Live-cell, single-molecule imaging resolves the dual nature of mRNA silencing by SARS-CoV-2 Nsp1
SARS-CoV-2 nonstructural protein 1 (Nsp1) is a conserved viral factor that potently silences host transcripts by binding ribosomes. Yet how it acts at the level of individual mRNAs remains unclear, with competing models arguing that Nsp1 blocks translation initiation, stalls ribosome elongation, cleaves mRNA, or does all three. Here, we use Nascent Chain Tracking to quantify in living cells how Nsp1 reshapes mRNA abundance and translation dynamics with single-molecule resolution. Using reporter transcripts that vary in UTR architecture, initiation mode, and open reading frame length, we find that Nsp1 mainly silences our reporter transcripts by reducing their numbers rather than inhibiting initiation. Two routes to Nsp1-mediated mRNA loss are identified, both requiring cap-dependent initiation: an early route acting during or shortly after initiation and a later route associated with downstream translation that depends in part on ZNF598. Together, these two mechanisms make Nsp1 an efficient and robust suppressor of host gene expression. We further find that reporters containing the SARS-CoV-2 UTRs remain susceptible to Nsp1-mediated mRNA loss, but the 5' and 3' UTRs cooperate to increase translation output. Neither viral UTR alone increased translation beyond the host reporter, whereas combining both UTRs substantially enhanced translation in the absence of Nsp1. Together, our results reveal that Nsp1 uses dual, cap-dependent routes to promote mRNA loss across the translation cycle, while the SARS-CoV-2 UTRs provide a cooperative translational advantage rather than robust protection from Nsp1.