prentice et al., 2004; Xu et al., 2006; Yang et al., 2003) . These analyses also indicate that the C-terminal domain of SARS-CoV nsp8 contains an RNA binding motif that is widely shared by helicases, telomereassociated proteins and other single strand nucleotide binding proteins (Imbert et al., 2006) . Nsp8 can also interact with nsp7, presumably for stabilization purposes, and the crystal structure of the nsp7-nsp8 super complex provided the first glance into the machinery of SARS replication and transcription complex (Zhai et al., 2005) . The ability of the nsp7-nsp8 complex to bind nucleic acid was first characterized by Zhai et al. (2005) , using random RNA and DNA sequences as substrates. In the same study, mutagenesis experiments targeting several positively charged residues demonstrated that the end of the extended substrate tail is crucial for nucleic acid affinity. Subsequent experimental assays concerning RNA with different sequences (Imbert et al., 2006) confirmed the role of the long helix motif of nsp8 in nucleic acid binding. In another species of positive-strand RNA viruses, the picornaviruses, both the cellular factors and the non-structural proteins are orchestrated for tuning the viral infection cycle (Brandt, 2005; Perera et al., 2007) . The cellular factor poly(rC) binding protein (PCBP2, also known as HnRNP K2) is reported to participate in both the initiation of translation and RNA replication (Graff et al., 1998). In addition, the mechanism of switching from viral protein synthesis to RNA replication was found to occur via proteolysis of the key regulator PCBP2 (Perera et al., 2007) . As a result of the action of the 3CD proteinase, the third domain of PCBP2 is truncated and viral translation is thus switched off. For coronaviruses, viral development in infected cells can follow similar stages subsequent to virus entry (Stertz et al., 2007) . By analyzing the viral RNA synthesis, it is reported that the plus strand synthesis of SARS-CoV in cells declines to undetectable levels by 12 hours post-infection (h.p.i.) (Sawicki et al., 2007) , when the infection stage moves from RNA synthesis to viral assembly. The native form of nsp8 in complex with nsp7 suggested that functional nsp8 interacts with nsp7 to form a channel-like hexadecamer, with the positively-charged electrostatic potential around the central channel significant for interaction with the negatively-charged RNA backbone (Zhai et al., 2005) (...truncated)


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Shuang Li, Qi Zhao, Yinjie Zhang, Yang Zhang, Mark Bartlam, Xuemei Li, Zihe Rao. New nsp8 isoform suggests mechanism for tuning viral RNA synthesis, Protein & Cell, 2010, pp. 198-204, Volume 1, Issue 2, DOI: 10.1007/s13238-010-0028-8