Aiemmin olen asettanut tämän nsp12- artikkelin (Kirchdorfer) jo. Tänään tarkistan nsp12 rakenneta ja siinä on sinkkikonfiguraatioita. atson niiden merkitystä tuossa polymeraasi ja interfaasiosassa. Jos löytyy selitystä. Ihan vain Googlehausta.
https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1001176
Zn2+ Inhibits Coronavirus and Arterivirus RNA Polymerase Activity In Vitro and Zinc Ionophores Block the Replication of These Viruses in Cell Culture
- Published: November 4, 2010
- https://doi.org/10.1371/journal.ppat.1001176 Author Summary
IntroductionZinc ions are involved in many different cellular processes and have proven crucial for the proper folding and activity of various cellular enzymes and transcription factors. Zn2+ is probably an important cofactor for numerous viral proteins as well. Nevertheless, the intracellular concentration of free Zn2+ is maintained at a relatively low level by metallothioneins, likely due to the fact that Zn2+ can serve as intracellular second messenger and may trigger apoptosis or a decrease in protein synthesis at elevated concentrations [1], [2], [3]. Interestingly, in cell culture studies, high Zn2+ concentrations and the addition of compounds that stimulate cellular import of Zn2+, such as hinokitol (HK), pyrrolidine dithiocarbamate (PDTC) and pyrithione (PT), were found to inhibit the replication of various RNA viruses, including influenza virus [4], respiratory syncytial virus (RSV) [5] and several picornaviruses [6], [7], [8], [9], [10], [11]. Although these previous studies provided limited mechanistic information, this suggests that intracellular Zn2+ levels affect a common step in the replicative cycle of these viruses.
n cell culture, PT stimulates Zn2+ uptake within minutes and inhibits RNA virus replication through a mechanism that has only been studied in reasonable detail for picornaviruses [11], [12]. In vitro studies with purified rhinovirus and poliovirus 3C proteases revealed that protease activity was inhibited by Zn2+ [13], [14], which is in line with the inhibition of polyprotein processing by zinc ions that was observed in cells infected with human rhinovirus and coxsackievirus B3 [11]. The replication of segmented negative-strand RNA viruses such as influenza virus, however, does not depend on polyprotein processing and the effect of PDTC-mediated Zn2+ import was therefore hypothesized to result from inhibition of the viral RNA-dependent RNA polymerase (RdRp) and cellular cofactors [4]. Moreover, an inhibitory effect of Zn2+ on the activity of purified RdRps from rhinoviruses and hepatitis C virus was noted, but not investigated in any detail [15], [16].
Details on the effect of zinc ions are currently largely unknown for nidoviruses. This large group of positive-strand RNA (+RNA) viruses includes major pathogens of humans and livestock, such as severe acute respiratory syndrome coronavirus (SARS-CoV), other human coronaviruses, the arteriviruses equine arteritis virus (EAV), and porcine reproductive and respiratory syndrome virus (PRRSV) [17], [18]. The common ancestry of nidoviruses is reflected in their similar genome organization and expression strategy, and in the conservation of a number of key enzymatic functions in their large replicase polyproteins [19]. A hallmark of the corona- and arterivirus replicative cycle is the transcription of a 5′- and 3′-coterminal nested set of subgenomic (sg) mRNAs from which the viral structural and accessory protein genes are expressed [20], [21].
Analogous to picornaviruses [13], [22], zinc ions were demonstrated to inhibit certain proteolytic cleavages in the processing of the coronavirus replicase polyproteins in infected cells and cell-free systems [23], [24]. In this study we report that the zinc-ionophore pyrithione (PT) in combination with Zn2+ is a potent inhibitor of the replication of SARS-coronavirus (SARS-CoV) and equine arteritis virus (EAV) in cell culture. To assess whether - besides a possible effect on proteolytic processing - nidovirus RTC subunits and RNA synthesis are directly affected by Zn2+, we employed in vitro systems for SARS-CoV and EAV RNA synthesis that are based on membrane-associated RTCs isolated from infected cells (from here on referred to as RTC assays) [25], [26]. In addition, we used in vitro recombinant RdRp assays to directly study the effect of zinc ions on the RdRps of SARS-CoV and EAV [27], [28].
Using these independent in vitro approaches, we were able to demonstrate that Zn2+ directly impairs nidovirus RNA synthesis, since it had a strong inhibitory effect in both RTC and RdRp assays. Interestingly, the Zn2+-mediated inhibition could be reversed through the addition of a Zn2+ chelator (MgEDTA). We therefore applied this compound to stop and restart the in vitro RNA-synthesizing activity at will. This convenient tool allowed us to study various mechanistic aspects of arteri- and coronavirus RNA synthesis in more detail. Additionally, the zinc-mediated inhibition of nidovirus RNA synthesis described here may provide an interesting basis to further explore the use of zinc-ionophores in antiviral therapy.
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Specific protein domains or pockets that contain zinc ions may be involved in protein-protein interactions, protein-RNA/DNA interactions, or conformational changes in enzyme structures. Zinc-binding domains commonly consist of at least three conserved cysteine and/or histidine residues within a stretch of ∼10–30 amino acids, such as in zinc-finger motifs and metalloproteases [2], [40], [41]. However, in RdRps there are only few precedents for the presence of zinc-binding pockets, such as those identified in the crystal structure of the Dengue RdRp [42]. Sequence analysis of the EAV nsp9 amino acid sequence revealed that it lacks patches rich in conserved cysteines and/or histidines. In contrast, inspection of the SARS-CoV nsp12 amino acid sequence revealed two such patches, namely H295-C301-C306-H309-C310 and C799-H810-C813-H816. A crystal structure for nsp12 is presently unavailable, but a predicted structure that represents the C-terminal two-thirds of the enzyme has been published [31]. Interestingly, in this model, C799, H810, C813 and H816 are in a spatial arrangement resembling that of the Zn2+ coordinating residues in the Zn2 zinc-binding pocket found in motif E of the Dengue virus RdRp (see Supplemental Fig. S3).
Clearly, an in-depth analysis of nidovirus RdRps, e.g. through structural analysis and subsequent mutational studies targeting aforementioned cysteines and histidines, is required to provide further insight into and a structural basis for the Zn2+-induced inhibitory effects on RdRp activity documented in this study. Such studies may, however, be complicated when Zn2+ binding proves to be very transient in nature and not detectable with currently available methods.
In summary, the combination of zinc ions and the zinc-ionophore PT efficiently inhibits nidovirus replication in cell culture. This provides an interesting basis for further studies into the use of zinc-ionophores as antiviral compounds, although systemic effects have to be considered [43], [44] and a water-soluble zinc-ionophore may be better suited, given the apparent lack of systemic toxicity of such a compound at concentrations that were effective against tumors in a mouse xenograft model [45]. In vitro, the reversible inhibition of the RdRp by Zn2+ has also provided us with a convenient research tool to gain more insight into the molecular details of (nido)viral RNA synthesis, and revealed novel mechanistic differences between the RdRps of SARS-CoV and EAV.
https://www.nature.com/articles/s41467-019-10280-3
https://www.ncbi.nlm.nih.gov/pubmed/31138817
Nat Commun. 2019 May 28;10(1):2342. doi: 10.1038/s41467-019-10280-3.
Structure of the SARS-CoV nsp12 polymerase bound to nsp7 and nsp8 co-factors.
Kirchdoerfer RN1, Ward AB2. Abstract
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