The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has driven extensive research into the molecular mechanisms underlying viral pathogenesis. The nucleocapsid (N) protein has emerged as a key player due to its roles in viral RNA packaging, replication, and modulation of host cell responses.1, 2 The N protein encompasses distinct functional domains, notably an N-terminal RNA-binding domain and a C-terminal dimerization domain, both pivotal for viral RNA encapsidation and assembly.3 Recent investigations have unveiled the dynamic interplay between the N protein and various host factors, shedding light on its involvement in modulating host immune responses and cellular processes.4, 5, 6, 7, 8, 9, 10

The N protein interacts with specific host and viral molecules during the viral life cycle. It has been shown that N forms the ribonucleoprotein (RNP) complex,11 the core of the virus,12 through liquid–liquid phase separation (LLPS), which is stimulated by RNA. This process facilitates the recruitment of viral proteins and interaction with host ribonucleoproteins and potentially influences viral replication and packaging.13, 14 Additionally, the N protein plays a crucial role in the viral life cycle by impairing the host stress response through binding host mRNAs and attenuating stress granules (SGs).15, 16, 17, 18, 19 This involves targeting the SG-nucleating component G3BP1, thereby preventing protein kinase R (PKR)-mediated activation of the integrated stress response and innate immunity.20

In addition to the canonical genomic and nine subgenomic RNAs, SARS-CoV-2 can also generate noncanonical viral RNA transcripts in abundance.21, 22, 23, 24 The noncanonical viral RNA transcripts are produced by recombination of the coronaviral genome, leading to the synthesis of RNA transcripts with various in-frame or out-of-frame open reading frames (ORFs).21, 25 Intriguingly, among the noncanonical viral RNA transcripts, some ORFs contain portions of the N protein, suggesting the presence of N-fusion proteins.21 Indeed, the capability of the noncanonical viral RNA transcripts to encode N-fusion proteins has been identified in other coronaviruses, such as the bovine coronavirus.25, 26 However, the functional implications of these N-fusion proteins in cellular processes remain poorly understood.

Here, we identified a novel SARS-CoV-2 protein, NFP, formed by the fusion of NSP1 at the N-terminal domain (NTD) junction of the N protein. While NFP shares some domains with the N protein, it has distinct structural features that require further study. This research, using biochemical assays, mass spectrometry, and mutagenesis, focuses on comparing NFP to the N protein, particularly its effects on SG inhibition, protein interactions, and cell cycle regulation. Our findings offer novel insights into the interplay between viral proteins and host cellular machinery, providing a foundation for exploring potential therapeutic targets against SARS-CoV-2.