No evidence of human genome integration of SARS-CoV-2 found by long-read DNA sequencing

Abstract

A recent study proposed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) hijacks the LINE-1 (L1) retrotransposition machinery to integrate into the DNA of infected cells. If confirmed, this finding could have significant clinical implications. Here, we applied deep (>50x) long-read Oxford Nanopore Technologies (ONT) sequencing to HEK293T cells infected with SARS-CoV-2, and did not find any evidence of the virus existing as DNA. By examining ONT data from separate HEK293T cultivars, we resolved the complete sequences of 78 L1 insertions arising in vitro in the absence of L1 overexpression systems. ONT sequencing applied to hepatitis B virus (HBV) positive liver cancer tissues located a single HBV insertion. These experiments demonstrate reliable resolution of retrotransposon and exogenous virus insertions via ONT sequencing. That we found no evidence of SARS-CoV-2 integration suggests such events in vivo are highly unlikely to drive later oncogenesis or explain post-recovery detection of the virus.

Somatic retrotransposition in the developing rhesus macaque brain

“…1B) and disproportionately few (23/61, 37.7%) of these were sense oriented to their host gene. Given modest genome-wide L1 integration site preferences, which mainly reflect the underlying distribution of AT-rich sequences, these patterns were likely dominated by post-integration selection, and are concordant with prior results obtained by human analyses (Sultana et al 2019;Smits et al 2021;Flasch et al 2019;Ewing and Kazazian 2010;Attig et al 2018;Smit 1999). Consistent with L1-mediated retrotransposition in humans and other mammals (Jurka 1997;Tang and Liang 2019;Moran et al 1996;Richardson et al 2017;Ewing et al 2020;Smits et al 2021), the L1 and Alu insertions generated TSDs with a median length of 15bp (Fig.…”

Retrotransposon instability dominates the acquired mutation landscape of mouse induced pluripotent stem cells

“…In principle, a single long read can completely resolve a de novo TE insertion present in a heterogeneous cell population, as well as the accompanying TPRT hallmarks 76 . Long-read sequencing can also discover TE insertions in repetitive genomic regions refractory to mapping with short-read approaches 35, 77, 78 .…”

Inflammation, immunity, and antigen persistence in post-acute sequelae of SARS-CoV-2 infection

“…A provocative hypothesis proposed by recent reports is the possibility that SARS-CoV-2 is capable of partial genomic integration, resulting in a somatic source of persistent antigen and sub-genomic RNA [30] . This speculation has been intensely disputed 31 , 32 , 33 , and will demand further examination as a potential source of persistent viral antigen that may be capable of driving protracted inflammation.…”