The Leader of Human Immunodeficiency Virus Type 1 Genomic RNA Harbors an Internal Ribosome Entry Segment That Is Active during the G ₂ /M Phase of the Cell Cycle

Abstract

The 5 leader of the human immunodeficiency virus type 1 (HIV-1) genomic RNA contains highly structured domains involved in key steps of the viral life cycle. These RNA domains inhibit cap-dependent protein synthesis. Here we report that the HIV-1 5 leader harbors an internal ribosome entry site (IRES) capable of driving protein synthesis during the G 2 /M cell cycle phase in which cap-dependent initiation is inhibited. The HIV-1 IRES was delineated with bicistronic mRNAs in in vitro and ex vivo assays. The HIV-1 leader IRES spans nucleotides 104 to 336 and partially overlaps the major determinants of genomic RNA packaging. These data strongly suggest that, as for HIV-1 transcription, IRES-mediated translation initiation could play an important role in virus replication during virus-induced G 2 /M cell cycle arrest.

Human Immunodeficiency Virus Type 1 (HIV-1) Induces the Cytoplasmic Retention of Heterogeneous Nuclear Ribonucleoprotein A1 by Disrupting Nuclear Import

“…pNL4-XX was described previously (1,30), the pMrev(Ϫ) and pMtat(Ϫ) constructs were provided by the NIH AIDS Reference and Reagent Program (31), the pNL4-3rev(Ϫ) Mason-Pfizer monkey virus (MPMV) CTE was provided by Mary-Louise Hammarskjold (NCI, NIH, Frederick, MD) (32), GFP and p62(1-520)-GFP constructs were provided by Ursula Stochaj (34), and the Myc epitope-tagged hnRNP A1 expressor (pMyc-A1) was provided by Benoit Chabot (25). The bicistronic constructs used to assess HIV-1-IRES activity (pRF and pRF 104 -336) have previously been described (35,36).…”

“…For IRES activity determinations, cells were harvested with passive lysis buffer (Promega, Madison, WI), and 20 g of cellular protein was used to assess IRES activity with the dual-luciferase reporter assay system (Promega) according to the manufactur-er's instructions. To calculate IRES activity, IRES-driven Firefly luciferase expression was normalized to cap-dependent Renilla luciferase activity as described (36). Band signal intensities were determined by densitometry using ImageJ software (NIH).…”

“…Indeed, hnRNP A1 regulates both cap-dependent and cap-independent translation initiation of a variety of cellular and viral RNAs (12,25,37,54,55). A recent report suggests that Gag protein synthesis can be driven by an internal ribosome entry site (IRES) (36). Thus, we sought to establish if hnRNP A1 acts as an IRES trans-activating factor for HIV-1 cap-independent translation initiation.…”

“…We used a well characterized bicistronic dual luciferase construct harboring the HIV-1 5Ј-IRES, nucleotides 104 -336 (numbering with respect to the pNL4.3 vRNA) responsible for driving the IRES-mediated translation initiation of Gag RNA (i.e. vRNA) (36) (Fig. 4A).…”

Translation initiation of the HIV-1 mRNA

“…86,88,89 The characterization of the HIV-1 IRES was done by using a series of bicistronic constructs that were verified to contain no cryptic promoter or spurious splice sites as they are often a caveat in this area of research 86,[88][89][90] In addition, translation of HIV-1 in a bicistronic context was shown to be resistant to picornaviral proteases that specifically inhibit cap-dependent translation initiation. 86,89,91 More recently, Monette et al have used transfection-infection experiments of DNA encoding a bicistronic RNA containing the HIV-1 5 0 UTR proving that its activity is not disrupted by poliovirus infection in 293 T cells.…”