The HIV 5' Gag Region Displays a Specific Nucleotide Bias Regulating Viral Splicing and Infectivity.

Bastian Grewe,Ralf Wagner,Bettina Tippler,Klaus Überla,Theresa Horstkötter,Han Xiao,Bianca Müller,Benedikt Asbach,Carolin Vogt,Jens Bohne

doi:10.3390/v13060997

Abstract

Alternative splicing and the expression of intron-containing mRNAs is one hallmark of HIV gene expression. To facilitate the otherwise hampered nuclear export of non-fully processed mRNAs, HIV encodes the Rev protein, which recognizes its intronic response element and fuels the HIV RNAs into the CRM-1-dependent nuclear protein export pathway. Both alternative splicing and Rev-dependency are regulated by the primary HIV RNA sequence. Here, we show that these processes are extremely sensitive to sequence alterations in the 5’coding region of the HIV genomic RNA. Increasing the GC content by insertion of either GFP or silent mutations activates a cryptic splice donor site in gag, entirely deregulates the viral splicing pattern, and lowers infectivity. Interestingly, an adaptation of the inserted GFP sequence toward an HIV-like nucleotide bias reversed these phenotypes completely. Of note, the adaptation yielded completely different primary sequences although encoding the same amino acids. Thus, the phenotypes solely depend on the nucleotide composition of the two GFP versions. This is a strong indication of an HIV-specific mRNP code in the 5′ gag region wherein the primary RNA sequence bias creates motifs for RNA-binding proteins and controls the fate of the HIV-RNA in terms of viral gene expression and infectivity.

Highlights

Most retroviruses are faced with the problem of expressing all genes from a single, polycistronic primary transcript, which serves as the genomic RNA to be packaged in newly formed viral particles [1]
Using iCLIP data and bioinformatics approaches, the term splicing code was established to describe and predict splicing events [8,9]. An extension of this concept, named mRNP code, takes into account how the primary RNA sequence dictates all steps of mRNA biogenesis including splicing, processing, translation, and localization based on differential protein recruitment [10,11,12]
RNA is folded into secondary structures and recognized by RNA-binding proteins (RBPs)

Summary

Introduction

Most retroviruses are faced with the problem of expressing all genes from a single, polycistronic primary transcript (pre-mRNA), which serves as the genomic RNA to be packaged in newly formed viral particles [1]. A variety of mechanisms have been described as to how the virus maintains its splicing ratios using HIV reporter constructs [3]. Many of these have served as blueprints to understand the regulation of cellular alternative splicing. Using iCLIP data and bioinformatics approaches, the term splicing code was established to describe and predict splicing events [8,9] An extension of this concept, named mRNP code, takes into account how the primary RNA sequence dictates all steps of mRNA biogenesis including splicing, processing, translation, and localization based on differential protein recruitment [10,11,12]

Methods

Results

Conclusion