A Temperature-Sensitive Lesion in the N-Terminal Domain of the Rotavirus Polymerase Affects Its Intracellular Localization and Enzymatic Activity

Abstract

Temperature-sensitive (ts) mutants of simian rotavirus (RV) strain SA11 have been previously created to investigate the functions of viral proteins during replication. One mutant, SA11-tsC, has a mutation that maps to the gene encoding the VP1 polymerase and shows diminished growth and RNA synthesis at 39°C compared to that at 31°C. In the present study, we sequenced all 11 genes of SA11-tsC, confirming the presence of an L138P mutation in the VP1 N-terminal domain and identifying 52 additional mutations in four other viral proteins (VP4, VP7, NSP1, and NSP2). To investigate whether the L138P mutation induces a ts phenotype in VP1 outside the SA11-tsC genetic context, we employed ectopic expression systems. Specifically, we tested whether the L138P mutation affects the ability of VP1 to localize to viroplasms, which are the sites of RV RNA synthesis, by expressing the mutant form as a green fluorescent protein (GFP) fusion protein (VP1 L138P -GFP) (i) in wild-type SA11-infected cells or (ii) in uninfected cells along with viroplasm-forming proteins NSP2 and NSP5. We found that VP1 L138P -GFP localized to viroplasms and interacted with NSP2 and/or NSP5 at 31°C but not at 39°C. Next, we tested the enzymatic activity of a recombinant mutant polymerase (rVP1 L138P ) in vitro and found that it synthesized less RNA at 39°C than at 31°C, as well as less RNA than the control at all temperatures. Together, these results provide a mechanistic basis for the ts phenotype of SA11-tsC and raise important questions about the role of leucine 138 in supporting key protein interactions and the catalytic function of the VP1 polymerase.IMPORTANCE RVs cause diarrhea in the young of many animal species, including humans. Despite their medical and economic importance, gaps in knowledge exist about how these viruses replicate inside host cells. Previously, a mutant simian RV (SA11-tsC) that replicates worse at higher temperatures was identified. This virus has an amino acid mutation in VP1, which is the enzyme responsible for copying the viral RNA genome. The mutation is located in a poorly understood region of the polymerase called the N-terminal domain. In this study, we determined that the mutation reduces the ability of VP1 to properly localize within infected cells at high temperatures, as well as reduced the ability of the enzyme to copy viral RNA in a test tube. The results of this study explain the temperature sensitivity of SA11-tsC and shed new light on functional protein-protein interaction sites of VP1.KEYWORDS rotavirus, temperature-sensitive mutant, polymerase, VP1, RNA synthesis, viroplasm R otaviruses (RVs) are segmented, double-stranded RNA (dsRNA) viruses and gastrointestinal pathogens of many animal species (1). In unvaccinated children, RVs cause severe watery diarrhea and vomiting, killing an estimated 215,000 children each

Understanding the penetrance of intrinsic protein disorder in rotavirus proteome

“…A priming loop (residues 557-567), also known as a ''primer grip," situated at the joining site of thumb and palm subdomains, has a flexible structure that interacts with triphosphates during initiation. The catalytic activity of VP1 is enhanced by the interaction between the residue Leu138 of NSP1 protein with NSP2/NSP5 nonstructural proteins under physiological condition [60]. Our analysis revealed that the mean PPID of the RdRp protein is rather low (3.1%) {Table 1}, this protein is having compact globular structure with few predicted MoRFs in its polymerase domain (residues 393-407 and 463-471) that may be responsible for the polymerase activity {Fig.…”

In Vitro Double-Stranded RNA Synthesis by Rotavirus Polymerase Mutants with Lesions at Core Shell Contact Sites

“…9B and C). The most extreme differences were detected in a modeled flexible surface-exposed loop (strain SA11 aa 346 to 358) of the PD (31). Specifically, residues 347, 349, and 352 were predicted to have reduced dynamical movement for all of the alanine mutants compared to WT VP1.…”

“…Molecular dynamics simulations were performed using the program GROMACS 2018 on a modified atomic model of VP1 (PDB accession no. 2R7Q), described by McKell et al (22,31). The UCSF Chimera software program was used for all visualizations and to generate mutants with Chimera's Rotamers tool, as previously described (32).…”

New Insights into the Effect of Residue Mutations on the Rotavirus VP1 Function Using Molecular Dynamic Simulations

“…The importance of amino acid residues that affect the activity of VP1 protein has been demonstrated [7,[10][11][12] and mutant VP1 proteins in specific positions were engineered in order to assess their capacity to synthesize dsRNA in vitro [10,11]. Authors reported that, based on their effect on the replication level, the induced mutations can be classified into three groups: (i) mutations with no significant effect on replication level; (ii) mutations significantly lower levels of dsRNA product; and (iii) mutation that enhanced the initiation capacity and product elongation rate of VP1 protein.…”