In a recent study posted to bioRxiv*, researchers evaluated the early replication kinetics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

Background

SARS-CoV-2 replication begins with cellular entry and release of viral RNA into the cytoplasm. The positive-stranded genomic RNA (gRNA) serves as a template for translating viral proteins and generating negative-stranded replication intermediates. This negative-stranded RNA is, in turn, a template for synthesizing additional positive-stranded RNA and shorter sub-genomic RNAs (sgRNAs).

SARS-CoV-2 modifies the host endoplasmic reticulum (ER) membrane to synthesize replication organelles (ROs), double-membrane vesicles enclosing viral RNAs. Several questions related to early SARS-CoV-2 replication remain unanswered, including the time required to complete viral RNA translation, initiate the replication, and synthesize gRNAs and sgRNAs.

The study and findings

The present study performed a time course analysis to evaluate SARS-CoV-2 replication kinetics. First, the researchers employed a single-molecule RNA fluorescence in situ hybridization (smRNA-FISH) technique to detect viral gRNA and sgRNA at a single-cell and -molecule level. As such, 40 fluorescent-labeled anti-sense oligonucleotides were developed based on spike gene sequence.

The authors determined that oligonucleotides were SARS-CoV-2-specific. Next, Vero E6 cells were infected with < 0.5 multiplicity of infection (MOI) of SARS-CoV-2 and subjected to smRNA-FISH with an sgRNA-spike (P1) probe. Cells were independently analyzed, as a positive control, by immunofluorescence to detect viral nucleocapsid (N) expression using α-N antibodies.

The probes and α-N antibodies respectively detected viral RNA and N protein in infected cells but not in non-infected cells. In addition, Vero E6 cells were infected with vesicular stomatitis virus (VSV) containing a codon-optimized sequence of spike open-reading frame (ORF) as a negative control. The P1 probe failed to detect VSV-spike in infected cells, implying that probes were specific to wildtype SARS-CoV-2 gene sequence.

The SARS-CoV-2-infected cells were subjected to smRNA-FISH and visualized at 20x magnification using high-speed high-resolution scanning fluorescence microscopy (HSHRS-FM). The authors observed small plaques characterized by probe-positive fluorescent clusters of cells around dead cells, indicating that the probe was selective for viral RNA in live infected cells.

Further, the researchers performed a time course analysis beginning at 0.5 hours post-infection (hpi). Cells were infected with 0.5 MOI of SARS-CoV-2 to preclude multiple viral particles from entering the same cell. Infected cells were visualized using HSHRS-FM at 20x magnification to quantify the proportion of infected cells. Approximately 7% of cells were infected by 6 hpi, 23% by 12 hpi, and more than 50% by 24 hpi, suggesting a delayed replication in the beginning.

Moreover, these cells were visualized using a fluorescence microscope with a wide-angle lens at higher magnification (60x) and 1.4 numerical aperture oil objective, allowing the detection of single molecules of SARS-CoV-2 RNA in infected cells. At 0.5 and 2 hpi, single molecules of RNA were visible in infected cells but not in non-infected cells, which were not identified at lower magnifications at these time points.

At 6 hpi, spots representing single molecules and larger patches of multiple RNAs were evident. By 12 hpi, SARS-CoV-2 RNA filled the entire cytoplasm. Next, the authors evaluated the ability of another probe (P2) derived from non-structural protein 12 (nsp12) gene sequences to detect gRNA alone since P1 could detect both gRNA and sgRNA.

The time course analysis was repeated using P1 and P2 probes together. At higher magnification (76x), most cells at 2 hpi and 3 hpi showed single RNA spots positive for P1 and P2 probes. At 3 hpi, the single RNA spot in some cells appeared to harbor both gRNAs and sgRNAs.

At the same time point, the authors observed a central region positive for P1 and P2 probes, encircled by P1-positive spots, implying that the center of the cluster likely harbored gRNA and the periphery contained sgRNA. This suggested that RNA clusters were SARS-CoV-2-induced ROs at 2 and 3 hpi.

Further microscopic examinations suggested that individual positive spots at < 2 hpi were likely single gRNAs in each cell. At later time points, the larger spots represented ROs with gRNA and sgRNA in the center and the periphery, respectively. This was followed by the formation of multiple daughter ROs at later time points.

Furthermore, the authors observed substantial heterogeneity in RNA appearance in infected cells. Most infected cells initially had one or few ROs per cell, which increased with time and filled the entire cytoplasm. The images obtained with the P2 probe were used to quantitate the progression of RNA replication since the probe hybridizes to gRNA alone. Replication stages were arbitrarily defined from stages 1 through 4.

Stage 1 was defined as one to five RO speckles per cell. Stages 2 and 3 were intermediates with the progressive increase in ROs. Stage 4 was the late stage, with ROs filling the entire cytoplasm. At 4 hpi, around 35% of infected cells showed SARS-CoV-2 replication at stages 1 – 3, with 2.5% of cells at stage 4. The proportion of cells with stage 4 viral replication increased over time, with 51% of cells at 6 hpi.

Conclusions

In summary, the study provided insights into SARS-CoV-2 replication kinetics at early time points at a single-cell and -molecule resolution and illustrated how ROs form and progress from a single RNA.

The rate of RNA replication was heterogeneous across cells, as the nature and the number of ROs were different in cells. The findings revealed that single gRNAs could be visualized within 30 minutes after infection, and ROs can appear within 2 hpi, rapidly progressing and filling the cytoplasm at later time points.

*Important notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information