Nuclear-Encoded Plastidal Carbonic Anhydrase Is Involved in Replication of Bamboo mosaic virus RNA in Nicotiana benthamiana

Abstract

On inoculation of Nicotiana benthamiana with Bamboo mosaic virus (BaMV), a gene with downregulated expression was found involved in the infection cycle of BaMV. To uncover how this downregulated gene affects the accumulation of BaMV in plants, we used loss- and gain-of-function experiments. Knockdown of this gene decreased the accumulation of BaMV coat protein to approximately 60% in both plants and protoplasts of N. benthamiana but had no effect on Potato virus X and Cucumber mosaic virus infection. The full-length gene was cloned and revealed as an N. benthamiana nuclear-encoded chloroplast carbonic anhydrase (CA) and so designated NbCA. As compared with the accumulation of BaMV RNAs in NbCA-knockdown protoplasts, both plus- and minus-strand RNAs were reduced. We further fused NbCA with Orange fluorescent protein to confirm its localization in chloroplasts on confocal microscopy. However, transiently expressed NbCA in chloroplasts did not considerably increase BaMV accumulation. The addition of exogenous CA may not have any additive effect on BaMV accumulation because of the natural abundance of CA in chloroplasts. In an in vitro replication assay, the addition of Escherichia coli-expressed NbCA enhanced exogenous template level (re-initiation and elongation) but not endogenous template level (only elongation). These results suggest that NbCA is possibly involved in re-initiation step of BaMV RNA replication. Further analysis indicated that proton concentration could influence the endogenous and exogenous template activities. Hence, our results implied that NbCA could be playing a role in harnessing proton concentration and favoring the replicase with the re-initiation template.

The Role of the Chloroplast in the Replication of Positive-Sense Single-Stranded Plant RNA Viruses

“…Analyses of the host factors assisting in BaMV replication have revealed at least three proteins, together with the abovementioned ChlPGK and Hsp90 may interact with the 3’ end of the viral genomic RNA. Among them are the eEF1a, glutathione S-transferase NbGSTU4 (from N. benthamiana ), and exonuclease XRN4, which all play a crucial role in the early stage of viral replication ( Cheng et al, 2013 ; Chen et al, 2017 ; Huang et al, 2017 ; Figure 1 ). For instance, XRN4 functions in reducing the activities of siRNA- and miRNA-mediated RNA silencing pathways in Arabidopsis ( Souret et al, 2004 ).…”

“…Oxidative stress may be attenuated via enzymatic activity by glutathione transferases (GSTs), and the 3′UTR-associated protein NbGSTU4 that in the presence of glutathione is able to neutralize oxidative stress inside chloroplasts, enabling efficient minus-strand RNA synthesis ( Huang et al, 2017 ). It has also been documented that the initiation and elongation of viral BaMV RNA synthesis is positively regulated by nuclear-encoded plastidic carbonic anhydrase (CA), with the knockdown of this gene reducing accumulation of the BaMV coat protein ( Chen et al, 2017 ). Furthermore, an increased level of CA was also observed by Obrepalska-Steplowska et al (2015) during peanut stunt virus infection in N. benthamiana , which suggests that the observed differences in the expression levels of chloroplast-related genes might be dependent on the virus infection process.…”

Discovery of Potent Covid-19 Main Protease Inhibitors Using Machine Learning Based Virtual Screening Strategy

“…This decrease in pH in turn restrict the binding of virus in the host cell and even viral replication. Moreover, inhibitors of carbonic anhydrase are also reported to have an activity against HIV infection [39,40]. Taken together, these findings provide evidences for the newly identified compounds that can potentially be developed as drugs for the management of Covid-19 infection.…”

Discovery of potent Covid‐19 main protease inhibitors using integrated drug‐repurposing strategy

“…This decrease in pH in turn restricts the binding of the virus in the host cell and even viral replication. Moreover, inhibitors of carbonic anhydrase are also reported to have activity against HIV infection 46,47 . It also highlights that hit compounds displayed significant structural similarity to that of recently discovered main protease inhibitors such as lurasidone sulfoxide, benzo[B]thiophene‐2‐carboxamidine 48,49 .…”

“…Moreover, inhibitors of carbonic anhydrase are also reported to have activity against HIV infection. 46,47 It also highlights that hit compounds displayed significant structural similarity to that of recently discovered main protease inhibitors such as lurasidone sulfoxide, benzo[B]thiophene-2-carboxamidine. 48,49 For instance, the Tanimoto coefficient value of more than 0.4 was observed in all the studied cases.…”