Novel Gag-Pol frameshift site in human immunodeficiency virus type 1 variants resistant to protease inhibitors

Abstract

Human immunodeficiency virus type 1 (HIV-1) variants resistant to protease inhibitors have been shown to contain a mutation in the p1/p6 Gag precursor cleavage site. At the messenger RNA level, this mutation generates a U UUU UUU sequence that is reminiscent of the U UUU UUA sequence required for ribosomal frameshifting and Gag-Pol synthesis. To test whether the p1/p6 cleavage site mutation was generating a novel frameshift site, HIV sequences were inserted in translation vectors containing a chloramphenicol acetyltransferase (CAT) reporter gene requiring -1 frameshifting for expression. All sequences containing the original HIV frameshift site supported the synthesis of CAT but expression was increased 3- to 11-fold in the presence of the mutant p1/p6 sequence. When the original frameshift site was abolished by mutation, expression remained unchanged when using constructs containing the mutant p1/p6 sequence, whereas it was decreased 2- to 4.5-fold when using wild-type p1/p6 constructs. Similarly, when introduced into HIV molecular clones, the p1/p6 mutant sequence supported Gag-Pol synthesis and protease activity in the absence of the original frameshift site, indicating that this sequence could also promote ribosomal frameshifting in virus-expressing cells.

FIG. 1

Nucleic acid sequences of the p1/p6 cleavage site mutation in HIV-1 protease inhibitor-resistant variants. (A) Variants obtained in the presence of protease inhibitors were sequenced in the p7/p1/p6 region, and DNA sequences were compared to that of the HIV-1 IIIB strain (5, 6, 17). The portion of the DNA sequence from HIV-1 IIIB is shown in its entirety as well as the deduced amino acid sequences (indicated in single-letter codes), read either in the Gag frame (top) or in the Pol frame (bottom). The arrows indicate the scissile bonds of the p7/p1 and p1/p6 cleavage sites. The sequences of variants obtained in the presence of palinavir (2011.40 and 2011.nL.23), BILA 1906 BS (1906.33), and BILA 2185 BS (2185.37) are shown, with sequence identity illustrated by a dash. All mutants contain a C-to-T transition at the p1/p6 junction. (B) Transcribed into RNA, the p7/p1/p6 sequence is predicted to give a stem-loop structure, with the p7/p1 and p1/p6 potential slippery sites (underlined) lying on either side. The dotted line shows a sequence possibly involved in transient pairing with 18S rRNA (see the text).

FIG. 2

Construction of a CAT expression vector for in vitro translation. A 93-bp DNA sequence encompassing the HIV p7/p1/p6 region was inserted in the beginning of the coding sequence of a CAT reporter gene which is under the control of a T7 promoter. The HIV and CAT sequences are not in the same reading frame, so a −1 frameshift in the HIV sequence is required to produce the CAT protein. The inserted HIV sequence is shown in boldface, with the original frameshift site (site a), the p1/p6 junction (site b), and the region in which an additional base pair was added to make the HIV and CAT sequences in frame (site c) underlined. The sequences of sites a, b, and c of all constructs used in this study are indicated in the bottom half of the figure, with added or modified nucleotides underlined. Apart from these three sites, all constructs were identical and were used in similar in vitro transcription and translation experiments.

FIG. 3

Construction and analysis of HIV frameshift mutant molecular clones. (A) A 930-bp DNA fragment containing part of the p7 gene, the entire p1 gene, and 3′ sequences leading into the reverse transcriptase gene from HIV-1 strain IIIB was inserted between the unique ApaI (A) and BST1107 (B) restriction sites of a modified NL4.3 vector called 2.12 to generate clone wt. Site-directed mutagenesis was then used as described previously (11) to introduce the p1/p6 cleavage site mutation in clone wtFF and to abolish the original frameshift site in clones wtk/o and wtFFk/o. The original frameshift site is represented by a solid box, the abolished frameshift site is represented by an open box, the wild-type p1/p6 cleavage site is also represented by a solid box, and the mutated p1/p6 cleavage site is represented by a shaded box. (B) Viral particles harvested upon transfection of molecular clones were analyzed by Western blotting using monoclonal antibodies directed either against the p24 protein (clone 39/5.1.23; ID Labs Inc.) or the p6 protein (23). Proteins of 55, 41, 24, and 6 kDa representing the p55^gag and p41 precursors and the mature p24 and p6 proteins, respectively, were detected. Two independent wtk/o clones and four independent wtFFk/o clones are shown. Immune reactivity was detected by a chemiluminescent substrate detection assay. The infectivities of viruses, determined by infection of C8166 cells with transfection supernatants, are shown as positive (+) or negative (−) at the bottom of the figure.

FIG. 4

Blocking of protease activity in mutant viral clones leads to the accumulation of polyprotein precursors. Viral particles were produced in the presence of the protease inhibitor palinavir (10 μM) and then harvested and analyzed by Western blotting using an anti-p24 monoclonal antibody. Proteins of 24, 55, and 160 kDa, representing the mature p24 protein and the p55^gag and p160^gag-pol precursors, respectively, were detected. Longer exposure was required for efficient p160^gag-pol detection (bottom panel). Gag-Pol/Gag ratios were estimated by comparing results obtained from density integration of the 160- and 55-kDa bands.