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The Type 6 IRESs are the most streamlined mechanism to date, as they adopt a three pseudoknot RNA structure to initiate factorless translation initiation by directly recruiting the ribosome and drive translation. The Halastavi \u00e1rva virus (HalV) IRES represents the most minimalistic subclass identified to date, whereby the IRES lacks specific pseudoknot domains that bind to the 40S subunit but instead recruits pre-assembled 80S ribosomes via a mechanism that is not fully understood. Here, we examined cellular conditions that can support HalV IRES translation. We demonstrated that the HalV IRES is translationally active in insect Sf21 lysates and Drosophila S2 cells, but inactive in mammalian RRL and wheat germ extract. Cells treated with heat shock or serum starvation suppressed HalV IRES activity, whereas virus infection robustly enhanced HalV IRES-mediated translation. Finally, the HalV IRES can support viral translation and replication using a heterologous viral replicon. These findings highlight the context-specific cellular conditions that allow ribosome assembly and translation by a factorless minimalist IRES.<\/jats:p>","DOI":"10.3390\/v18050492","type":"journal-article","created":{"date-parts":[[2026,4,23]],"date-time":"2026-04-23T12:04:42Z","timestamp":1776945882000},"page":"492","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Functional Analysis of the Halastavi \u00e1rva Virus (HalV) Internal Ribosome Entry Site"],"prefix":"10.3390","volume":"18","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-6139-5390","authenticated-orcid":false,"given":"Subash","family":"Chapagain","sequence":"first","affiliation":[{"name":"Department of Biochemistry and Molecular Biology, Life Sciences Institute, University of British Columbia, Vancouver, BC V6T 1Z4, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8685-5135","authenticated-orcid":false,"given":"Lauren F.","family":"Woodburn","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biology, Life Sciences Institute, University of British Columbia, Vancouver, BC V6T 1Z4, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Natalie C. J.","family":"Strynadka","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biology, Life Sciences Institute, University of British Columbia, Vancouver, BC V6T 1Z4, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eric","family":"Jan","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biology, Life Sciences Institute, University of British Columbia, Vancouver, BC V6T 1Z4, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,4,23]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"283","DOI":"10.1146\/annurev-virology-100114-055014","article-title":"A Cap-to-Tail Guide to mRNA Translation Strategies in Virus-Infected Cells","volume":"3","author":"Jan","year":"2016","journal-title":"Annu. Rev. 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