Molecular Approaches To Antiviral Development For Viral Hepatitis and Other Viral Diseases

National Institute Of Diabetes And Digestive And Kidney Diseases

Investigators

Linked publications, trials & patents

Abstract

Hepatitis delta virus (HDV) is a human pathogen of global public health concern, causing acute and chronic liver disease. While HDV causes the most severe form of viral hepatitis, there is currently no licensed therapy that directly targets viral replication. In addition, much is unknown about the HDV replication cycle. HDV produces three RNAs: genome, antigenome and mRNA. The mRNA encodes a single viral protein, the hepatitis delta antigen (HDAg), which forms homo-octamers that bind to the genome to form a ribonucleoprotein (RNP) complex. During active replication, the RNP recruits various host proteins and adopts them for various aspects of the replication cycle. One such protein is RNA polymerase II (RNAP II), a DNA-dependent RNA polymerase that HDV co-opts to replicate its RNA via an unknown mechanism. AirID is a highly versatile and efficient in vitro proximity-based biotinylation system that identifies proteins closely interacting, either transiently or permanently, with a protein of interest. We fused AirID to sHDAg and showed that the fusion protein retains its ability to complex with unmodified sHDAg. We used this fusion protein to biotinylate host proteins in close proximity to HDAg with or without replicating HDV genome. The biotinylated proteins were then enriched with streptavidin and analyzed by mass spectrometry. Using this method, we identified and validated proteins that were previously found to interact with HDAg, as well as proteins yet unknown for HDAg interaction. Intriguingly, several of the proteins are known to complex with RNAPII, aiding in active RNA elongation and preventing stalling. siRNA knock-down experiments confirmed these proteins’ proviral interaction with the HDV replication cycle, as knock-down significantly decreased levels of HDV RNA. In addition, the proteins were found to colocalize with HDAg in cells with replicating HDV in unique nuclear structures via confocal microscopy. This novel finding may lead us to better understand how an RNA virus, HDV, co-opts host DNA-dependent RNAP II for its replication, and new approaches for therapeutic development against HDV. With the emergence of COVID-19 in 2020, our lab pivoted part of our efforts to antiviral development for SAR-CoV-2 infection. Since the fusion process is relatively conserved among envelope viruses, we previously tested the two HCV fusion inhibitors described above against SARS-CoV-2. They were active against SARS-CoV-2 in cell culture but did not exhibit potent antiviral effects in animal model. We also screened other structurally related compounds to one of the fusion inhibitors against SARS-CoV-2. Among them, lonafarnib (LNF), an approved drug inhibitor of cellular farnesyltransferase, was effective against SARS-CoV-2 and its variants in various cell culture models. More importantly, LNF at clinically relevant dose significantly suppressed viral levels in the respiratory tract and improve pulmonary pathology and clinical parameters in a SARS-CoV-2 animal model. LNF, an approved oral drug with excellent human safety data, is a promising antiviral against SARS-CoV-2 that warrants further clinical assessment for treatment of COVID-19 and potentially other viral infections.