Yeast and the AIDS virus: the odd couple

Abstract

Despite being simple eukaryotic organisms, the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe have been widely used as a model to study human pathologies and the replication of human, animal, and plant viruses, as well as the function of individual viral proteins. The complete genome of S. cerevisiae was the first of eukaryotic origin to be sequenced and contains about 6,000 genes. More than 75% of the genes have an assigned function, while more than 40% share conserved sequences with known or predicted human genes. This strong homology has allowed the function of human orthologs to be unveiled starting from the data obtained in yeast. RNA plant viruses were the first to be studied in yeast. In this paper, we focus on the use of the yeast model to study the function of the proteins of human immunodeficiency virus type 1 (HIV-1) and the search for its cellular partners. This human retrovirus is the cause of AIDS. The WHO estimates that there are 33.4 million people worldwide living with HIV/AIDS, with 2.7 million new HIV infections per year and 2.0 million annual deaths due to AIDS. Current therapy is able to control the disease but there is no permanent cure or a vaccine. By using yeast, it is possible to dissect the function of some HIV-1 proteins and discover new cellular factors common to this simple cell and humans that may become potential therapeutic targets, leading to a long-lasting treatment for AIDS.

Figure 1

The two-hybrid assay,  checking for interactions between two proteins, here called Bait and Prey. (a) Gal4 transcription factor gene produces two domain proteins (BD and AD), which are essential for transcription of the reporter gene (LacZ). (b) and (c), Two fusion proteins are prepared: Gal4BD + Bait and Gal4AD + Prey. None of them is usually sufficient to initiate the transcription (of the reporter gene) alone. (d) When both fusion proteins are produced and the Bait part of the first interacts with Prey part of the second, transcription of the reporter gene occurs (adapted from http://www.wikimediafoundation.org/).

Figure 2

Organization of Pro and Pol proteins. Schematic representations of the mature Pro and Pol proteins and their precursors are drawn for examples from several retroviruses. The sequences representing the mature proteins PR, RT, and IN are indicated. Rectangles, precursor proteins, with solid vertical lines marking major cleavage sites and thick horizontal bars indicating mature proteins. DTG or DSG and YMDD indicate the conserved active site residues in PR and RT. The RNase H domain of RT is also indicated (ASLV: avian sarcoma leucosis virus, MMTV: murine mammary tumor virus, MLV: murine leukemia virus, and FIV: feline immunodeficiency virus).

Figure 3

Tertiary structure of RT, IN, and PR: (a) RT: p66 in red and p51 in green; (b) catalytic core of IN in blue. Aminoacid 143 in red, 97 and 120 in green, and 64 and 116 in yellow. (c) Dimeric PR in complex with a protease inhibitor.

Figure 4

Drop test experiment: 3 μL droplets of plasmid-containing standard yeast suspensions containing about 20 000 ura+ colony-forming units were deposited on the appropriate selective media to allow expression of the HIV-1 integrase in yeast. When the drops had dried, plates were incubated for 3 days, and the effect of integrase expression on yeast was determined by visual observation of the Petri dishes. Lethality is observed when IN is expressed in yeast (2) but not in the presence of empty plasmid (1) or when expressing inactive D116A or E152A IN (3 and 4). JSC302 and W303-1A: RAD52+, W839-5C: identical to W303-1A except for RAD52 gene disruption. AB2: diploid obtained from W303-1A (adapted from [39]).

Figure 5

IN expressed in yeast and in human cell. (a) Expression of IN in yeast several times after induction. Cytoplasmic localisation: 6-7 h; perinuclear localisation: 8–10 h; nuclear localisation: 30 h. IN-GFP in green, nucleus in blue surrounded by dashed lanes. Barr = 1 μm. (b) Expression of IN in H9 cells 20 h after transfection. Left, MTOC in red after immunodetection of γ-tubulin (arrows). Right, IN in green immunodetected with anti-IN antibody (arrows). (c) Expression of IN in H9 cells at higher magnitude. IN in green, α-tubulin in red. Barr = 8 μm (Desfarges, unpublished results).

Figure 6

Integration assay in yeast. Adapted from [46].

Figure 7

Frameshift suppression in the synthesis of Gag-Pro-Pol. Shown are the nucleotide sequences at the frameshift site and the amino acids encoded in the Gag-Pro-Pol precursors of the indicated viruses. The upper amino acid sequence is read from either the gag or pro reading frame, and the lower sequence is read from either the pro or pol reading frame, as shown. The boxes represent the indicated reading frames. The colored arrows indicate the position of the nucleotide (shown in color) that is read in both reading frames, the Vs represent the positions in the sequence that encode PR processing sites, and the numbers represent the number of nucleotides between the frameshift site and the end of the reading frame (irrespective of the nucleotide that is read in both frames). The nucleotides in the boxes include the beginning and ending codons in the reading frames shown (ASLV: avian sarcoma leucosis virus and MMTV: murine mammary tumor virus).