Biology subjects

Alleman, E. M.

Publications and source records attributed to Alleman, E. M..

3 recordsLinked to original sources

bioRxiv2026

A codon-sensitive conformational switch gates commitment to translation start sites

Human translation initiation requires single-nucleotide precision to establish the reading frame, yet initiation at non-AUG codons plays key roles in gene expression. How the initiation machinery balances precision with this regulated flexibility remains unclear. Here, we define a conformational branchpoint governed by the human initiation factor eIF5 that gates commitment to start codons. Using single-molecule and structural approaches, we demonstrate that eIF5 reversibly occupies two conformations, which depends on a strictly conserved loop in the protein that monitors start codon identity. AUG codons favor the conformation that is stabilized by an eIF5-stimulated GTP hydrolysis step, which commits the complex to the start site. Non-AUG codons favor a standby conformation that destabilizes eIF5 and likely overlaps the binding site of an ancient structural homolog. This branchpoint complements enforcement of start codon fidelity by upstream steps and intrinsically controls the efficiency of non-AUG initiation.

biophysics↗

bioRxiv2025

The Sindbis virus nsP3 opal codon protects viral RNA and fitness by maintaining replication spherule integrity

Most alphaviruses maintain an in-frame opal stop codon that interrupts their non-structural polyprotein (nsP) ORF between nsP3 and nsP4 in both vertebrate and insect hosts. We show that the nsP3 opal stop codon confers a replicative advantage to Sindbis virus (SINV) in RNAi-competent mosquito cells and in Aedes aegypti mosquitoes, but not in cells or mosquitoes lacking RNAi. Mutation of the opal stop codon delays processing of the viral nsP polyprotein, disrupts viral replication spherule integrity, and renders viral RNA susceptible to Dicer 2 cleavage, resulting in higher antiviral siRNA responses against SINV. Similarly, these defects caused by opal codon mutations lead to increased viral RNA detection and enhanced immune signaling in vertebrate cells. Thus, a single stop codon in alphaviruses mediates a multipotent viral strategy to evade innate immune defenses across diverse hosts. TeaserA conserved ORF-interrupting stop codon helps alphaviruses avoid triggering innate antiviral immunity across diverse hosts.

microbiology↗

bioRxiv2024

A conserved opal termination codon optimizes a temperature-dependent tradeoff between protein production and processing in alphaviruses

Alphaviruses are enveloped, single-stranded, positive-sense RNA viruses that often require transmission between arthropod and vertebrate hosts for their sustained propagation. Most alphaviruses encode an opal (UGA) termination codon in nonstructural protein 3 (nsP3) upstream of the viral polymerase, nsP4. The selective constraints underlying the conservation of the opal codon are poorly understood. Using primate and mosquito cells, we explored the role and selective pressure on the nsP3 opal codon through extensive mutational analysis in the prototype alphavirus, Sindbis virus (SINV). We found that the opal codon is highly favored over all other codons in primate cells under native 37{o}C growth conditions. However, this preference is diminished in mosquito and primate cells grown at a lower temperature. Thus, the primary determinant driving the selection of the opal stop codon is not host genetics but the passaging temperature. We show that the opal codon is preferred over amber and ochre termination codons because it results in the highest translational readthrough and polymerase production. However, substituting the opal codon with sense codons leads to excessive full-length polyprotein (P1234) production, which disrupts optimal nsP polyprotein processing, delays the switch from minus-strand to positive-strand RNA production, and significantly reduces SINV fitness at 37{degrees}C; this fitness defect is relieved at lower temperatures. A naturally occurring suppressor mutation unexpectedly compensates for a delayed transition from minus to genomic RNA production by also delaying the subsequent transition between genomic and sub-genomic RNA production. Our study reveals that the opal stop codon is the best solution for alphavirus replication at 37{o}C, producing enough nsP4 protein to maximize replication without disrupting nsP processing and RNA replication transitions needed for optimal fitness. Our study uncovers the intricate strategy dual-host alphaviruses use at a single codon to optimize fitness.

microbiology↗