ADP Ribosylation Factor 1 Plays an Essential Role in the Replication of a Plant RNA Virus

Abstract

bEukaryotic positive-strand RNA viruses replicate using the membrane-bound replicase complexes, which contain multiple viral and host components. Virus infection induces the remodeling of intracellular membranes. Virus-induced membrane structures are thought to increase the local concentration of the components that are required for replication and provide a scaffold for tethering the replicase complexes. However, the mechanisms underlying virus-induced membrane remodeling are poorly understood. RNA replication of red clover necrotic mosaic virus (RCNMV), a positive-strand RNA plant virus, is associated with the endoplasmic reticulum (ER) membranes, and ER morphology is perturbed in RCNMV-infected cells. Here, we identified ADP ribosylation factor 1 (Arf1) in the affinity-purified RCNMV RNA-dependent RNA polymerase fraction. Arf1 is a highly conserved, ubiquitous, small GTPase that is implicated in the formation of the coat protein complex I (COPI) vesicles on Golgi membranes. Using in vitro pulldown and bimolecular fluorescence complementation analyses, we showed that Arf1 interacted with the viral p27 replication protein within the virus-induced large punctate structures of the ER membrane. We found that inhibition of the nucleotide exchange activity of Arf1 using the inhibitor brefeldin A (BFA) disrupted the assembly of the viral replicase complex and p27-mediated ER remodeling. We also showed that BFA treatment and the expression of dominant negative Arf1 mutants compromised RCNMV RNA replication in protoplasts. Interestingly, the expression of a dominant negative mutant of Sar1, a key regulator of the biogenesis of COPII vesicles at ER exit sites, also compromised RCNMV RNA replication. These results suggest that the replication of RCNMV depends on the host membrane traffic machinery. E ukaryotic positive-strand RNA [(ϩ)RNA] viruses replicate their genomes using membrane-bound replicase complexes, which contain multiple viral and host components. A growing number of host proteins that affect viral RNA replication have been identified using genome-wide and proteomics analyses in several animal and plant viruses (1-13). These host proteins are involved in translation, template selection, and the assembly of the viral replication complex (VRC) on intracellular membranes, which serve as the site of viral RNA replication (14). However, the functions of host proteins remain largely unknown.The replication compartments of (ϩ)RNA viruses are derived from various cellular organelle membranes, such as the endoplasmic reticulum (ER), mitochondria, chloroplasts, peroxisomes, and the Golgi apparatus (15-17). The formation of viral replication compartments generally involves the emergence of spherules, vesicles, and multivesicular bodies associated with various organelles (15, 17). Although viral proteins play an essential role in the formation of replication compartments containing VRCs, host factors also regulate this process (14,15,18). Tomato bushy stunt virus (TBSV) coopts the proteins of the endosomal sorting compl...

Harnessing host ROS-generating machinery for the robust genome replication of a plant RNA virus

“…2 and 3). Because most, if not all, p27-containing perinuclear ER aggregate structures are believed to be the sites of de novo viral RNA synthesis (28), the presented data suggest that a large proportion of NbRBOHB molecules are retargeted to the viral replication site from the PM through interaction with p27. The ER membrane is known to associate with the PM (45), and therefore it is possible that p27 recognizes NbRBOHB at or near the ER-PM contact sites and The relative accumulation levels of viral RNAs were determined as described in the legend to Fig.…”

“…Alternatively, p27 may recruit RBOH proteins from the Golgi apparatus, where a population of RBOH molecules resides (3). In this context, it should be noted that p27 binds to Arf1, which is a key regulator of vesicle trafficking from the Golgi apparatus, and interferes with host secretory pathways (28,37). The data presented here suggest that BMV, another plant (+)RNA virus, also uses the host ROS-generating machinery for viral RNA replication (Fig.…”

“…At 2 d after inoculation, inoculated leaves from three different plants infected with the same inoculum were pooled, and total RNA was extracted using plant RNA purification reagents (Invitrogen), treated with RQ1 RNasefree DNase (Promega), purified by phenol-chloroform extraction, and precipitated with ethanol. Viral RNAs were detected by Northern blotting, as described previously (27,28).…”

“…RNA extraction from N. benthamiana leaves was performed using an RNeasy Plant Mini-Kit (Qiagen). Reverse-transcription PCR (RT-PCR) was carried out as described earlier (28). Superscript III reverse transcriptase (Invitrogen) and oligo (dT) were used for RT.…”

“…p27 and p88 pol target the endoplasmic reticulum (ER) membranes and form a viral replicase complex with co-opted multiple host factors (24)(25)(26). Among them are host heat shock proteins, small GTPase ADP ribosylation factor 1 (Arf1), and a PA-generating enzyme phospholipase D (PLD) hijacked by RCNMV for robust viral replication (27)(28)(29). Interestingly, affinity-purified RCNMV replicase complex from a model plant N. benthamiana (30) contains several key players in plant stress responses such as RBOH proteins and CDPK (29), suggesting that these host proteins have direct regulatory functions during a plant virus infection.…”