Eight-amino-acid sequence at the N-terminus of SARS-CoV-2 nsp1 is involved in stabilizing viral genome replication.

Coronavirus disease 19 is caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) enveloped virus with a single-stranded positive-sense ribonucleic acid (RNA) genome. The CoV non-structural protein (nsp) 1 is a multifunctional protein that undergoes translation shutoff, messenger RNA (mRNA) cleavage, and RNA binding. The C-terminal region is involved in translational shutoff and RNA cleavage. The N-terminal region of SARS-CoV-2 nsp1 is highly conserved among isolated SARS-CoV-2 variants. However, the I-004 variant, isolated during the early SARS-CoV-2 pandemic, lost eight amino acids in the nsp1 region. In this study, we showed that the eight amino acids are important for viral replication in infected interferon-incompetent cells and that the recombinant virus that lost these amino acids had low pathogenicity in the lungs of hamster models. The loss of eight amino acids-induced mutations occurred in the 5' untranslated region (UTR), suggesting that nsp1 contributes to the stability of the viral genome during replication.

Establishment of a new reverse genetics system for respiratory syncytial virus under the control of RNA polymerase II.

A reverse genetics system for the respiratory syncytial virus (RSV), which causes acute respiratory illness, is an effective tool for understanding the pathogenicity of RSV. To date, a method dependent on T7 RNA polymerase is commonly used for RSV. Although this method is well established and recombinant RSV is well rescued from transfected cells, the requirement for artificial supply of T7 RNA polymerase limits its application. To overcome this, we established a reverse genetics system dependent on RNA polymerase II, which is more convenient for the recovery of recombinant viruses from various cell lines. First, we identified human cell lines with high transfection efficiency in which RSV can replicate effectively. Two human cell lines, Huh-7 and 293T, permitted the propagation of recombinant green fluorescent protein-expressing RSV. Our minigenome system revealed that efficient transcription and replication of RSV occurred in both Huh-7 and 293T cells. We then confirmed that recombinant green fluorescent protein-expressing RSV was rescued in both Huh-7 and 293T cells. Furthermore, the growth capability of viruses rescued from Huh-7 and 293T cells was similar to that of recombinant RSV rescued using the conventional method. Thus, we succeeded in establishing a new reverse genetics system for RSV that is dependent on RNA polymerase II.