It is well known that viruses already present in host cells block secondary infections by the same or closely related viruses through superinfection exclusion (SIE). Since its initial discovery in 1929, SIE has been observed in infections of many viruses, suggesting that it plays an important role in virus survival and reproduction. However, the molecular mechanism of SIE remains poorly understood. Previously research in the Qu lab unveils that for the small positive sense RNA virus known as turnip crinkle virus (TCV), the p28 auxiliary replication protein is responsible for inducing SIE targeting this virus, and is itself the SIE target encoded by the superinfecting TCV. It is postulated that p28 could switch from the auxiliary replication function to the replication-thwarting SIE function in a concentration-dependent manner. The goal of my dissertation research was to further elucidate the molecular mechanism of p28-mediated SIE in TCV infections.
I first carried out deletion and site-directed mutagenesis of the p28 protein to identify the key regions and amino acid (aa) residues contributing to SIE elicitation. Using the agro-infiltration delivery system, I tested the SIE-elicitation abilities of two sets of p28 deletion and single-aa mutants, tagged with GFP and HA respectively. As a result, the region spanning aa 171-182 was found to be a key SIE-induction region, while aa 2-50 and 126-155 played less critical but reinforcing roles. By further interrogating the last four residues of aa 171-182, I discovered that substituting alanine (A) for the original residues of 181 (valine, V) and 182 (phenylalanine, F), respectively, modestly compromised SIE in transiently expressed p28. When introduced into TCV replicons, these two mutations – V181A and F182A, decoupled the SIE and replication functions of p28 diametrically. Specifically, V181A did not affect replication but diminished SIE, while F182A abolished replication but retained SIE.
Moreover, disrupting the replication function of p28 but not p88, the F182A mutation also decoupled the replication functions of these two TCV replication proteins. Using western blotting analysis, it was found that the F182A mutation decreased the cellular concentration of transiently-expressed p28 protein. Furthermore, this protein reduction was correlated to the loss of p28 foci indicative of intermolecular aggregations. Collectively, these results further indicated the close correlation between p28 aggregation and SIE-elicitation.
After investigating the four C-terminal aa residues of the SIE-inducing region, chapter 3 focuses on studying the N-terminal four aa 171-174. Introduction of point-mutations changing the acid-base property of aa 171 directly into TCV replicons diminished replications of TCV replicons. While trans-complementation tests, demonstrated that these mutations disrupted replication functions of both p28 and p88. As for the mutants that replicated robustly, we further examined their SIE-elicitation abilities. As a result, the four mutant replicons compromised SIE in varying extents. This work further bolstered the finding that replication and SIE functions of p28 could be decoupled by single-aa mutations.
In chapter 4, describes the mapping of the minimal region required for eliciting SIE through formation of p28 aggregates, I interrogated the intermolecular interactions of a series of p28 deletion mutants with bimolecular fluorescence complementation (BIFC) analysis. In interaction tests with themselves and with wild-type (WT) p28, mutants Δ2-125 and Δ2-155 exhibited divergent interaction manners and fluorescence patterns, indicating aa 126 as the N-terminal boundary for the nucleation ability of p28. Moreover, by further deleting the C-terminal 60 aa from these two mutants respectively, yielding constructs [126-190] and [156-190], it was found that [126-190], but not [156-190], was able to form foci-like structures. In addition, the protein structure prediction of p28 revealed that there were three α-helices localizing within the aa 126-190 domain, forming a clustered structure relatively independent from the rest part of p28. These results together indicated that the minimal domain for nucleation of p28 aggregates was aa 126-190. It is worth noting that this region was also identified as critical for SIE-elicitation function of p28 in chapter 2.
Mechanistic understanding of SIE not only would provide valuable insights into evolutionary rationale of SIE, but more importantly, it is expected to provide molecular tools for improvements of plant disease management strategies and for anti-viral therapeutics for animals, including human beings. My dissertation research made progresses in understanding molecular mechanism of SIE with respect to the following aspects: i) identified three key domains in p28 that were responsible for SIE-induction; ii) discovered single-aa mutations that decoupled the replication and SIE capacities of p28; iii) determined that the replication facilitation functions of p28 and p88 were independent; iv) further confirmed that TCV SIE was dependent on cellular p28 concentration; v) determined the minimal region required for nucleation of p28.