Combination of two rare mutations in the SARS-CoV-2 M gene in patients with severe and prolonged COVID-19

Abstract Background The SARS-CoV-2 virus significantly changed our knowledge about coronaviruses. The interplay between SARS-CoV-2 and the human host, the infection ranges from asymptomatic to lethal, and differences in the degree of disease severity are important examples. Methods In this retrospective study, 24 nasopharyngeal swabs from 21 out of 457 patients with SARS-CoV-2 infection were analysed by whole-genome sequencing. The principal selection criteria were the duration of infection and disease severity. Results Two co-occurring rare mutations in the SARS-CoV-2 M gene were detected in six samples. Three of these samples were collected from an immunocompromised patient with fatal outcome, two from an immunocompetent patient, and one from a patient with severe disease and fatal outcome, all with a prolonged course of infection. Conclusions Although this interesting finding was demonstrated in a small number of patients, the results increase the knowledge regarding the significance of mutations in the M gene of SARS-CoV-2 in the context of persistent infection and viral escape mechanisms.


Introduction
The SARS-CoV-2 pandemic has raised many questions regarding viral evolution, viral-host interactions, antiviral host responses and therapy-based responses.One of the most intriguing topics is the greatly varying course of infection from asymptomatic to life-threatening.A large number of studies have shown that an individual's immune response towards a new pathogen and the mutability of the virus are two crucial influencing factors.Although immune mechanisms are complex and dynamic, viral biology seems to be more relevant.Multiple viral mutations have been reported since the onset of the pandemic, as a result of viral evolution, therapy and prophylaxis [1].Although most of them are localised in the spike (S) protein RBD, mutations have also been reported in the nucleocapsid (N), envelope (E) and non-structural and accessory proteins [2].
The membrane (M) protein is one of the major viral proteins responsible for virus particle assembly through interactions with N, E and S proteins.It consists of extravirion part, three transmembrane helices and intravirion domain (b-sheet) [3].Currently, the M protein is considered relatively conserved, and mutations in the M gene are rare [4,5].Therefore, it is considered a promising target for antiviral therapy [3].
The aim of our retrospective study was to find out viral mutations in SARS-CoV-2 that can be related to the duration and severity of disease.SARS-CoV-2 viral RNA from 24 samples was evaluated by whole genome sequencing.In six samples, belonging to three patients with prolonged infection and different disease outcome we detected viral variant B.1.258carrying combination of two rare mutations, located in the SARS-CoV-2 M-gene, M:S4F (C26533T) and M:H155Y (C26985T).We suggest that these mutations in M-gene might provide evolutionary advantage to the viral variant.

Materials and methods
PCR was performed using naso/oropharyngeal swabs obtained from hospitalised patients (n ¼ 457) in COVID-19 wards at Lozenetz University Hospital during 1 November 2020 to 10 June 2021.The IVD LiliF TM COVID-19 Multi Real-Time RT-PCR Kit was used for PCR analysis.The samples were stored at À80 C for further examination.A total of 24 samples from 21 patients with prolonged and/or severe COVID-19 were selected for whole-genome sequencing on the MiSeq Illumina platform using the MiSeq Reagent Micro Kit v2, with the NEBNext V R ARTIC SARS-CoV-2 Library Prep Kit (NEB Inc., Hitchin, UK), by instructions of the manufacturers.Initial data analysis was performed using the MiSeq Reporter software (v2.6), followed by bioinformatic analysis using Galaxy https://usegalaxy.eu/(online), the BWA-MEM tool (Galaxy Version 0.7.17.2),Ivar tools (Galaxy Version 1.3.1)and Nextclade Version 2.8.1 (online).Criteria for variant calling were: mapping quality QC (quality score) !60, base QC !30, coverage > Â20.Sequence data were submitted to the SRA repository at the NCBI BioProject ID: PRJNA925289 https://www.ncbi.nlm.nih.gov/sra/PRJNA925289 (Table 1).The study was approved by the local institutional ethics committee and conducted in accordance with the Declaration of Helsinki.

Results
A repetitive combination of two mutations in the SARS-CoV-2 M gene, M:S4F (C26533T) and M:H155Y (C26985T), was detected in only six of the 24 samples (Figure 1).Three of these six samples were taken from an immunocompromised patient with persistent SARS-CoV-2 infection and positive PCR samples for over 90 days (sequenced samples taken on days 15, 34 and 73 after the onset of symptoms), two were from an immunocompetent patient with moderate form of the disease but positive PCR for over 35 days (samples from days 0 and 27), and one (day 23) was from the single assessment of a patient with a severe disease course and fatal outcome on day 32 after the onset (Table 1).The samples were obtained between 28 November 2020 and 25 January 2021.The viral variant belonging to clade 20A (Nextstrain), line B.1.258,was detected only in these six samples (Supplementary Table 1).The following three additional mutations, which had already been identified and announced for this early subline, were present in just these six samples: ORF1a: I2501T (T7767C), ORF1b: H1213Y (C17104T) and S: N439K (C22879A).

Discussion
The SARS-CoV-2 pandemic raised many questions on the diagnosis, clinical management and therapy of COVID-19.One of the most significant queries is the viral adaptation to the antiviral treatment and to the host immune system assuring long-lasting shedding.In contrast to the spike glycoprotein, M-protein is evolutionary conservative, thus being of interest for the development of new antiviral agents.Besides, it is essential in the virion formation and may elicit a measurable antibody response.
The M protein of SARS-CoV-2 consists of 222 amino acids encoded by 669 nucleotides [6].Its N-terminal extravirion end participates in virus-host interaction, the three transmembrane helices ensure dimer formation, and a C-terminal intravirion domain (b-sheet) is supposed to interact with the N, E and S structural proteins [3].The M protein plays a key role in virus assembly and morphogenesis (membrane budding) [7].Mutations in the M gene have rarely been reported.For example, M:S4F and M:H155Y have been detected at the frequencies of 1% and 2%, respectively.The first identification of these SARS-CoV-2 mutants is March 2020 in Saudi Arabia and USA, respectively [8].
In the present study, we report the simultaneous occurrence of two mutations in the SARS-CoV-2 M protein in six samples with B.1.528viral variant detected.The first one M:S4F (C26533T) is localised in the N-terminal region, outside the viral envelope (extravirion), and the second one, M:H155Y (C26985T) in the b-Sheet domain (intravirion).Both mutations lead to the substitution of polar to nonpolar amino acids.Changes in amino acid polarity are expected to result in structural alterations of the M protein [9].As previously shown, mutations in the M gene might impact virus host cell interactions by modulating cooperation with the remaining structural proteins [7,8].The mutations we describe, most probably do not impair protein stability, but may even have a positive impact.This is supported by the fact that they were present in the sequenced samples during the entire period of prolonged viral persistence in two of the three patients (the third patient had only one sample examined in the laboratory).To our knowledge, the detailed characteristics or co-occurrence of these mutations has not been previously reported.
In the N-terminus (amino acids 1-20), where the first mutation (M:S4F) is located, linear B cell epitopes may elicit a strong IgM response and IgG antibodies against the M protein in acute and to a lesser extent in convalescent COVID-19 [10].
The second mutation, M:H155Y (C26985T), is located in the intravirion part, a region that is not known to interact with other structures or to be involved in dimer formation.We cannot define whether both mutations arose spontaneously or were carried by the initial viral variant, because the patients were hospitalised at least a week after the onset of symptoms.The location of M:S4F suggests that it might provide a benefit towards viral escape (spontaneous mutation after the onset) or impaired affinity of antigen-antibody interaction (initial viral variant).Besides, M:S4F participates in the structural transition between the short and the long form of the Mprotein [7,9].Therefore, we cannot exclude fine structural modifications with still unknown consequence.By the information available in the literature, it is difficult to determine whether and how this mutation may affect the virus or disease pathogenesis.
According to our results and the observation that the Ser residue at position 4 of the M gene is a unique feature of SARS-CoV-2 compared to other coronaviruses [4], the potential of the conserved M protein as a target for vaccine development needs to be revised.In conclusion, we describe two co-occurring mutations in the SARS-CoV-2 M gene of the B.1.528viral variant in co-morbid patients with prolonged COVID-19 infection and positive PCR results for over 22 days after the onset of symptoms.Although this interesting finding was demonstrated in a small number of patients, the results contribute to the knowledge regarding the significance of mutations in the M gene.Further studies will determine whether these mutations provide an evolutionary advantage to the particular viral variant through escape from the host immune defence.

Figure 1 .
Figure 1.Visualisation of SARS-CoV-2 membrane (M) protein amino acid substitutions.(A) An overview of the structure of SARS-CoV-2 M protein.Different domains are represented by different blockstransmembrane domain one (TM1), TM2, TM3 and b-sheet domain.With numbers 1 and 222 are marked the beginning and the end of the protein.Mutations at positions 4 and 155 are highlighted with red markers.On (B), the mutations we found are shown depending on the membrane orientation of M protein with red circles in the extraand intravirion parts.(Protter: interactive protein feature visualisation and integration with experimental proteomic data.Omasits U, Ahrens CH, M€ uller S, Wollscheid B. Bioinformatics.2014;30(6):884-886.DOI:10.1093/bioinformatics/btt607).