The Human Immunodeficiency Virus Type 1 Ribosomal Frameshifting Site Is an Invariant Sequence Determinant and an Important Target for Antiviral Therapy

Abstract

Human immunodeficiency virus type 1 (HIV-1) utilizes a distinctive form of gene regulation as part of its life cycle, termed programmed ؊1 ribosomal frameshifting, to produce the required ratio of the Gag and Gag-Pol polyproteins. We carried out a sequence comparison of 1,000 HIV-1 sequences at the slippery site (UUUUUUA) and found that the site is invariant, which is somewhat surprising for a virus known for its variability. This prompted us to prepare a series of mutations to examine their effect upon frameshifting and viral infectivity. Among the series of mutations were changes of the HIV-1 slippery site to those effectively utilized by other viruses, because such mutations would be anticipated to have a relatively mild effect upon frameshifting. The results demonstrate that any change to the slippery site reduced frameshifting levels and also dramatically inhibited infectivity. Because ribosomal frameshifting is essential for HIV-1 replication and it is surprisingly resistant to mutation, modulation of HIV-1 frameshifting efficiency potentially represents an important target for the development of novel antiviral therapeutics.Due to their compact genomes, viruses often develop novel modes of gene expression in order to synthesize multiple proteins from a single RNA species. For example, human immunodeficiency virus type 1 (HIV-1) uses a unique mode of gene regulation termed programmed Ϫ1 ribosomal frameshifting. The structural and enzymatic components of the virus are synthesized as polyproteins with a common N terminus. The open reading frame (ORF) encoding the structural 55-kDa Gag protein is located at the 5Ј end of the mRNA. The 160-kDa Gag-Pol polyprotein is translated only as a result of a programmed Ϫ1 ribosomal frameshift event (Fig. 1A) (17). The pol ORF, which encodes the enzymatic proteins, including viral protease (PR), reverse transcriptase, and integrase, is 3Ј to the gag gene and out of reading frame with respect to the gag ORF. Programmed Ϫ1 ribosomal frameshifting allows synthesis of the required ratio of Gag to Gag-Pol, which is highly regulated in retroviruses, with frameshifting occurring at levels of 5 to 10% for HIV-1 (17, 30). These polyprotein precursors are incorporated into viral particles and are subsequently cleaved during viral maturation by the viral PR.In vitro and in vivo studies demonstrate that there are two cis-acting elements located within the overlapping region of the HIV-1 gag and pol genes that are critical for translational frameshifting to occur (Fig. 1B). The first is the heptamer UUUUUUA, where frameshifting occurs and which computer analysis indicates is invariant (see Results). The second is a structural RNA motif downstream of the heptamer that assumes a stem-loop and possibly a pseudoknot structure (9,11,12,26). An 8-nucleotide spacer separates these two regions.Other viruses as well as additional retroviruses also employ ribosomal frameshifting during viral gene expression (3, 12). The consensus viral slippery sequence is X XXY YYZ, where spaces indicate the ...

Specific Effects of Ribosome-Tethered Molecular Chaperones on Programmed −1 Ribosomal Frameshifting

“…Changes in PRF efficiency alter those ratios, inhibiting virion morphogenesis (9). In particular, retroviruses appear to be very susceptible to such changes (4,21,33,37). Thus, elucidation of the molecular mechanisms underlying PRF can aid in the rational design of antiviral therapeutics (reviewed in reference 8).…”

A loop 2 cytidine-stem 1 minor groove interaction as a positive determinant for pseudoknot-stimulated –1 ribosomal frameshifting

“…The efficiency of this frameshifting event varies between 2% and 15% among different viruses, with the majority of elongating ribosomes terminating translation normally at a P1 stop codon (4)(5)(6). Although it is unknown to what extent the frameshifting efficiency in plant luteoviruses influences viral replication and infectivity, studies in the human retrovirus HIV-1, Moloney murine leukemia virus, and the yeast L-A double-stranded RNA virus reveal that an optimal level of frameshift stimulation is essential for maintaining high levels of virus infectivity and propagation (7)(8)(9)(10).…”

Secondary Structure of the HIV-1 Genome

“…The second helix is formed by the pairing of the slippery sequence itself and the third helix anchors the others in a discrete structural element. It is suggested that this three helix structure explains why mutating the slippery sequence eliminates frameshifting, as this prohibits base pairing in the second helix [10]. In this instance the incorporation of the mutagenic ana lysis notably improves the argument and highlights the importance of including complementary Priority PaPer evaluation | Stephenson & Lever approaches and comparison with other genomes in order to increase confidence when attributing functionality to structural elements.…”