Functional Characterization of Circulating Mumps Viruses with Stop Codon Mutations in the Small Hydrophobic Protein

Abstract

Between 2015 and 2017, routine molecular surveillance in the United States detected multiple mumps viruses (MuVs) with mutations in the small hydrophobic (SH) gene compared to a reference virus of the same genotype. These mutations include an unusual pattern of uracil-to-cytosine hypermutations and other mutations resulting in the generation of premature stop codons or disruption of the canonical stop codon. The mumps virus SH protein may serve as a virulence factor, based on evidence that it inhibits apoptosis and innate immune signaling in vitro and that recombinant viruses that do not express the SH protein are attenuated in an animal model. In this study, mumps viruses bearing variant SH sequences were isolated from contemporary outbreak samples to evaluate the impact of the observed mutations on SH protein function. All isolates with variant SH sequences replicated in interferon-competent cells with no evidence of attenuation. Furthermore, all SH-variant viruses retained the ability to abrogate induction of NF-κB-mediated innate immune signaling in infected cells. Ectopic expression of variant mumps SH genes is consistent with findings from infection experiments, indicating that the observed abrogation of signaling was not mediated by other viral factors that may modulate innate immune signaling. Molecular surveillance is an important public health tool for monitoring the diversity of circulating mumps viruses and can provide insights into determinants of disease. These findings, in turn, will inform studies employing reverse genetics to elucidate the specific mechanisms of MuV pathogenesis and potential impacts of observed sequence variants on infectivity, fitness, and virulence.IMPORTANCE Mumps virus (MuV) outbreaks occur in the United States despite high coverage with measles, mumps, rubella (MMR) vaccine. Routine genotyping of laboratory-confirmed mumps cases has been practiced in the United States since 2006 to enhance mumps surveillance. This study reports the detection of unusual mutations in the small hydrophobic (SH) protein of contemporary laboratory-confirmed mumps cases and is the first to describe the impact of such mutations on SH protein function. These mutations are predicted to profoundly alter the amino acid sequence of the SH protein, which has been shown to antagonize host innate immune responses; however, they were neither associated with defects in virus replication nor attenuated protein function in vitro, consistent with detection in clinical specimens. A better understanding of the forces governing mumps virus sequence diversity and of the functional consequences of mutations in viral proteins is important for maintaining robust capacity for mumps detection and disease control.

Keywords: SH protein; altered termination; genomics; host-pathogen interactions; molecular epidemiology; mumps virus; next-generation sequencing; paramyxovirus; surveillance studies; vaccine; vaccine preventable.

FIG 1

(A) Amino acid alignment of a panel of contemporary, noncanonical SH amino acid sequences aligned against the SH amino acid sequence of the reference virus MuVi/Sheffield.GBR/01.05[G]. MuV SH protein topology is indicated by the superimposed shaded boxes; the ectodomain, transmembrane domain, and cytoplasmic domain are shown in orange, gray, and blue, respectively. (B) The replication kinetics of virus isolates bearing variant SH genes were evaluated through paired experiments at an MOI of 0.1 in Vero cells, which have interferon signaling defects and are routinely used for MuV isolation, and in interferon-competent A549 cells. Supernatant was sampled at the indicated time points and infectious virus titers from four biological replicates of each condition were determined for all samples by plaque assay on Vero cells. The limit of detection is indicated by a dashed line. Results shown are representative of three independent experiments. The well-characterized isolate MuVi/Iowa.USA/00.06 bearing a canonical SH sequence was included for comparison to contemporary viruses.*, P < 0.05, Vero versus A549; ns, not significant (two-way ANOVA, by virus, with post hoc Sidak correction for multiple comparisons using GraphPad Prism v8).

FIG 2

(A) A549 cells were transfected (X-tremeGENE HP DNA transfection reagent, Roche) with pCMV-β-Gal and p(PRDII)5tkΔ(−39)lucter, which carries a luciferase gene expressed under the control of an NF-κB-inducible promoter. Transfected A549 cells were inoculated at 24 h posttransfection with MuVs at an MOI of 0.5 and, 16 h later, were stimulated with either TNF-α or IL-1β or were not stimulated. Cell lysates were harvested 4 h poststimulation for quantification of luminescence and beta-galactosidase activity using commercially available assay systems (OneGlo Luciferase Assay System and Beta-Galactosidase Enzyme Assay System, respectively; Promega). Luminescence values from each sample were normalized based on beta-galactosidase activity and were expressed in relative light units (RLU). The fold change of NF-κB induction was estimated by calculating the ratio between RLU from stimulated versus unstimulated controls for each condition. The SH amino acid sequence of MuVi/Hawaii.USA/24.17/2 is identical to the reference strain MuVi/Sheffield/GBR/01.05 [G]; this condition was included as a positive control for SH-mediated abrogation of NF-κB signaling. Statistical analysis was performed by one-way ANOVA followed by post hoc Holm-Sidak correction for multiple comparisons in GraphPad Prism v8. (B) Noncanonical SH sequences were cloned for ectopic expression through high-fidelity assembly (NEBuilder HiFi Assembly Mix; New England BioLabs) into expression vector pCMV-HA-N. Plasmid constructs were verified by Sanger sequencing (BigDye v3.1, ABI). A549 cells were transfected with the indicated plasmids (X-tremeGENE HP DNA transfection reagent, Sigma-Aldrich) and lysates were harvested at 24 h posttransfection. Expression of N-terminally tagged SH proteins was confirmed by immunoblot following SDS-PAGE of lysates (20 μg total protein) in denaturing conditions (12% Bis-Tris protein gel in MES buffer, Invitrogen) and transfer to nitrocellulose using the iBlot system (Invitrogen). Membranes were probed for HA (H3663, Sigma-Aldrich) and endogenous β-actin (A3854, Sigma-Aldrich), which served as a loading control. Sources and characteristics of the cloned SH sequences are summarized in text and in graphic form, respectively. (C) A549 cells were cotransfected with reporter plasmids and either null vector or SH expression vectors, as described in panel A. Cells were stimulated with either TNF-α or IL-1β at 24 h posttransfection and then harvested for detection of luminescence and beta-galactosidase activity.

FIG 3

SH substitutions observed in seven sequenced MuV strains bearing hypervariable SH genes, relative to MuVi/Iowa.USA/00.06. Strains were geographically and temporally similar to MuVi/Hawaii.USA/44.17/12, the source of noncanonical SH₂ in Fig. 2B. All sequenced isolates shared the same substitutions in this region of the MuV genome. Substitutions were classified as synonymous or coding (amino acid substitution is noted); U-to-C substitutions are noted by color. For instances where multiple substitutions contribute to the coding result at a single codon, they were considered separately for all reported calculations.