The efficiency of virus internalization into target cells is a major determinant of infectivity. SARS-CoV-2 internalization occurs via S-protein-mediated cell binding followed either by direct fusion with the plasma membrane or endocytosis and subsequent fusion with the endosomal membrane. Despite the crucial role of virus internalization, the precise kinetics of the processes involved remains elusive. We developed a pipeline, which combines live-cell microscopy and advanced image analysis, for measuring the rates of multiple internalization-associated molecular events of single SARS-CoV-2-virus-like particles (VLPs), including endosome ingression and pH change. Our live-cell imaging experiments demonstrate that only a few minutes after binding to the plasma membrane, VLPs ingress into RAP5-negative endosomes via dynamin-dependent scission. Less than two minutes later, VLP speed increases in parallel with a pH drop below 5, yet these two events are not interrelated. By co-imaging fluorescently labeled nucleocapsid proteins, we show that nucleocapsid release occurs with similar kinetics to VLP acidification. Neither Omicron mutations nor abrogation of the S protein polybasic cleavage site affected the rate of VLP internalization, indicating that they do not confer any significant advantages or disadvantages during this process. Finally, we observe that VLP internalization occurs two to three times faster in VeroE6 than in A549 cells, which may contribute to the greater susceptibility of the former cell line to SARS-CoV-2 infection. Taken together, our precise measurements of the kinetics of VLP internalization-associated processes shed light on their contribution to the effectiveness of SARS-CoV-2 propagation in cells.

HIV is a lentivirus characterized by its cone shaped mature core. Visualization and structural examination of HIV requires the purification of virions to high concentrations. The yield and integrity of these virions are crucial for ensuring a uniform representation of all viral particles in subsequent analyses. In this study, we present a method for the purification of HIV virions which minimizes the forces applied to virions while maximizing the efficiency of collection. This method, which relies on virion sedimentation simulations, allows us to capture between 1000 and 5000 HIV virions released from individual HEK293 cells after transfection with the NL4.3 HIV backbone. We utilized this approach to investigate HIV core formation from several constructs: pNL4-3(RT:D₁₈₅A&D₁₈₆A) with an inactive reverse transcriptase, NL4.3(IN: V₁₆₅A&R₁₆₆A) with a type-II integrase mutation, and NL4.3(Ψ: Δ(105–278)&Δ(301–332)) featuring an edited Ψ packaging signal. Notably, virions from NL4.3(Ψ: Δ(105–278)&Δ(301–332)) displayed a mixed population, comprising immature virions, empty cores, and cores with detectable internal density. Conversely, virions derived from NL4.3(IN: V₁₆₅A&R₁₆₆A) exhibited a type II integrase mutant phenotype characterized by empty cores and RNP density localized around the cores, consistent with previous studies. In contrast, virions released from pNL4-3(RT:D₁₈₅A&D₁₈₆A) displayed mature cores containing detectable RNP density. We suggest that the sedimentation simulations developed in this study can facilitate the characterization of enveloped viruses.