Ultrastructural modifications induced by an attenuated SARS-CoV-2-related pangolin coronavirus GX_P2V(short_3UTR) in Vero cells

Abstract

Abstract Background Positive-strand RNA viruses, such as SARS-CoV-2, manipulate host cell endomembranes to form viral replication organelles (vROs) for replication and protection. Pangolin coronavirus GX_P2V(short_3UTR), a cell-culture-adapted SARS-CoV-2-related coronavirus with a 104-nucleotide deletion in its 3´-terminus untranslated region, is highly attenuated in both in vitro and in vivo infection models. The mechanism underlying this attenuation remains unclear.Methods Vero cells were infected with GX_P2V(short_3UTR) and analyzed using transmission electron microscopy at various time points post-infection.Results Our study demonstrated that GX_P2V(short_3UTR) enters cells via endocytosis, leading to the formation of delayed vROs, composed of double-membrane vesicle, convoluted membranes, and double-membrane spherules. These structures were only observed after 12 hours post-infection. At 24 hours post-infection, vROs were readily identifiable, including the formation of annular lamellae due to nuclear pore stacking. By 48 hours post-infection, infected cells exhibited a characteristic feature of a complex reticulovesicular network. Similar to SARS-CoV-2, GX_P2V(short_3UTR) were found to bud within endoplasmic reticulum-Golgi compartments, accumulate in autophagy-like vesicles and multivesicular bodies, and egress via the lysosomal pathway. Notably, we did not observe any large vacuoles containing highly dense viral particles, which had been reported in SARS-CoV-2-infected cells.Conclusions Pangolin coronavirus GX_P2V(short_3UTR) undergoes a typical SARS-CoV-2-like life cycle in Vero cells. The delayed formation of vROs and the sparsely populated viral vacuoles in infected cells could contribute to the attenuation of pangolin coronavirus GX_P2V(short_3UTR).