Scientists at the University of California San Diego and UC Riverside have further studied the molecular pathway used by the SARS-CoV-2 virus to infect human lung cells and subsequently identified a key host-cell player that may prove a new and enduring therapeutic target for treating COVID-19. The findings “PCIF1-mediated deposition of 5′-cap N6,2′-O-dimethyladenosine in ACE2 and TMPRSS2 mRNA regulates susceptibility to SARS-CoV-2 infection” appear in PNAS.

To enter and infect host cells, the SARS-CoV-2 virus deploys its spike proteins to bind to the angiotensin-converting enzyme (ACE2) cell surface receptor, triggering expression of transmembrane serine protease 2 (TMPRSS2), which results in the generation of new virus particles that help further the disease COVID-19.

Much research has been conducted to find ways to inhibit or disrupt the ACE2/TMPRSS2 pathway, to make it more difficult for the SARS-CoV-2 virus to replicate and spread. In the new study, Rana and colleagues highlight the role of another enzyme, one that may provide a new therapeutic target and the possibility of broader, sustained protection against both current COVID-19 variants and those yet to emerge.

Promising role for PCIF1

The enzyme is called phosphorylated CTD-interacting factor 1 or PCIF1, which regulates cellular entry through the mediation of N6,2-O-dimethyladenosine (m6Am) activity, an evolutionarily conserved and abundant mRNA modification. The researchers found that PCIF1 promotes the stability of both ACE2 and TMPRSS2 mRNAs, sustaining two key entry factors for SARS-CoV-2 and other coronaviruses.

“…we investigated the potential roles of N6,2′-O-dimethyladenosine (m6Am), one of the most abundant modifications of eukaryotic messenger ribonucleic acid (mRNAs), in SARS-CoV-2 infection of human cells,” write the investigators.

“Using genome-wide m6Am-exo-seq, RNA sequencing analysis, and Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing, we demonstrate that phosphorylated C-terminal domain (CTD)-interacting factor 1 (PCIF1), a cap-specific adenine N6-methyltransferase, plays a major role in facilitating infection of primary human lung epithelial cells and cell lines by SARS-CoV-2, variants of concern, and other coronaviruses.

“We show that PCIF1 promotes infection by sustaining expression of the coronavirus receptors angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) via m6Am-dependent mRNA stabilization. In PCIF1-depleted cells, both ACE2/TMPRSS2 expression and viral infection are rescued by re-expression of wild-type, but not catalytically inactive, PCIF1.

“These findings suggest a role for PCIF1 and cap m6Am in regulating SARS-CoV-2 susceptibility and identify a potential therapeutic target for prevention of infection.”

“Essentially, it’s as if once SARS-CoV-2 has opened the door to a cell, PCIF1 helps keep the door open,” said senior author Tariq Rana, PhD, Distinguished Professor of Pediatrics in the UC San Diego School of Medicine and a faculty member of both the Institute of Genomic Medicine and Moores Cancer Center at UC San Diego Health.

Rana and colleagues validated their findings using primary normal human bronchial cells, which line the passages of the lungs and act as a defensive barrier to pathogens. They also found, not described in this publication, positive correlations between PCIF1 and ACE2/TMPRSS2 expression levels in human lung tissues.

Fundamentally, explained the researchers, the results point to a new approach to reducing or blocking SARS-CoV-2 infections. Currently, Paxlovid (a combination of two antiviral drugs) is used to treat early cases of COVID-19. It works by directly targeting the virus itself, but may lose efficacy as the virus mutates and new drug-resistant variants of concern arise.

The new findings advocate for drug development that targets host-cell factors, such as PCIF1 and TMPRSS2.

“In doing so, there is less potential for drug resistance,” said Rana. “And in combination with viral-targeted agents, there could be a synergistic effect that more broadly and effectively protects against the coronavirus, both current strains and those emerging.”

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