Geminivirus C2 protein might be the key player for geminiviral co-option of SCF-mediated ubiquitination

Abstract

Viruses are obligate intracellular parasites, and need to create a suitable cell environment for viral propagation to complete their life cycle. In order to achieve this, viruses must usurp or interfere with the cellular machinery. Ubiquitination, a post-translational modification that controls numerous cellular processes, has proven to be a common target for viruses. Recently, geminivirus C2 protein has been shown to interact with the CSN complex and disrupt its activity over CULLIN1, interfering with the function of the CULLIN1-based SCF ubiquitin E3 ligases. Interestingly, over-expression of a given F-box protein may circumvent the general SCF malfunction caused by C2. This result raises the tantalizing idea that geminiviruses might be not only hampering, but also redirecting the activity of SCF complexes, thus co-opting the SCF-mediated ubiquitination pathway. We hypothesize that the mechanism of C2-facilitated co-option of SCF-mediated ubiquitination might not be exclusive for geminiviruses, but rather a common strategy for viruses.

Geminivirus C2 Protein Subverts SCF-Mediated Ubiquitination

Viruses are obligate intracellular parasites, which must infect cells to complete their life cycle. To this end, they are forced to usurp, counteract or redirect the cellular machinery, exploiting cellular biology in order to create a suitable cell environment for viral infection. Ubiquitination, a highly dynamic post-translational modification that controls most of the protein degradation events in eukaryotes, is a hub regulator of multiple cellular processes, and as such is an attractive target for viruses.^1,2

Geminiviruses are plant viruses with circular, single-stranded DNA genomes that infect a wide range of plant species and cause extensive losses in crop production worldwide. These viruses have highly reduced genomes, encoding a limited number of multifunctional proteins (ranging from 6 to 8, depending on the species). Strikingly, provided with this apparently light weaponry, these pathogens efficiently subvert the cell to create a niche for viral propagation, triggering changes that are reflected at all levels, from gene expression to cellular organization.³

C2 (also known as L2, AC2, AL2 or TrAP, for Transcriptional Activator Protein) is a pathogenicity factor encoded by geminiviruses. Not surprisingly, C2 is a multifunctional protein, playing a plethora of different roles during the infection: it has been shown to act as an activator of transcription of viral genes,⁴ to repress host defences,⁵ and to suppress gene silencing.⁶ Interestingly, C2 from different geminiviruses (Tomato yellow leaf curl Sardinia virus-TYLCSV, Tomato yellow leaf curl virus-TYLCV and Beet curly top virus-BCTV) is able to interact with CSN5, catalytic subunit of the CSN complex.⁷ The CSN (or COP9 signalosome) is a conserved essential eukaryotic complex whose best characterized function is the removal of the ubiquitin-like RUB moiety from cullins, the scaffold proteins of the Cullin-RING-Ligases (CRLs) ubiquitin E3 ligases. Since CRLs activity is upregulated by conjugation of RUB to the cullin component,⁸ the CSN complex is one of the regulators of the activity of CRLs, being essential for their function in vivo. Expression of C2 in transgenic Arabidopsis plants compromises the activity of the CSN over CUL1, which accumulates in its rubylated form. As a result, responses regulated by CUL1-based CRLs, named SCF (for Skp1/Cullin1/F-box) complexes (including plant responses to the jasmonates, auxins, gibberellins, ethylene and ABA in the guard cells) are altered in these plants, consistently with a hindered SCF function.⁷ Given that SCFs are key regulators of numerous fundamental cellular processes, the capability of geminiviruses to selectively interfere with these complexes might represent a novel and powerful strategy in viral infections.

The Overexpression of an F-Box Protein May Circumvent the General SCF Malfunction Caused by C2

According to previous results, C2 would be capable of hindering the activity of several SCF complexes in the plant cell, probably conferring some biological advantage for the viral infection such as leading to a suppression of hormone-mediated plant defence responses. It is an attractive hypothesis, however, that the virus might be not only impairing the function of the SCF complexes, but rather also redirecting them from unwanted towards selected target proteins. It has been shown by Denti et al. that the malfunction of SCF^SKP2 in cultured cells in which CSN5 had been knocked down could be rescued by over-expression of the F-box adaptor protein, Skp2; a similar effect has been recently described in Arabidopsis,¹⁰ where overexpression of TIR1 partially complemented the csn2-5 auxin resistance phenotype. Accordingly, it would be possible that C2 or other geminiviral protein(s) might be triggering the expression of particular F-box proteins in the infected cell that could be incorporated into the SCF complexes, overcoming the general impairment. In order to test this idea, we looked for genes encoding F-box proteins upregulated in transgenic Arabidopsis plants expressing TYLCSV C2.⁷ Transcriptomic analysis of these plants showed that the expression of EID1, a gene encoding the F-box subunit of the SCF^EID1 complex, which acts as an inhibitor of PhyA,¹¹ was upregulated. eid1 mutants display shorter hypocotyls than wild-type plants when grown under far red light conditions;¹¹ consistently with the function of EID1 in an SCF complex, the cul1 mutant axr6-3 shows hypersensitivity to far-red light, also developing shorter hypocotyls than wild-type plants.¹² As a means to assay the functionality of this protein in our Arabidopsis transgenic lines expressing TYLCSV, TYLCV of BCTV C2 (also named L2), we grew them under far red light and measured the hypocotyl length after six days. Figure 1 shows that the hypocotyls of C2 plants grown under these conditions are longer than those of wild-type plants, suggesting that the over-produced EID1 protein is exerting its function as part of the SCF^EID1 complex. This result raises the tantalizing idea that C2 may modulate the function of some SCF E3 ligases by triggering the overexpression of the corresponding F-box protein substrate adaptor, thus co-opting the SCF-mediated ubiquitination pathway.

Figure 1.

Hypocotyl length of 10-day-old wild-type (WT) or transgenic Arabidopsis seedlings expressing TYLCSV, TYLCV or BCTV C2 (C2-TS, C2-TM and L2-BC respectively) grown under far-red light. N ≥ 30; bars represent standard deviation. Asterisks indicate a statistically significant difference when compared to the wild-type value according to Mann-Whitney rank sum test.

C2-Facilitated Co-Option of SCF-Mediated Ubiquitination: A Common Strategy for Viruses?

Other plant viruses, nanoviruses and poleroviruses, have been shown to co-opt the SCF machinery by encoding their own viral F-box proteins, which are assembled into plant SCF complexes,^13–15 triggering the ubiquitination of plant proteins that interfere with viral infection (pRB and AGO4, respectively). The comparison between these viruses and geminiviruses lead us to hypothesize that, on one hand, in order to boost the effect of their encoded F-box proteins, nanoviruses and poleroviruses could have also developed means, yet to be described, to interfere with the rubylation/derubylation cycle of cullins. On the other hand, it would be feasible that geminiviruses could be re-directing the SCF ubiquitination machinery themselves, not only through the promotion of over-production of certain host-encoded F-box proteins, but also by encoding their own adaptor proteins. Interestingly enough, the geminiviral protein V2 has been recently described to harbour an F-box motif and interact with the plant SKP1.¹⁶ If this viral protein is successfully assembled into an SCF complex, as these preliminary results suggest, it might trigger the ubiquitination of its interactor SGS3, which may underlie its role as silencing suppressor. In such a scenario, the changes in CUL1 rubylation state triggered by C2 might help maximize the effect of V2 on its target host protein.

The co-option of CRLs is not limited to plant viruses; on the contrary, multiple examples are provided by animal viruses.¹⁷ One of the best studied cases is that of the HIV protein Vpr, which loads the host protein UNG2 onto the CUL4-based CRL DCAF1 for ubiquitination,¹⁸ probably inducing cell cycle arrest to promote infection. In this context, maintaining the RUB/NEDD8 modification of CUL4, which would lead to enhanced activity of the DCAF1 complex, would be extremely beneficial for the pathogen. Intriguingly, Vpr also interacts with the CSN subunit CSN6.¹⁹

It is a tempting idea, derived from the effect of geminivirus C2 on CUL1 and the studied cases of viral co-option of CRLs, that the interference with the activity of the CSN complex might not be exclusive for geminiviruses. On the contrary, we conjecture that other viruses may have evolved similar mechanisms as a result of convergent evolution, and therefore this line of attack might be revealed as a common strategy for viruses in the following years. Further studies in different models will be required to determine whether this is the case.

Addendum to: Lozano-Duran R, Rosas-Diaz T, Gusmaroli G, Luna AP, Taconnat L, Deng XW, Bejarano ER. Geminiviruses subvert ubiquitination by altering CSN-mediated de-rubylation of SCF E3 ligase complexes and inhibit jasmonate signalling. Plant Cell. 2011;23:1014–1032. doi: 10.1105/tpc.110.080267.