ABSTRACT
The relationship between pangolin-CoV and SARS-CoV-2 has been a subject of debate. Further evidence of a special relationship between the two viruses can be found by the fact that all known COVID-19 viruses have abnormally hard outer shell (low M disorder; i.e., low content of intrinsically disordered residues in the membrane (M) protein) that so far was found in CoVs associated with burrowing animals, such as rabbits and pangolins, in which transmission involves virus remaining in buried feces for a long time. While a hard outer shell is necessary for viral survival, a harder inner shell could also help. For this reason, the N disorder range of pangolin-CoVs, not bat-CoVs, more closely matches that of SARS-CoV-2 especially when Omicron is included. The low N disorder (i.e., low content of intrinsically disordered residues in the nucleocapsid (N) protein), first observed in pangolin-CoV-2017 amd later in Omicron, is associated with attenuation according to the Shell-Disorder-Model. Our experimental study revealed that pangolin-CoV-2017 and SARS-CoV-2 Omicron (XBB.1.16 subvariant) show similar attenuations with respect to viral growth and plaque formation. Subtle differences have been observed that are consistent with disorder-centric computational analysis.
Subject(s)
Oculocerebrorenal Syndrome , Combat Disorders , COVID-19ABSTRACT
SARS-CoV-2-related pangolin coronavirus GX_P2V(short_3UTR) is highly attenuated, but can cause mortality in a specifically designed human ACE2-transgenic mouse model, making it an invaluable surrogate model for evaluating the efficacy of drugs and vaccines against SARS-CoV-2.
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
We previously reported that a SARS-CoV-2 related pangolin coronavirus GX_P2V can induce neutralizing antibodies against SARS-CoV-2 in a golden hamster model. The two viruses GX_P2V and SARS-CoV-2 have genomes with high homology and both use ACE2 as their receptor, but have distinct receptor-binding domains (RBDs) of their spike proteins. The shared neutralizing epitopes of these two viruses are unknown. Here we describe a novel method of soluble expression of GX_P2V RBD in E. coli by tagging RBD with a carboxy-terminal fragment of the SADS coronavirus nucleocapsid protein. The recombinant GX_P2V RBD have human ACE2-binding activities. Mice immunized with this recombinant RBD produced significant titers of neutralizing antibodies against GX_P2V pseudoviruses, not SARS-CoV-2 pseudoviruses. Our data suggest that the GX_P2V RBD has none or limited neutralizing epitopes against SARS-CoV-2, thus neutralizing antibodies against SARS-CoV-2 from GX_P2V infected golden hamsters is likely RBD-independent.
Subject(s)
Coronavirus InfectionsABSTRACT
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.
ABSTRACT
Since the infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in several somatic cells, little is known about the infection of SASRS-CoV-2 and its related pangolin coronavirus (GX_P2V). Here we present for the first time that SARS-CoV-2 pseudovirus and GX_P2V could infect lung progenitor and even anterior foregut endoderm cells causing these cells death, which differentiated from human embryonic stem cells (hESCs). The infection and replication of SARS-CoV-2 and GX_P2V were inhibited when treated with whey protein of breastmilk and Remdesivir, confirming that these two viruses could infect lung progenitor and even anterior foregut endoderm. Moreover, we found that SARS-CoV-2 pseudovirus could infect endoderm and ectoderm. We found that whey protein blocked SARS-CoV-2 infecting these cells. In line with the SARS-CoV-2 results, GX_P2V could also infected endoderm and ectoderm, and also was inhibited by Remdesivir treatment. Although expressing coronavirus related receptor such as ACE2 and TMPRSS2, mesoderm cells are not permissive for SARS-CoV-2 and GX_P2V infection, which needed further to study the mechanisms. Interestingly, we also found that hESCs, which also express ACE2 and TMPRSS2 markers, are permissive for GX_P2V but not SARS-CoV-2 pseudovirus infection and replication, indicating the widespread cell types for GX_P2V infection. Heparin treatment blocked efficiently viral infection. These results provided insight that these stem cells maybe provided a stable repository of coronavirus function or genome. The potential consequence of SARS-CoV-2 and animal coronavirus such as GX_P2V infection in hESCs, germ layer and induced progenitors should be closely monitored.