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the crystal structure analysis revealed that Arg298 is a key residue nsp9 to be associated with nsp7, nsp8 and nsp10 (von Brunn et
in maintaining dimerization (Shi et al., 2008). Mutations in nsp3 al., 2007). Furthermore, there is evidence supporting the role of
and nsp10 have been known to adversely affect nsp5-mediated nsp9 in reducing the disorder of nsp8 N-terminus in an analytical
polyprotein processing (Donaldson et al., 2007; Stokes et al., ultracentrifugation experiment (Sutton et al., 2004).
2010). Nsp10 is one of the most well conserved nsps among differ-
Nsp6 produces two products of approximately 23 and ent CoVs. It contains two zinc fingers acting as a zinc binding
25 kDa, and analysis into its transmembrane topology revealed domain and acts as a co-factor for nsp14 and nsp16 to elicit
six-membrane-spanning segments and a conserved hydropho- 3′–5′ exoribonuclease and 2′-O-methyltransferase (2′-O-MTase)
bic domain at the C-terminus on the cytosolic side (Baliji et al., activities, respectively. Moreover, nsp9 and nsp10 was found to be
2009). In SARS-CoV, nsp3, nsp 4 and nsp 6 are required to induce membrane-associated in the replicase complex by immunofluo-
double-membrane vesicles (DMVs) in virus infected cells (Ange- rescence (van der Meer et al., 1998). Using the mouse hepatitis
lini et al., 2013). Mutations in MHV nsp3 has been reported to virus strain A59 (MHV-A59), a single amino-acid mutation in
impair DMV-formation (Stokes et al., 2010). IBV nsp6 has been nsp10 (Q65E) was found to be defective in negative-strand syn-
shown to generate autophagosomes from the ER, indicating that thesis of viral RNA and activation of Mpro (Donaldson et al.,
it can induce autophagy in cells when expressed (Cottam et al., 2007). The structure of both monomeric and dodecameric forms
2011). of nsp10 has been solved for SARS-CoV by X-ray crystallography
Nsp7 forms the hexadecameric complex with nsp8. Alone, (Su et al., 2006). The monomeric nsp10 contains a helical-hairpin
nsp7 consists of an antiparallel three-helix bundle with an α-helix at the N-terminus, followed by an irregular β-sheet surrounded
at the C-terminus. The C-terminal portion of nsp7 is flexible and by additional helices, and finally a C-terminal subdomain loop.
can associate with nsp8. This interaction between nsp7 and nsp8 The first zinc-finger module of nsp10 is CCHC-type with a
within the hexadecameric complex is stabilized by the aliphatic C-(X)2-C-(X)5-H-(X)6-C sequence motif, while the second
side-chains on the N-terminus of the three-helix bundle of nsp7 zinc-binding site is a CCCC-type with a C-(X)2-C-(X)7-C-(X)-
and the hydrophobic interactions involving the C-terminal helix C sequence motif (Su et al., 2006). Recently, the key residues on
of nsp7 and the C-terminus of nsp 8 (Zhai et al., 2005). nsp10 surface responsible for its interactions with nsp14 have
Many positive-sense RNA viruses utilize the Vpg (viral been identified (Bouvet et al., 2014). Interestingly, the same
protein genome-linked) oligonucleotides as a primer for the nsp10 surface interacting with nsp14 has been demonstrated to
RNA-dependent RNA polymerase (RdRP) (Pettersson et al., overlap with nsp10-mediated activation of nsp16 2′-O-MTase,
1978; Steil et al., 2010). Since the CoV RNA synthesis is primer- indicating that nsp10 is a major regulator of CoV replicase func-
dependent (Cheng et al., 2005), and the CoV genome does not tion (Bouvet et al., 2014).
encode a Vpg, nsp8 serves as a primase by synthesizing short Nsp11 forms the C-terminus of pp1a. This 13-residue oligo-
primers for the RdRP and a second RdRP in SARS-CoV (Imbert peptide in SARS-CoV sits at the ribosomal frame-shifting site
et al., 2006). In synthesizing primers in a manganese-dependent and is produced when no ribosomal frame-shifting occurs at the
reaction, nsp8 selectively synthesizes primers specific to the slippery sequence. So far, nsp11 protein has not been detected in
5′-(G/U)CC-3′ sites on the template RNA. By observing the 3D infected cells. Nsp11 shares its N-terminus with nsp12, the RdRP.
structure of nsp8, the inner diameter within the hexadecameric Obviously, since most of nsp11 coding sequence overlaps with
complex is approximately 30Å, and can accommodate double- the RNA frame-shifting sequence and nsp12 coding sequence, no
stranded RNA, suggesting that the hexadecameric complex may functional role for nsp11 has been assigned.
act as a processivity factor for RdRP (Zhai et al., 2005). Alongside Nsp12 is arguably the most conserved nsp of the CoV replicase
Nsp7, Nsp8 is also known to form a stable complex with NSP12, transcriptase. The RdRP activity of nsp12 was confirmed in 2005
independent of the UTRs of viral RNA and other viral proteins when a bacterially expressed nsp12 could extend an oligouri-
(Tan et al., 2018). dylate (U) primer hybridized to a poly(A) template (Cheng et al.,
Nsp9 plays an essential role in viral replication by forming a 2005). Phylogenetic clustering studies have found CoV nsp12 to
homodimer which binds ssRNA (Egloff et al., 2004). Structur- be closely related to RdRPs of other positive-sense RNA viruses
ally, nsp9 comprises a single beta-barrel with an OB-fold and a whose genome RNAs have 5′-end covalently linked to viral protein
C-terminal extension related to the subdomains of the Mpro genome-linked (VPg) (Koonin, 1991). RdRPs of VPg-containing
of SARS-CoV (Sutton et al., 2004). Crystal structure solved viruses share a conserved sequence motif G implicated in the
for SARS-CoV nsp9 suggests that this protein may be dimeric recognition of primer-template RNA complex (Barrette-Ng et al.,
(Sutton et al., 2004), and this has been recently confirmed by 2002; Thompson and Peersen, 2004); this same motif was also
Hu and colleagues (2017) on IBV nsp9. Indeed, IBV nsp9 forms conserved in CoVs, implying that nsp12 contains VPg-like activi-
a homodimer via interactions across the hydrophobic surface ties (Gorbalenya et al., 2002). Nsp12 contains 932 residues and
containing two parallel alpha helices near the C-terminal of nsp9. conserved RdRP motifs which function as the catalytic domain
Moreover, dimeric nsp9 resembles SARS-CoV nsp9, indicating at the C-terminus (Gorbalenya et al., 1989; Koonin, 1991). A
that the dimerization may be conserved among all CoVs. Although 3D-homology model proposed by Xu and colleagues (2003)
the function of nsp9 remains elusive other than a ssRNA-binding for SARS-CoV nsp12 showed a cupped right-hand palm-finger-
protein, it is presumed to be involved in viral RNA synthesis, thumb structure surrounding a nucleic acid-binding tunnel. It is
since yeast-2-hybrid and coimmunoprecipitation have found important to note that the expression of nsp12 requires ribosomal