Recurrent SARS-CoV-2 mutations in immunodeficient patients.

Long-term severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections in immunodeficient patients are an important source of variation for the virus but are understudied. Many case studies have been published which describe one or a small number of long-term infected individuals but no study has combined these sequences into a cohesive dataset. This work aims to rectify this and study the genomics of this patient group through a combination of literature searches as well as identifying new case series directly from the COVID-19 Genomics UK (COG-UK) dataset. The spike gene receptor-binding domain and N-terminal domain (NTD) were identified as mutation hotspots. Numerous mutations associated with variants of concern were observed to emerge recurrently. Additionally a mutation in the envelope gene, T30I was determined to be the second most frequent recurrently occurring mutation arising in persistent infections. A high proportion of recurrent mutations in immunodeficient individuals are associated with ACE2 affinity, immune escape, or viral packaging optimisation. There is an apparent selective pressure for mutations that aid cell-cell transmission within the host or persistence which are often different from mutations that aid inter-host transmission, although the fact that multiple recurrent de novo mutations are considered defining for variants of concern strongly indicates that this potential source of novel variants should not be discounted.

SARS-CoV-2 VARIANTS EVOLVED CONVERGENT STRATEGIES TO REMODEL THE HOST RESPONSE

Background: Five variants of concern (VOCs) have dominated COVID-19 disease etiology since 2020-Alpha, Beta, Gamma, Delta, and Omicron-possessing over 150 defining genomic alterations. Here, we used global proteomic and genomic approaches to study the host responses and selective forces driving VOC evolution. Method(s): We infected Calu-3 human lung epithelial cells with 5 VOCs and 2 wave 1 (W1) controls and performed mass spectrometry abundance proteomics, phosphoproteomics, and mRNA sequencing at 10 and 24 hours post infection. We additionally performed affinity purification mass spectrometry (APMS) by individually expressing all VOC mutant viral proteins (52) and corresponding W1 forms in human cells to quantify differential virus-host protein-protein interactions. Data was integrated using network modeling and bioinformatics to pinpoint VOC-specific differences. Four novel mutant viruses were developed using reverse genetics technology to validate the impact of specific genomic alterations. Result(s): We discovered VOCs evolved convergent molecular strategies to remodel the host response by modulating viral RNA and protein levels (most notably of N, Orf9b, and Orf6), altering nucleocapsid phosphorylation, and rewiring virus-host protein complexes. Integrative systems analyses revealed that Alpha, Beta, Gamma, and Delta ultimately converged in the suppression of interferon stimulated genes (ISGs) relative to W1 viruses, but Omicron BA.1 did not, and Delta induced more pro-inflammatory genes compared to other VOCs. Altered regulation of ISGs correlated with the expression of viral innate immune antagonist proteins, including Orf6, N, and Orf9b;for example, Omicron BA.1 depicted a 2-fold decrease in Orf6 expression. We identified mutations that alter expression of Orf9b (N D3L and N -3A del) and the novel VOC protein N* (N R203K/G204R), and confirmed Orf6 innate immune antagonism using recombinant virus technology. Remarkably, Omicron BA.4 and BA.5 regained strengthened innate immune antagonism compared to BA.1, which again correlated with enhanced Orf6 expression, though dampened in BA.4 by a mutation (D61L) that we discovered disrupts the Orf6-nuclear pore interaction. Conclusion(s): Collectively, our findings suggest SARS-CoV-2 convergent evolution overcomes human innate immune barriers, laying the groundwork to understand future coronavirus evolution associated with immune escape and enhanced human-to-human transmission.

Exhaled Breath Aerosol Shedding by Highly Transmissible Versus Prior SARS-CoV-2 Variants.

BACKGROUND: Aerosol inhalation is recognized as the dominant mode of SARS-CoV-2 transmission. Three highly transmissible lineages evolved during the pandemic. One hypothesis to explain increased transmissibility is that natural selection favors variants with higher rates of viral aerosol shedding. However, the extent of aerosol shedding of successive SARS-CoV-2 variants is unknown. We aimed to measure the infectivity and rate of SARS-CoV-2 shedding into exhaled breath aerosol (EBA) by individuals during the Delta and Omicron waves and compared those rates with those of prior SARS-CoV-2 variants from our previously published work. METHODS: COVID-19 cases (n = 93, 32 vaccinated and 20 boosted) were recruited to give samples, including 30-minute breath samples into a Gesundheit-II exhaled breath aerosol sampler. Samples were quantified for viral RNA using RT-PCR and cultured for virus. RESULTS: Alpha (n = 4), Delta (n = 3), and Omicron (n = 29) cases shed significantly more viral RNA copies into exhaled breath aerosols than cases infected with ancestral strains and variants not associated with increased transmissibility (n = 57). All Delta and Omicron cases were fully vaccinated and most Omicron cases were boosted. We cultured virus from the EBA of one boosted and three fully vaccinated cases. CONCLUSIONS: Alpha, Delta, and Omicron independently evolved high viral aerosol shedding phenotypes, demonstrating convergent evolution. Vaccinated and boosted cases can shed infectious SARS-CoV-2 via EBA. These findings support a dominant role of infectious aerosols in transmission of SARS-CoV-2. Monitoring aerosol shedding from new variants and emerging pathogens can be an important component of future threat assessments and guide interventions to prevent transmission.

SARS-CoV-2, HIV, and HPV: Convergent evolution of selective regulation of cGAS-STING signaling.

Recognizing aberrant cytoplasmic double-stranded DNA and stimulating innate immunity is essential for the host's defense against viruses and tumors. Cyclic GMP-AMP (cGAMP) synthase (cGAS) is a cytosolic DNA sensor that synthesizes the second messenger 2'3'-cGAMP and subsequently activates stimulator of interferon genes (STING)-mediated activation of TANK-binding kinase 1 (TBK1)/interferon regulatory factor 3 (IRF3) and the production of type I interferon (IFN-I). Both the cGAS-STING-mediated IFN-I antiviral defense and the countermeasures developed by diverse viruses have been extensively studied. However, recent studies have revealed a convergent evolutionary feature of severe acute respiratory syndrome coronavirus 2 and human immunodeficiency virus (HIV) viral proteins in terms of the selective regulation of cGAS-STING-mediated nuclear factor-κB (NF-κB) signaling without any effect on cGAS-STING-mediated TBK1/IRF3 activation and IFN production. The potential beneficial effect of this cGAS-STING-mediated, NF-κB-dependent antiviral effect, and the possible detrimental effect of IFN-I in the pathogenesis of coronavirus disease 2019 and HIV infection deserve more attention and future investigation.

DNA virus oncoprotein HPV18 E7 selectively antagonizes cGAS-STING-triggered innate immune activation.

Cellular infections by DNA viruses trigger innate immune responses mediated by DNA sensors. The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon gene (STING) signaling pathway has been identified as a DNA-sensing pathway that activates interferons in response to viral infection and, thus, mediates host defense against viruses. Previous studies have identified oncogenes E7 and E1A of the DNA tumor viruses, human papillomavirus 18 (HPV18) and adenovirus, respectively, as inhibitors of the cGAS-STING pathway. However, the function of STING in infected cells and the mechanism by which HPV18 E7 antagonizes STING-induced Interferon beta production remain unknown. We report that HPV18 E7 selectively antagonizes STING-triggered nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation but not IRF3 activation. HPV18 E7 binds to STING in a region critical for NF-κB activation and blocks the nuclear accumulation of p65. Moreover, E7 inhibition of STING-triggered NF-κB activation is related to HPV pathogenicity but not E7-Rb binding. HPV18 E7, severe acute respiratory syndrome coronavirus-2 open reading frame 3a, human immunodeficiency virus-2 viral protein X, and Kaposi's sarcoma-associated herpesvirus KSHV viral interferon regulatory factor 1 selectively inhibited STING-triggered NF-κB or IRF3 activation, suggesting a convergent evolution among these viruses toward antagonizing host innate immunity. Collectively, selective suppression of the cGAS-STING pathway by viral proteins is likely to be a key pathogenic determinant, making it a promising target for treating oncogenic virus-induced tumor diseases.

Convergent Evolution in SARS-CoV-2 Spike Creates a Variant Soup from Which New COVID-19 Waves Emerge.

The first 2 years of the COVID-19 pandemic were mainly characterized by recurrent mutations of SARS-CoV-2 Spike protein at residues K417, L452, E484, N501 and P681 emerging independently across different variants of concern (Alpha, Beta, Gamma, and Delta). Such homoplasy is a marker of convergent evolution. Since Spring 2022 and the third year of the pandemic, with the advent of Omicron and its sublineages, convergent evolution has led to the observation of different lineages acquiring an additional group of mutations at different amino acid residues, namely R346, K444, N450, N460, F486, F490, Q493, and S494. Mutations at these residues have become increasingly prevalent during Summer and Autumn 2022, with combinations showing increased fitness. The most likely reason for this convergence is the selective pressure exerted by previous infection- or vaccine-elicited immunity. Such accelerated evolution has caused failure of all anti-Spike monoclonal antibodies, including bebtelovimab and cilgavimab. While we are learning how fast coronaviruses can mutate and recombine, we should reconsider opportunities for economically sustainable escape-proof combination therapies, and refocus antibody-mediated therapeutic efforts on polyclonal preparations that are less likely to allow for viral immune escape.

Analysis of co-occurring and mutually exclusive amino acid changes and detection of convergent and divergent evolution events in SARS-CoV-2.

The inflation of SARS-CoV-2 lineages with a high number of accumulated mutations (such as the recent case of Omicron) has risen concerns about the evolutionary capacity of this virus. Here, we propose a computational study to examine non-synonymous mutations gathered within genomes of SARS-CoV-2 from the beginning of the pandemic until February 2022. We provide both qualitative and quantitative descriptions of such corpus, focusing on statistically significant co-occurring and mutually exclusive mutations within single genomes. Then, we examine in depth the distributions of mutations over defined lineages and compare those of frequently co-occurring mutation pairs. Based on this comparison, we study mutations' convergence/divergence on the phylogenetic tree. As a result, we identify 1,818 co-occurring pairs of non-synonymous mutations showing at least one event of convergent evolution and 6,625 co-occurring pairs with at least one event of divergent evolution. Notable examples of both types are shown by means of a tree-based representation of lineages, visually capturing mutations' behaviors. Our method confirms several well-known cases; moreover, the provided evidence suggests that our workflow can explain aspects of the future mutational evolution of SARS-CoV-2.

Perspectives: SARS-CoV-2 Spike Convergent Evolution as a Guide to Explore Adaptive Advantage.

Viruses rapidly co-evolve with their hosts. The 9 million sequenced SARS-CoV-2 genomes by March 2022 provide a detailed account of viral evolution, showing that all amino acids have been mutated many times. However, only a few became prominent in the viral population. Here, we investigated the emergence of the same mutations in unrelated parallel lineages and the extent of such convergent evolution on the molecular level in the spike (S) protein. We found that during the first phase of the pandemic (until mid 2021, before mass vaccination) 31 mutations evolved independently ≥3-times within separated lineages. These included all the key mutations in SARS-CoV-2 variants of concern (VOC) at that time, indicating their fundamental adaptive advantage. The omicron added many more mutations not frequently seen before, which can be attributed to the synergistic nature of these mutations, which is more difficult to evolve. The great majority (24/31) of S-protein mutations under convergent evolution tightly cluster in three functional domains; N-terminal domain, receptor-binding domain, and Furin cleavage site. Furthermore, among the S-protein receptor-binding motif mutations, ACE2 affinity-improving substitutions are favoured. Next, we determined the mutation space in the S protein that has been covered by SARS-CoV-2. We found that all amino acids that are reachable by single nucleotide changes have been probed multiple times in early 2021. The substitutions requiring two nucleotide changes have recently (late 2021) gained momentum and their numbers are increasing rapidly. These provide a large mutation landscape for SARS-CoV-2 future evolution, on which research should focus now.

Poxviruses and paramyxoviruses use a conserved mechanism of STAT1 antagonism to inhibit interferon signaling.

The induction of interferon (IFN)-stimulated genes by STATs is a critical host defense mechanism against virus infection. Here, we report that a highly expressed poxvirus protein, 018, inhibits IFN-induced signaling by binding to the SH2 domain of STAT1, thereby preventing the association of STAT1 with an activated IFN receptor. Despite encoding other inhibitors of IFN-induced signaling, a poxvirus mutant lacking 018 was attenuated in mice. The 2.0 Å crystal structure of the 018:STAT1 complex reveals a phosphotyrosine-independent mode of 018 binding to the SH2 domain of STAT1. Moreover, the STAT1-binding motif of 018 shows similarity to the STAT1-binding proteins from Nipah virus, which, similar to 018, block the association of STAT1 with an IFN receptor. Overall, these results uncover a conserved mechanism of STAT1 antagonism that is employed independently by distinct virus families.

Phylogenetic Analyses of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) B.1.1.7 Lineage Suggest a Single Origin Followed by Multiple Exportation Events Versus Convergent Evolution.

The emergence of new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) herald a new phase of the pandemic. This study used state-of-the-art phylodynamic methods to ascertain that the rapid rise of B.1.1.7 "Variant of Concern" most likely occurred by global dispersal rather than convergent evolution from multiple sources.

Long-Term Evolution of SARS-CoV-2 in an Immunocompromised Patient with Non-Hodgkin Lymphoma.

Recent studies have shown that persistent SARS-CoV-2 infections in immunocompromised patients can trigger the accumulation of an unusual high number of mutations with potential relevance at both biological and epidemiological levels. Here, we report a case of an immunocompromised patient (non-Hodgkin lymphoma patient under immunosuppressive therapy) with a persistent SARS-CoV-2 infection (marked by intermittent positivity) over at least 6 months. Viral genome sequencing was performed at days 1, 164, and 171 to evaluate SARS-CoV-2 evolution. Among the 15 single-nucleotide polymorphisms (SNPs) (11 leading to amino acid alterations) and 3 deletions accumulated during this long-term infection, four amino acid changes (V3G, S50L, N87S, and A222V) and two deletions (18-30del and 141-144del) occurred in the virus Spike protein. Although no convalescent plasma therapy was administered, some of the detected mutations have been independently reported in other chronically infected individuals, which supports a scenario of convergent adaptive evolution. This study shows that it is of the utmost relevance to monitor the SARS-CoV-2 evolution in immunocompromised individuals, not only to identify novel potentially adaptive mutations, but also to mitigate the risk of introducing "hyper-evolved" variants in the community. IMPORTANCE Tracking the within-patient evolution of SARS-CoV-2 is key to understanding how this pandemic virus shapes its genome toward immune evasion and survival. In the present study, by monitoring a long-term COVID-19 immunocompromised patient, we observed the concurrent emergence of mutations potentially associated with immune evasion and/or enhanced transmission, mostly targeting the SARS-CoV-2 key host-interacting protein and antigen. These findings show that the frequent oscillation in the immune status in immunocompromised individuals can trigger an accelerated virus evolution, thus consolidating this study model as an accelerated pathway to better understand SARS-CoV-2 adaptive traits and anticipate the emergence of variants of concern.