Biology subjects

Sabsay, K. R.

Publications and source records attributed to Sabsay, K. R..

2 recordsLinked to original sources

bioRxiv2023

Deep mutational scanning reveals the functional constraints and evolutionary potential of the influenza A virus PB1 protein

The influenza virus polymerase is central to influenza virus evolution. Adaptive mutations within the polymerase are often a prerequisite for efficient spread of novel animal-derived viruses in human populations. The polymerase also determines fidelity, and therefore the rate at which the virus will acquire mutations that lead to host range expansion, drug resistance, or antigenic drift. Despite its importance to viral replication and evolution, our understanding of the mutational effects and associated constraints on the influenza RNA-dependent RNA polymerase (RdRp) is relatively limited. We performed deep mutational scanning of the A/WSN/1933(H1N1) PB1, generating a library of 95.4% of amino acid substitutions at 757 sites. After accuracy filters, we were able to measure replicative fitness for 13,354 (84%) of all possible amino acid substitutions, and 16 were validated by results from pairwise competition assays. Functional and structural constraints were better revealed by individual sites involved in RNA or protein interactions than by major subdomains defined by sequence conservation. Mutational tolerance, as defined by site entropy, was correlated with evolutionary potential, as captured by diversity in available H1N1 sequences. Of 29 beneficial sites, many have either been identified in the natural evolution of PB1 or shown experimentally to have important impacts on replication and adaptation. Accessibility of amino acid substitutions by single nucleotide mutation was a key factor in determining whether mutations appeared in natural PB1 evolution. Our work provides a comprehensive map of mutational effects on a viral RdRp and a valuable resource for subsequent studies of influenza replication and evolution.

microbiology↗

bioRxiv2023

Evolution of transient RNA structure-RNA polymerase interactions in respiratory RNA virus genomes

RNA viruses are important human pathogens that cause seasonal epidemics and occasional pandemics. Examples are influenza A viruses (IAV) and coronaviruses (CoV). When emerging IAV and CoV spill over to humans, they adapt to evade immune responses and optimize their replication and spread in human cells. In IAV, adaptation occurs in all viral proteins, including the viral ribonucleoprotein (RNP) complex. RNPs consists of a copy of the viral RNA polymerase, a double-helical coil of nucleoprotein, and one of the eight segments of the IAV RNA genome. The RNA segments and their transcripts are partially structured to coordinate the packaging of the viral genome and modulate viral mRNA translation. In addition, RNA structures can affect the efficiency of viral RNA synthesis and the activation of host innate immune response. Here, we investigated if RNA structures that modulate IAV replication processivity, so called template loops (t-loops), vary during the adaptation of pandemic and emerging IAV to humans. Using cell culture-based replication assays and in silico sequence analyses, we find that the sensitivity of the IAV H3N2 RNA polymerase to t-loops increased between isolates from 1968 and 2017, whereas the total free energy of t-loops in the IAV H3N2 genome was reduced. This reduction is particularly prominent in the PB1 gene. In H1N1 IAV, we find two separate reductions in t-loop free energy, one following the 1918 pandemic and one following the 2009 pandemic. No destabilization of t-loops is observed in the IBV genome, whereas analysis of SARS-CoV-2 isolates reveals destabilization of viral RNA structures. Overall, we propose that a loss of free energy in the RNA genome of emerging respiratory RNA viruses may contribute to the adaption of these viruses to the human population.

microbiology↗