Preprint Article Version 1 This version is not peer-reviewed

Selenium-Dependent Readthrough of the Conserved 3’-Terminal UGA Stop Codon of HIV-1 Nef

Version 1 : Received: 25 May 2020 / Approved: 26 May 2020 / Online: 26 May 2020 (13:16:09 CEST)

How to cite: Premadasa, L.; Dailey, G.; Ruzicka, J.; Taylor, E. Selenium-Dependent Readthrough of the Conserved 3’-Terminal UGA Stop Codon of HIV-1 Nef. Preprints 2020, 2020050432 (doi: 10.20944/preprints202005.0432.v1). Premadasa, L.; Dailey, G.; Ruzicka, J.; Taylor, E. Selenium-Dependent Readthrough of the Conserved 3’-Terminal UGA Stop Codon of HIV-1 Nef. Preprints 2020, 2020050432 (doi: 10.20944/preprints202005.0432.v1).

Abstract

The HIV-1 nef gene terminates in a 3’-UGA stop codon, which is highly conserved in the main group of HIV-1 subtypes, along with a downstream potential coding region that could extend the nef protein by 33 amino acids, if readthrough of the stop codon occurs. Antisense tethering interactions (ATIs) between a viral mRNA and a host selenoprotein mRNA are a potential viral strategy for the capture of a host selenocysteine insertion sequence (SECIS) element (Taylor et al, 2016) [1]. This mRNA hijacking mechanism could enable the expression of virally encoded selenoprotein modules, via translation of in-frame UGA stop codons as selenocysteine (SeC). Here we show that readthrough of the 3’-terminal UGA codon of nef occurs during translation of HIV-1 nef expression constructs in transfected cells. This was accomplished via fluorescence microscopy image analysis and flow cytometry of HEK 293 cells, transfected with engineered GFP reporter gene plasmid constructs, in which GFP can only be expressed by translational recoding of the UGA codon. SiRNA knockdown of thioredoxin reductase 1 (TR1) mRNA resulted in a 67% decrease in GFP expression, presumably due to reduced availability of the components involved in selenocysteine incorporation for the stop codon readthrough, thus supporting the proposed ATI. Addition of 20 nM sodium selenite to the media significantly enhanced stop codon readthrough in the pNefATI1 plasmid construct, by >100%, supporting the hypothesis that selenium is involved in the UGA readthrough mechanism.

Subject Areas

antisense; HIV-1 nef; stop codon readthrough; selenium; thioredoxin reductase

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