Single-cell RNA sequencing of lungs of SARS-CoV-2–infected African green monkeys reveals virus replication and host response dynamics in vivo.

Summary: Detailed knowledge about the dynamics of SARS-CoV-2 infection is important for unraveling the viral and host factors that contribute to COVID-19 pathogenesis. Old-World nonhuman primates recapitulate mild-moderate COVID-19 cases, thereby serving as important pathogenesis models. We compared African green monkeys inoculated with SARS-CoV-2 or inactivated virus to study the dynamics of virus replication throughout the respiratory tract. RNA sequencing of single cells from the lungs and mediastinal lymph nodes allowed a high-resolution analysis of virus replication and host responses over time. Viral replication was mainly localized to the lower respiratory tract, with evidence of replication in the pneumocytes. Macrophages were found to play a role in initiating a pro-inflammatory state in the lungs, while also interacting with infected pneumocytes. Our dataset provides a detailed view of changes in host and virus replication dynamics over the course of mild COVID-19 and serves as a valuable resource to identify therapeutic targets.

Single-cell RNA sequencing has soared in popularity in recent years. The ability to deeply profile the states of individual cells during the course of disease or infection has helped to expand our knowledge of coordinated responses. However, significant challenges arise when performing this analysis in high containment settings such as biosafety level 3 (BSL-3), BSL-3+ and BSL-4. Working in containment is necessary for many important pathogens, such as Ebola virus, Marburg virus, Lassa virus, Nipah and Hendra viruses. Since standard operating procedures (SOPs) for inactivation are extensive and may compromise sample integrity, we tested whether the removal of single-cell sequencing libraries from containment laboratories using existing inactivation protocols for nucleic acid extraction (Trizol, RLT buffer, or AVL buffer) was feasible. We have demonstrated that the inactivation does not affect sample quality and can work with existing methods for inactivation.

The unprecedented research effort associated with the emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) included several extensive proteomic studies that identified host proteins that interact with individual viral gene products. However, in most cases, the consequences of those virus–host interactions for virus replication were not experimentally pursued, which is a necessary step in determining whether the interactions represent pro- or anti-viral events. One putative interaction commonly identified in multiple studies was between the host adaptor protein complex 3 (AP-3) subunit B1 (AP3B1) and the SARS-CoV-2 envelope protein (E). AP3B1 is one subunit of AP-3 required for the biogenesis of lysosomal-related organelles (LROs), and its function impacts important disease processes including inflammation and vascular health. Thus, interactions between AP3B1 and SARS-CoV-2 might influence the clinical outcomes of infection. To determine if AP3B1 has a role in the SARS-CoV-2 replication cycle, we first confirmed the interaction in virus-infected cells using immunoprecipitation (IP) and immunofluorescence assays (IFA). AP3B1 is required by multiple viruses to aid in the replication cycle and therefore may be a therapeutic target. However, we found that the overexpression of AP3B1 suppressed SARS-CoV-2 replication, whereas the siRNA-mediated depletion of AP3B1 increased the release of infectious virus, suggesting an antiviral role for AP3B1. Together, our findings suggest that AP3B1 is an intrinsic barrier to SARS-CoV-2 replication through interactions with the viral E protein. Our work justifies further investigations of LRO trafficking in SARS-CoV-2 target cells and their role in viral pathogenesis.

Tripartite motif-containing protein 5α (TRIM5α) functions as a cellular antiviral restriction factor with exquisite specificity towards the capsid lattices of retroviruses. The relative avidity of TRIM5α binding to retrovirus capsids directly impacts primate species susceptibility to infection, but the antiviral role of TRIM5α is thought limited to retroviruses. In contrast to this current understanding, here we show that both human and rhesus TRIM5α possess potent antiviral function against specific flaviviruses through interaction with the viral protease (NS2B/3) to inhibit virus replication. Importantly, TRIM5α was essential for the antiviral function of IFN-I against sensitive flaviviruses in human cells. However, TRIM5α was ineffective against mosquito-borne flaviviruses (yellow fever, dengue, and Zika viruses) that establish transmission cycles in humans following emergence from non-human primates. Thus, TRIM5α is revealed to possess remarkable plasticity in recognition of diverse virus families, with potential to influence human susceptibility to emerging flaviviruses of global concern.