Significance: We discovered that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) expresses a small viral noncoding RNA, named CoV2-miR-O7a (for SARS-CoV-2 miRNA-like ORF7a-derived small RNA). CoV2-miR-O7a associates with the cellular RNA interference machinery and has the potential to regulate host transcripts, likely via target slicing. The production of CoV2-miR-O7a relies on cellular machinery and the formation of a strong hairpin within ORF7a sequences. This newly described CoV2-miR-O7a may contribute to SARS-CoV-2 pathogenesis and could become a target for therapeutic intervention.

Like their host cells, many viruses express noncoding RNAs (ncRNAs). Despite the technical challenge of ascribing function to ncRNAs, diverse biological roles for virally expressed ncRNAs have been...

Pumilio proteins (PUM1 and PUM2) are essential post-transcriptional regulators of gene expression found across plants, animals and yeast. They bind Pumilio response elements (PREs) on messenger RNAs (mRNAs) to modulate mRNA stability and translation. PUMs have been implicated in diverse cellular processes, including stem cell maintenance, neurogenesis and cell cycle regulation. They have also been reported to negatively regulate innate immunity genes and to participate in viral RNA sensing. Previous high-throughput interactome studies revealed that PUMs bind severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA. We found that SARS-CoV-2 transcripts contain multiple conserved PREs, some of which are preferentially bound by PUM2. Surprisingly, altering PUM levels does not affect the production of progeny virions. However, depletion of PUM1 slightly increases intracellular viral RNA levels, suggesting that PUM1 either plays a mild antiviral role against SARS-CoV-2 or regulates host factors that promote viral replication. Notably, PUM1 also negatively regulates innate immunity gene expression both at steady state and during SARS-CoV-2 infection. Our findings support a complex immunomodulatory role for PUM1, acting both as a negative regulator of innate immunity genes and a mild inhibitor of SARS-CoV-2 RNA accumulation. However, in cell culture, these roles appear negligible based on viral progeny output. Whether the multiple PREs found in the SARS-CoV-2 genome contribute to evasion of PUM1 activity remains an open question.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease (COVID-19), continues to be a pressing health concern. In this study, we investigated the impact of SARS-CoV-2 infection on host microRNA (miRNA) populations in three human lung-derived cell lines, as well as in nasopharyngeal swabs from SARS-CoV-2 infected individuals. We did not detect any major and consistent differences in host miRNA levels after SARS-CoV-2 infection. However, we unexpectedly discovered a viral miRNA-like small RNA, named vmiR-5p (for viral miRNA), derived from the SARS-CoV-2 ORF7a transcript. Its abundance ranges from low to moderate as compared to host miRNAs. vmiR-5p functionally associates with Argonaute proteins — core components of the RNA interference pathway — leading to downregulation of host transcripts. One such host messenger RNA encodes Basic Leucine Zipper ATF-Like Transcription Factor 2 (BATF2), which is linked to interferon signaling. We demonstrate that vmiR-5p production relies on cellular machinery, yet is independent of Drosha protein, and is enhanced by the presence of a strong and evolutionarily conserved hairpin formed within the ORF7a sequence. Significance statement We discovered that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) expresses a small viral non-coding RNA, named vmiR-5p (for viral miRNA), derived from the ORF7a transcript. vmiR-5p associates with the cellular RNA interference machinery to regulate host transcripts likely via target silencing. The production of vmiR-5p relies on cellular machinery and the formation of a strong hairpin within ORF7a sequences. This newly-described vmiR-5p may contribute to SARS-CoV-2 pathogenesis and could become a target for therapeutic intervention.

Pumilio proteins (PUM1 and PUM2) are essential post-transcriptional regulators of gene expression found across plants, animals, and yeast. They bind Pumilio Response Elements (PREs) on messenger RNAs (mRNAs) to modulate mRNA stability and translation. PUMs have been implicated in diverse cellular processes, including stem cell maintenance, neurogenesis, and cell cycle regulation. They have also been reported to negatively regulate innate immunity genes and to participate in viral RNA sensing. Previous high-throughput interactome studies revealed that PUMs bind SARS-CoV-2 RNA. We found that SARS-CoV-2 transcripts contain multiple conserved PREs, some of which are preferentially bound by PUM2. Surprisingly, altering PUM levels does not affect the production of progeny virions. However, depletion of PUM1 slightly increases intracellular viral RNA levels, suggesting that PUM1 either plays a mild antiviral role against SARS-CoV-2 or regulates a host factor that promotes viral replication. Notably, PUM1 also negatively regulates innate immunity gene expression both at steady state and during SARS-CoV-2 infection. Our findings support a complex immunomodulatory role for PUM1, acting both as a negative regulator of innate immunity genes and a mild inhibitor of SARS-CoV-2 RNA accumulation. However, in cell culture, these roles appear negligible based on viral progeny output. Whether the multiple PREs found in the SARS-CoV-2 genome contribute to evasion of PUM1 activity remains an open question.

Viral non-coding RNAs (ncRNAs) often perform multiple functions during persistent infection, but how they interface with host RNA-binding proteins remains incompletely understood. Herpesvirus saimiri (HVS), a T-tropic γ-herpesvirus that induces aggressive lymphomas in primates, during its latency expresses abundant small nuclear RNAs HSUR1 and HSUR2. HSUR1 is known to trigger target-directed microRNA decay (TDMD) of host miR-27a, and HSUR2 can function as a miRNA-dependent adaptor; here we show that both HSURs also bind host Pumilio proteins (PUM1 and PUM2) via perfectly conserved Pumilio response elements (PREs). PUM proteins are potent post-transcriptional regulators that bind PREs usually located in 3′ UTRs of mRNAs to repress protein production. In Jurkat T cells, wild-type HSUR1/2, but not PRE-mutant variants, shift cells from a naïve-like toward an effector/memory-like state. This PRE-dependent shift is evidenced by increased surface expression of CXCR3, a chemokine receptor of activated effector T cells, and CD57, a marker of terminally differentiated T and NK cells. Our data support a model in which HSURs sequester a fraction of PUM proteins. Consistently, depletion of PUM1 or PUM2 phenocopies HSUR-induced changes, with more abundant PUM1 exerting the dominant effect. Consistently, depletion of PUM1 or PUM2 phenocopies HSUR-induced changes, with more abundant PUM1 exerting the dominant effect. In BJAB B cells, HSUR1 still drives PRE-dependent gene regulation, but the affected genes only partially overlap with those in Jurkat cells, and some transcripts even show reversed direction of change, revealing strong cell-type specificity. By identifying PUM1/2 as key host partners of HSUR ncRNAs, this work uncovers an underappreciated role for PUM proteins in shaping T- and B-cell states and reveals an additional way in which γ -herpesviral ncRNAs reprogram lymphocytes to promote viral persistence.