Rapid propagation of low-fitness drug-resistant mutants of human immunodeficiency virus type 1 by a streptococcal metabolite sparsomycin.

Here we report that sparsomycin, a streptococcal metabolite, enhances the replication of HIV-1 in multiple human T cell lines at a concentration of 400 nM. In addition to wild-type HIV-1, sparsomycin also accelerated the replication of low-fitness, drug-resistant mutants carrying either D30N or L90M within HIV-1 protease, which are frequently found mutations in HIV-1-infected patients on highly active antiretroviral therapy (HAART). Of particular interest was that replication enhancement appeared profound when HIV-1 such as the L90M-carrying mutant displayed relatively slower replication kinetics. The presence of sparsomycin did not immediately select the fast-replicating HIV-1 mutants in culture. In addition, sparsomycin did not alter the 50% inhibitory concentration (IC50) of antiretroviral drugs directed against HIV-1 including nucleoside reverse transcriptase inhibitors (lamivudine and stavudine), non-nucleoside reverse transcriptase inhibitor (nevirapine) and protease inhibitors (nelfinavir, amprenavir and indinavir). The IC50s of both zidovudine and lopinavir against multidrug resistant HIV-1 in the presence of sparsomycin were similar to those in the absence of sparsomycin. The frameshift reporter assay and Western blot analysis revealed that the replication-boosting effect was partly due to the sparsomycin's ability to increase the -1 frameshift efficiency required to produce the Gag-Pol transcript. In conclusion, the use of sparsomycin should be able to facilitate the drug resistance profiling of the clinical isolates and the study on the low-fitness viruses.

Transcriptional activators in yeast.

Eukaryotic transcription activation domains (ADs) are not well defined on the proteome scale. We systematicallly tested approximately 6000 yeast proteins for transcriptional activity using a yeast one-hybrid system and identified 451 transcriptional activators. We then determined their transcription activation strength using fusions to the Gal4 DNA-binding domain and a His3 reporter gene which contained a promoter with a Gal4-binding site. Among the 132 strongest activators 32 are known transcription factors while another 35 have no known function. Although zinc fingers, helix-loop-helix domains and several other domains are highly overrepresented among the activators, only few contain characterized ADs. We also found some striking correlations: the stronger the activation activity, the more acidic, glutamine-rich, proline-rich or asparagine-rich the activators were. About 29% of the activators have been found previously to specifically interact with the transcription machinery, while 10% are known to be components of transcription regulatory complexes. Based on their transcriptional activity, localization and interaction patterns, at least six previously uncharacterized proteins are suggested to be bona fide transcriptional regulators (namely YFL049W, YJR070C, YDR520C, YGL066W/Sgf73, YKR064W and YCR082W/Ahc2).

Fungal phenalenones inhibit HIV-1 integrase.

A phenalenone compound, atrovenetinone methyl acetal, was isolated from a culture broth of Penicillium sp. FKI-1463 as an HIV-1 integrase inhibitor, and it showed anti-HIV activity in vitro. HIV-1 integrase inhibition and anti-HIV activity of two other natural phenalenones were also studied. Among the tested compounds, funalenone inhibited HIV-1 integrase with an IC50 value of 10 microM and showed the best selectivity (anti-HIV, IC50=1.7 microM; cytotoxicity, IC50=87 microM).

Analysis of gene network regulating yeast multidrug resistance by artificial activation of transcription factors: involvement of Pdr3 in salt tolerance.

We established a strategy to constitutively activate Zn(2)Cys(6)-type protein by fusing its DNA-binding domain with the VP16 trans-activation domain. To explore gene network regulating yeast multidrug resistance, the strategy was applied to Pdr1, Pdr3 and Yrr1, known to regulate multidrug resistance, as well as three uncharacterized Yrr1-related transcription factors. DNA microarray analysis revealed that all of the six mutants induce typical drug transporter genes including SNQ2 and YOR1, suggesting redundancy in regulation. On the other hand, each displays a unique spectrum of targets, which is coincident with the phylogenetic tree of the transcription factors and presumably reflects their functional specification. Indeed, careful analysis of target genes specific to each transcription factor led us to reveal an unexpected role for Pdr3 in salt tolerance. The strategy would thus contribute not only to identify target genes but to reveal redundancy and specificity in complex gene regulatory networks.

Barrier proteins remodel and modify chromatin to restrict silenced domains.

Transcriptionally active and inactive domains are frequently found adjacent to one another in the eukaryotic nucleus. To better understand the underlying mechanisms by which domains maintain opposing transcription patterns, we performed a systematic genomewide screen for proteins that may block the spread of silencing in yeast. This analysis identified numerous proteins with efficient silencing blocking activities, and some of these have previously been shown to be involved in chromatin dynamics. We isolated subunits of Swi/Snf, mediator, and TFIID, as well as subunits of the Sas-I, SAGA, NuA3, NuA4, Spt10p, Rad6p, and Dot1p complexes, as barrier proteins. We demonstrate that histone acetylation and chromatin remodeling occurred at the barrier and correlated with a block to the spread of silencing. Our data suggest that multiple overlapping mechanisms were involved in delimiting silenced and active domains in vivo.

Gag non-cleavage site mutations contribute to full recovery of viral fitness in protease inhibitor-resistant human immunodeficiency virus type 1.

It is well documented that human immunodeficiency virus type 1 (HIV-1) Gag cleavage site mutations (CSMs) emerge in conjunction with various HIV-1 mutations for protease inhibitor (PI) resistance and improve viral replication capacity, which is reduced by acquisition of the resistance. However, CSMs are not the only mutations that emerge in Gag during treatment; many mutations other than CSMs (non-CSMs) have been found to accumulate in the Gag region. In the present study we demonstrate the important role of Gag non-CSMs with regard to viral fitness recovery. We selected three Gag-protease sequences with different PI resistance-associated mutations and CSMs from patients with antiretroviral treatment failure. To clarify the significance of CSMs and non-CSMs, four types of recombinant viruses with different patterns in each sequence were constructed. These were the GP type (patient-derived Gag and protease), the P type (HXB2 Gag and patient-derived protease), the GP(-c) type (CSMs removed from the GP type), and the P(+c) type (CSMs in the HXB2 Gag frame and patient-derived protease). By comparison of these four types of recombinant viruses in each patient-derived Gag-protease sequence, we found that non-CSMs, which had no systematic pattern, make a significant contribution to viral fitness recovery. Our findings demonstrate a delicate interaction between the in vivo evolution of Gag and protease to evade drug selective pressure and the importance of Gag in evaluating drug-resistant viruses.

Novel enzyme-linked minisequence assay for genotypic analysis of human immunodeficiency virus type 1 drug resistance.

We constructed a novel tool for genotypic analysis of human immunodeficiency virus type 1 (HIV-1) drug resistance by using an enzyme-linked minisequence assay (ELMA). ELMA is a combination of hybridization and a 1-base extension reaction, and we designed the assay to detect five mutations conferring nucleoside analogue resistance (M41L, D67N, K70R, T215Y, and M184V) and six mutations conferring protease inhibitor resistance (D30N, M46I, G48V, V82A, I84V, and L90M). At all detection points, ELMA demonstrated high sensitivity and specificity, sufficient for clinical use. Compared to that obtained by direct sequencing, the genotypic information obtained by ELMA is limited to the targeted loci for which it was designed. However, ELMA proves advantageous in several respects. The assay does not require expensive equipment, such as an autosequencer, and can be performed in regular clinical diagnostic laboratories. Therefore ELMA can be a candidate for a drug resistance monitoring assay to be introduced in developing countries. In addition, ELMA demonstrated higher sensitivity in the detection of minor resistant populations. We successfully detected a minor virus population (10%) by the assay. The high sensitivity and specificity of the assay recommend it as a first screening assay for drug resistance surveillance.

Mutagenically separated PCR assay for rapid detection of M41L and K70R zidovudine resistance mutations in CRF01_AE (subtype E) human immunodeficiency virus type 1.

A rapid zidovudine (ZDV) resistance genotypic assay was developed based on the mutagenically separated PCR (MS-PCR) technique to detect two ZDV-resistant mutations, M41L and K70R in CRF01_AE (subtype E). Endpoint dilution analysis revealed that the newly constructed MS-PCR assay could successfully detect three to nine copies of human immunodeficiency virus type 1 template RNA. The test against wild-type and mutant template mixtures in different ratios demonstrated that the assay could detect 10% minor population, at least. Fifty-one subtype E clinical samples were analyzed by the newly constructed MS-PCR assay and direct nucleotide sequencing. The concordance of the two assays was 92 and 100% in codons 41 and 70, respectively. The MS-PCR assay is a rapid, simple, and inexpensive assay that is highly sensitive in detecting mutant targets, including minor populations. Thus, it could be used as a powerful tool for epidemiological surveillance of drug-resistant mutations in developing countries.

Roles for the two-hybrid system in exploration of the yeast protein interactome.

Comprehensive analysis of protein-protein interactions is a challenging endeavor of functional proteomics and has been best explored in the budding yeast. The yeast protein interactome analysis was achieved first by using the yeast two-hybrid system in a proteome-wide scale and next by large-scale mass spectrometric analysis of affinity-purified protein complexes. While these interaction data have led to a number of novel findings and the emergence of a single huge network containing thousands of proteins, they suffer many false signals and fall short of grasping the entire interactome. Thus, continuous efforts are necessary in both bioinformatics and experimentation to fully exploit these data and to proceed another step forward to the goal. Computational tools to integrate existing biological knowledge buried in literature and various functional genomic data with the interactome data are required for biological interpretation of the huge protein interaction network. Novel experimental methods have to be developed to detect weak, transient interactions involving low abundance proteins as well as to obtain clues to the biological role for each interaction. Since the yeast two-hybrid system can be used for the mapping of the interaction domains and the isolation of interaction-defective mutants, it would serve as a technical basis for the latter purpose, thereby playing another important role in the next phase of protein interactome research.