Structural domains within the HIV‐1 mRNA and the ribosomal protein S25 influence cap‐independent translation initiation

Abstract

The 5′leader of the HIV-1 genomic RNA is a multifunctional region that folds into secondary/tertiary structures that regulate multiple processes during viral replication including translation initiation. In this work we examine the internal ribosome entry site (IRES) located in the 5′leader that drives translation initiation of the viral Gag protein under conditions that hinder cap-dependent translation initiation. We show that activity of the HIV-1 IRES relies on ribosomal protein S25 (eS25). Additionally, a mechanistic and mutational analysis revealed that the HIV-1 IRES is modular in nature and that once the 40S ribosomal subunit is recruited to the IRES, translation initiates without the need of ribosome scanning. These findings elucidate a mechanism of initiation by the HIV-1 IRES whereby a number of highly structured sites present within the HIV-1 5′leader leads to the recruitment of the 40S subunit directly at the site of initiation of protein synthesis.

Translational Control in Virus-Infected Cells

“…IRESs were first discovered in poliovirus and EMCV where they allow translation initiation of uncapped, single-stranded (+)-sense RNA viral genomes immediately upon release into the cytoplasm (Jang et al 1988;Pelletier and Sonenberg 1988). Subsequently, IRESs have been identified in many virus genomes includ-ing retroviruses like the human immunodeficiency virus (HIV)-1, which uses an IRES to express the Gag protein late in infection when capdependent initiation is impaired (Brasey et al 2003;Amorim et al 2014;Carvajal et al 2016). Some DNA virus genomes like KSHV even contain IRES elements to facilitate translation of polycistronic mRNA (Othman et al 2014).…”

“…Indeed, significant insight into how ribosome composition impacts translation has been gleaned from viral models. Two 40S subunit-associated proteins (eS25, RACK1) dispensable for cap-dependent initiation, but required for IRES activity, have been identified (Landry et al 2009;Hertz and Thompson 2011a;Majzoub et al 2014;Olivares et al 2014;Carvajal et al 2016). Whereas RACK1 is required by specific IRESs (dicistroviral 5 0 -IRES, HCV), it is not compulsory for the IGR IRES (Majzoub et al 2014).…”

“…Whereas RACK1 is required by specific IRESs (dicistroviral 5 0 -IRES, HCV), it is not compulsory for the IGR IRES (Majzoub et al 2014). In contrast, eS25 is essential for 40S subunit recruitment by numerous IRESs, including the dicistrovirus IGR and picornavirus, flavivirus, retrovirus, and cellular IRESs (Landry et al 2009;Hertz et al 2013;Olivares et al 2014;Carvajal et al 2016). Unexpectedly, eS25 is also important for ribosomal shunting, suggesting a potential shared mechanism between these two noncanonical initiation strategies (Hertz et al 2013).…”

Does functional specialization of ribosomes really exist?

“…Another example for the likely effects from changes in ribosome numbers comes from the study of mRNAs containing internal ribosome entry sites (IRES). For example, the hepatitis C virus (HCV) IRES has reported sensitivity to the depletion of Rps25, Rpl10A/uL1, Asc1/RACK1, Rps5/uS7, and Rpp1/2 (Landry et al 2009;Majzoub et al 2014;Bhat et al 2015;Campos et al 2017;Shi et al 2017), depletion of Rpp1/2 disrupts replication of footand-mouth disease virus (Martínez-Azorín et al 2008), and Rps25 is required by HIV and the Dicistroviridae (Landry et al 2009;Carvajal et al 2016), a family of viruses that also requires Asc1/RACK1 (Majzoub et al 2014). Further, in plants, the replication of turnip mosaic virus is inhibited by depletion of Rps6, Rps2/uS5, Rpl19, Rpl13, Rpl7/uL30, or Rpp1/2 (Yang et al 2009;Rajamäki et al 2017).…”

Regulation of Ribosomal Proteins on Viral Infection

“…RPS25 likely functions in a downstream step, such as loading of viral mRNA into 40S subunit, start codon recognition, or 60S subunit joining [31,36]. When cap-dependent translation initiation is hindered, the 5 leader of human immunodeficiency virus type 1 (HIV-1) genomic RNA can fold into IRES to recruit 40S subunit and drive translation initiation of viral Gag protein, in which the activity of IRES is also dependent on RPS25 [37]. RACK1, as ribosome scaffold protein, is also required for viral IRES-2-mediated translation, and the effect of RACK1 may associate with eIF3 to assemble a translation preinitiation complex [38].…”