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).
ABSTRACT
SARS-CoV-2 related coronaviruses (SARS-CoV-2r) from Guangdong and Guangxi pangolins have been implicated in the emergence of SARS-CoV-2 and future pandemics. We previously reported the culture of a SARS-CoV-2r GX_P2V from Guangxi pangolins. Here we report the GX_P2V isolate rapidly adapted to Vero cells by acquiring two genomic mutations: an alanine to valine substitution in the nucleoprotein and a 104-nucleotide deletion in the hypervariable region (HVR) of the 3'-terminus untranslated region (3'-UTR). We further report the characterization of the GX_P2V variant in in vitro and in vivo infection models. In cultured Vero and BGM cells, the GX_P2V variant produced minimal cell damage and small plaques. The GX_P2V variant infected golden hamsters and BALB/c mice but was highly attenuated. Golden hamsters infected intranasally had a short duration of productive infection. These productive infections induced neutralizing antibodies against pseudoviruses of GX_P2V and SARS-CoV-2. Collectively, our data show that the GX_P2V variant is highly attenuated in in vitro and in vivo infection models. Attenuation of the variant is likely due to the 104-nt deletion in the HVR in the 3'-UTR. This study furthers our understanding of pangolin coronaviruses pathogenesis and provides novel insights for the design of live attenuated vaccines against SARS-CoV-2.