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
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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)