Before late 2019, six coronaviruses were known to infect humans. The emergence of SARS-CoV-2, closely related to SARS-CoV-1, added a seventh. The coronavirus spike (S) protein, crucial for infection, has two domains: S1 (receptor binding) and S2 (membrane fusion). SARS-CoV-2 uses angiotensin-converting enzyme 2 (ACE2) as its receptor. The S1–S2 junction is processed by furin, unlike SARS-CoV-1 which uses TMPRSS2. Analysis of SARS-CoV-2 isolates revealed increased genetic variation, notably the D614G mutation in the S1 domain, associated with enhanced viral infectivity. This study investigates the mechanism behind this enhanced infectivity, focusing on the D614G mutation's impact on S protein properties and viral entry.

The literature review section highlights previous research on coronaviruses, SARS-CoV-1 and SARS-CoV-2, focusing on the structure and function of the spike protein, its interaction with ACE2, and the role of furin cleavage in viral entry. It also cites studies that identified the D614G mutation and its correlation with increased viral infectivity. Several studies are referenced to support these points, including research on the structure of the SARS-CoV-2 spike protein and its interaction with ACE2, and studies showing the enhanced infectivity of the D614G variant.

The study utilized murine leukemia virus (MLV)-based pseudoviruses (PVs) expressing enhanced green fluorescent protein (eGFP) and pseudotyped with SARS-CoV-2 S protein (either D614 or G614). HEK293T cells expressing human ACE2 were used for entry assays. The impact of TMPRSS2 expression on entry was also assessed. Virus-like particles (VLPs) containing native SARS-CoV-2 proteins (M, N, E, and S) were also produced and analyzed. Western blotting, flow cytometry, and qPCR were employed to quantify S protein levels, S1 shedding, and PV entry efficiency. ACE2 binding assays and neutralization assays using human immune plasma were conducted to assess the impact of D614G on ACE2 affinity and neutralization sensitivity. Statistical analysis using one-way ANOVA, two-sided multiple comparison tests, and two-sided unpaired Student's t-tests were performed.

The study's key findings demonstrate that the D614G mutation enhances pseudovirus infectivity. This is correlated with decreased S1 shedding and increased S protein density in the virion, observed in both pseudoviruses and virus-like particles. Quantitative analysis showed approximately 4.17 times more total S protein (S1+S2) in G614 pseudoviruses compared to D614 pseudoviruses. The S1:S2 ratio was also significantly higher in G614. Importantly, the D614G mutation did not affect S protein affinity for ACE2 or increase pseudovirus resistance to neutralization. The results consistently showed increased infectivity due to enhanced S protein incorporation, not increased binding affinity or neutralization resistance.

The results indicate that the D614G mutation's enhanced infectivity is primarily due to the increased incorporation of functional spike proteins into the virion, leading to higher viral density. This increased density likely contributes to more efficient transmission. The observation that D614G doesn't enhance ACE2 binding or neutralize resistance suggests other factors contribute to the phenotypic changes. The study suggests the possibility of investigating the impact of the D614G mutation on disease course. The acquisition of a furin-cleavage site during SARS-CoV-2's evolution is also discussed, contrasting it with SARS-CoV-1.

This study demonstrates that the D614G mutation in SARS-CoV-2 enhances viral infectivity by increasing the density of functional spike proteins on the virion surface, without altering ACE2 binding or neutralization sensitivity. This finding has significant implications for understanding SARS-CoV-2 transmission and pathogenesis. Further research could focus on investigating the specific mechanisms underlying the increased S protein incorporation and the impact of D614G on viral fitness in different host contexts.

The study primarily used in vitro assays, which may not fully reflect the complexities of in vivo infection. The limited sample size in certain aspects of the study needs to be considered. Additionally, while the study suggests a potential link between D614G and increased transmission, it does not definitively prove causation. Further in vivo studies are necessary to fully validate the findings.