Across the world, scientists are working frantically to develop effective therapies and vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection.
A new study published on the preprint server bioRxiv* in October 2020 reports the discovery of important viral genomic features that are crucial to its replication in the human airway cells, and its enhancing mutations which increase the rate of replication and determine its target cells.
This discovery suggests that SARS-CoV-2's small envelope (E) glycoprotein has important specific functions in human respiratory cells, as well as in vivo, and further indicates that residues at positions 5 and 6 play key roles in E activities in human cells.
Study: Distinct Phenotypes of SARS-CoV-2 Isolates Reveal Viral Traits Critical for Replication in Primary Human Respiratory Cells. Image Credit: Orpheus FX / Shutterstock
Viral Spike Protein and Cell Entry
The virus gains host cell entry via its spike glycoprotein. This surface protein binds the host cell angiotensin-converting enzyme (ACE2) as its receptor for cell entry and membrane fusion. This latter process is vital for its internalization and depends on a host protease's function, which cleaves the spike into its two subunits following ACE2 attachment.
The furin cleavage site at the two subunits interface allows furin protease to prime the spike protein beforehand, thus enhancing the efficiency of viral entry via fusion.
Other viral proteins, such as the membrane, nucleocapsid, and envelope proteins, have their own structural and functional significance. The virus's entry into the host cell triggers viral genomic RNA translation, by which the viral nonstructural proteins are produced, namely, nsp 1-16.
Viral Replicase-Transcriptase Complex
Among these are the essential components of the viral replicase-transcriptase complex, as well as other viral factors that enable immune evasion. The coronavirus replication complex has sophisticated proofreader genes that stabilize the genome against errors, but many mutations have been published already, despite this feature. Some are associated with replication or infectivity benefits, such as the D614G mutation in the spike protein, which has caused the containing strains to become dominant rapidly.
Identifying Mutations via Passaging
The current study focused on 14 strains of SARS-CoV-2 isolated from patients infected in the pandemic's first Swiss wave. These were from the different European clades of the period (March to May 2020), and each had its own genetic departures. With a limited number of passages in Vero cells and mature primary human bronchial epithelial cells, rare additional mutations also appeared.
The researchers first isolated the virus from samples taken from patients in the first wave, mostly from those with a low cycle threshold. The 62% isolation rate from 21 samples with a Ct below 25, vs. the 1 isolate from 46 samples with Ct above 25 agree with earlier figures of the rate of isolation of infectious virus from such samples.
They found that the isolates represented the major European clades of the period. Of the mutations induced by passaging, most were in S, E, or the nonstructural proteins, with different mutations emerging despite similar passaging conditions.
In 6/14 isolates, E residues were altered during growth in Vero cells, but only 2/14 showed furin cleavage site deletion on passaging. This number of passages here was low, and more substitutions would probably have occurred otherwise.
The researchers characterized these isolates as representing the early strains, concerning their adaptation to and replication in the human host. They found that all isolates grew well, with some differences in plaque size. The differences intensified with the analysis of multiple cycles of growth.
This showed that most viruses produced final titers of 106 or 107 plaque-forming units (PFUs) in 72 hours, but some underwent attenuation to 10- or 100-times lower titers. Some also showed a slower rate of growth.
In a culture of primary human bronchial epithelial cells, they found that virus inoculation produced different growth rates over 72 hours. Of the 14 isolates, 5 grew to over 107 PFU, but two were strongly attenuated to up to 100-fold lower titers. Two did not replicate at all. Five of them showed intermediate growth, at 10-fold lower rates.
Factors Determining Host-Cell Specific Replication Efficiency
The researchers also discovered that while two isolates showed equally efficient replication in either Vero cells or bronchial cells, three preferred the latter, showing very attenuated growth in Vero cells. Each of the three showed unique combined substituent residues in ORF3a and nsp2. The authors suggest this could indicate a link between these changes and the specificity of the host cell.
Another three replicated with very high efficiency in the former but not the latter, all having gene variants at position 5 or 6 of the E protein. One strain grew very slowly in the latter but moderately well in the Vero cells, probably because over 80% had a deletion of the furin cleavage site.
Therefore, the virus is very specific with respect to its hosts, with G614 showing greater replication efficiency than D614. Replication data shows the link of several amino acids with disease phenotypes, indicating that these residues carried viral traits required for efficient replication.
Global Prevalence of Functional Mutations
The study shows that variants in ORF3a and nsp2 occur in a fifth of the isolates even after passaging, agreeing with worldwide estimates. The 5/6 position variants in the E protein and the furin cleavage site deletion in the spike protein were absent in worldwide samples but increased on passaging in Vero cells. In other words, isolate variants that increased the replication efficiency to one or other of the culture hosts were positively selected for when passaged in Vero cells.
Comparative Passaging Shows Functional Traits
The furin cleavage site is found in samples from all over the world but can be deleted during Vero cell passaging. The researchers found that passaging four different replicates with different frequencies of this site in both types of cultures caused one strain to replicate in both, namely, the strain which showed the deletion in ~24% of instances. The deletion frequency typically went up from ~20% to 50% to 80% in Vero cell passaging. The deletion was lost when passaged in bronchial epithelium, every recovered sequence showing the original cleavage site.
The deletion site occurred in over 81% with another strain, but the virus showed efficient replication in Vero cells and kept the furin cleavage site deletion at the same level. In the other cell type, it grew very weakly. The latter, when sequenced, showed severe attenuation, and the deletion was lost.
Implications and Future Directions
This study shows the furin cleavage site to be vital in the bronchial cells. This comparative study showed the key role played by the furin cleavage site, and envelope protein positions 5/6 as deciding the infectivity and efficient replication of the virus in human respiratory cells.
Moreover, the G614 mutation was linked to higher replication efficiency compared to D614. Previous pseudotypes bearing the spike protein of SARS-CoV-2 showed greater infectivity compared to the original variant. This is supported by reverse genetics studies. The authors say this helps understand how the D614G mutation drove the rapid spread of the virus.
Thirdly, natural ORF3a, and nsp2 mutations were linked to cell tropism, with these strains preferring human bronchial cells for efficient replication while the rest showed poor replication in Vero cells.
These variants occur in a fifth of all worldwide sequences, and more research is required to understand the functions of the nsp2. It may be involved in many trafficking and translation activities of the virus in the host cell. ORF3a has an ion channel structure that promotes apoptosis and inflammation.
One common ORF3a variant has come about after human introduction since early isolates lack it. The presence of this mutation alters protein sequences in two possible reading frame products. More studies will show the functional connection between variants and the extent of tissue tropism shown by these viruses.
The study also shows amino acid substituents in E emerging from passaging the original samples through Vero cells. Mutations at E positions 5/6 (V5G/A or S6W) correlate with efficient replication only in Vero cells, being highly weakened in bronchial cells.
Prior work on SARS-CoV indicates E-mutation variants are attenuated in human cells and in vivo and can be used to construct vaccine strains. The E protein is probably central to viral replication within human respiratory cells, especially these two positions.
The last point relates to the need to monitor stock preparations given the increased frequency of furin cleavage site deletions on passaging the virus through Vero cells. Otherwise, this could lead to false conclusions when passaged strains are compared for phenotypes.
The authors comment, “Our work identifies both key features in the SARS-CoV-2 genome that are essential for its growth in the human respiratory epithelium, as well as variants derived during human circulation that determine efficient replication and cell tropism.”
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.