Rapid assessment of SARS-CoV-2 evolved variants using virus-like particles

Abstract

Newly evolved SARS-CoV-2 variants are driving ongoing outbreaks of COVID-19 around the world. Efforts to determine why these viral variants have improved fitness are limited to mutations in the viral spike (S) protein and viral entry steps using non-SARS-CoV-2 viral particles engineered to display S. Here we show that SARS-CoV-2 virus-like particles can package and deliver exogenous transcripts, enabling analysis of mutations within all structural proteins and rapid dissection of multiple steps in the viral life cycle. Identification of an RNA packaging sequence was critical for engineered transcripts to assemble together with SARS-CoV-2 structural proteins S, nucleocapsid (N), membrane (M) and envelope (E) into non-replicative SARS-CoV-2 virus-like particles (SC2-VLPs) that deliver these transcripts to ACE2- and TMPRSS2-expressing cells. Using SC2-VLPs, we tested the effect of 30 individual mutations within the S and N proteins on particle assembly and entry. While S mutations unexpectedly did not affect these steps, SC2-VLPs bearing any one of four N mutations found universally in more-transmissible viral variants (P199L, S202R, R203M and R203K) showed increased particle production and up to 10-fold more reporter transcript expression in receiver cells. Our study provides a platform for rapid testing of viral variants outside a biosafety level 3 setting and identifies viral N mutations and viral particle assembly as mechanisms to explain the increased spread of current viral variants, including Delta (N:R203M).

Stimulation of dysregulated IFN-β responses by aberrant SARS-CoV-2 small viral RNAs

“…The SARS-CoV-2 packaging signal generator is estimated to reside within an internal region encompassing nsp15 and nsp16 (Syed et al, 2021), indicating that 5′ end svRNAs are unlikely to be packaged into released virions as defective interfering particles. Instead, exosomes are recognized as a key vehicle to transfer various genetic materials including small RNAs including miRNAs (Valadi et al, 2007).…”

SARS-CoV-2 Variants Associated with Vaccine Breakthrough in the Delaware Valley through Summer 2021

“…In contrast, several substitutions were estimated to be less likely to appear in vaccine breakthrough samples, including nucleocapsid mutation P199L (detected in B.1.2, B.1.526, and B.1.596; odds ratio of 0.5; 95% credible interval of 0.24–0.88), and spike mutation D253G (detected in B.1.526; odds ratio of 0.5; 95% credible interval of 0.23–0.89). The spike D253G substitution has been implicated in MAb evasion ( 45 ), and nucleocapsid P199L may alter the assembly of SARS-CoV-2 VLPs ( 46 ), functions possibly linked to their depletion in vaccine breakthrough.…”

Genomic and Virological Characterization of SARS-CoV-2 Variants in a Subset of Unvaccinated and Vaccinated U.S. Military Personnel

“…Most research has focused on analyzing mutations in the spike (S) protein to better understand viral fitness of emerging SARS-CoV-2 variants. However, a recent study found that four mutations in the nucleocapsid (N) protein, which is an important protein for viral replication, improve the viruses' ability to form infectious particles (29). These four mutations in N, which are universally found in more transmissible SARS-CoV-2 variants, increased mRNA delivery and expression and two of the mutations (S202R and R203M) produced more infectious virus (29).…”

“…However, a recent study found that four mutations in the nucleocapsid (N) protein, which is an important protein for viral replication, improve the viruses' ability to form infectious particles (29). These four mutations in N, which are universally found in more transmissible SARS-CoV-2 variants, increased mRNA delivery and expression and two of the mutations (S202R and R203M) produced more infectious virus (29). Interestingly, the Delta variant contains the R203M mutation and the Omicron variant contains the R203K mutation, which provide a possible explanation for the increased fitness of these variants.…”