A fission yeast cell-based system for multidrug resistant HIV-1 proteases.

BACKGROUND: HIV-1 protease (PR) is an essential enzyme for viral production. Thus, PR inhibitors (PIs) are the most effective class of anti-HIV drugs. However, the main challenge to the successful use of PI drugs in patient treatment is the emergence of multidrug resistant PRs (mdrPRs). This study aimed to develop a fission yeast cell-based system for rapid testing of new PIs that combat mdrPRs. RESULTS: Three mdrPRs were isolated from HIV-infected patients that carried seven (M7PR), ten (M10PR) and eleven (M11PR) PR gene mutations, respectively. They were cloned and expressed in fission yeast under an inducible promoter to allow the measurement of PR-specific proteolysis and drug resistance. The results showed that all three mdrPRs maintained their abilities to proteolyze HIV viral substrates (MA↓CA and p6) and to confer drug resistance. Production of these proteins in the fission yeast caused cell growth inhibition, oxidative stress and altered mitochondrial morphologies that led to cell death. Five investigational PIs were used to test the utility of the established yeast system with an FDA-approved PI drug Darunavir (DRV) as control. All six compounds suppressed the wildtype PR (wtPR) and the M7PR-mediated activities. However, none of them were able to suppress the M10PR or the M11PR. CONCLUSIONS: The three clinically isolated mdrPRs maintained their viral proteolytic activities and drug resistance in the fission yeast. Furthermore, those viral mdrPR activities were coupled with the induction of growth inhibition and cell death, which could be used to test the PI activities. Indeed, the five investigational PIs and DRV suppressed the wtPR in fission yeast as they did in mammalian cells. Significantly, two of the high level mdrPRs (M10PR and M11PR) were resistant to all of the existing PI drugs including DRV. This observation underscores the importance of continued searching for new PIs against mdrPRs.

HIV-1 Protease in the Fission Yeast Schizosaccharomyces pombe.

BACKGROUND: HIV-1 protease (PR) is an essential viral enzyme. Its primary function is to proteolyze the viral Gag-Pol polyprotein for production of viral enzymes and structural proteins and for maturation of infectious viral particles. Increasing evidence suggests that PR cleaves host cellular proteins. However, the nature of PR-host cellular protein interactions is elusive. This study aimed to develop a fission yeast (Schizosaccharomyces pombe) model system and to examine the possible interaction of HIV-1 PR with cellular proteins and its potential impact on cell proliferation and viability. RESULTS: A fission yeast strain RE294 was created that carried a single integrated copy of the PR gene in its chromosome. The PR gene was expressed using an inducible nmt1 promoter so that PR-specific effects could be measured. HIV-1 PR from this system cleaved the same indigenous viral p6/MA protein substrate as it does in natural HIV-1 infections. HIV-1 PR expression in fission yeast cells prevented cell proliferation and induced cellular oxidative stress and changes in mitochondrial morphology that led to cell death. Both these PR activities can be prevented by a PR-specific enzymatic inhibitor, indinavir, suggesting that PR-mediated proteolytic activities and cytotoxic effects resulted from enzymatic activities of HIV-1 PR. Through genome-wide screening, a serine/threonine kinase, Hhp2, was identified that suppresses HIV-1 PR-induced protease cleavage and cell death in fission yeast and in mammalian cells, where it prevented PR-induced apoptosis and cleavage of caspase-3 and caspase-8. CONCLUSIONS: This is the first report to show that HIV-1 protease is functional as an enzyme in fission yeast, and that it behaves in a similar manner as it does in HIV-1 infection. HIV-1 PR-induced cell death in fission yeast could potentially be used as an endpoint for mechanistic studies, and this system could be used for developing a high-throughput system for drug screenings.

Regulation of unbalanced redox homeostasis induced by the expression of wild-type HIV-1 viral protein R (NL4-3Vpr) in fission yeast.

The wild-type viral protein R (Vpr) of human immunodeficiency virus type 1 exerts multiple effects on cellular activities during infection, including the induction of cell cycle G2 arrest and the death of human cells and cells of the fission yeast Schizosaccharomyces pombe. In this study, wild-type Vpr (NL4-3Vpr) integrated as a single copy gene in S. pombe chromosome was used to investigate the molecular impact of Vpr on cellular oxidative stress. NL4-3Vpr triggered an atypical response in early (14-h), and a wellregulated oxidative stress response in late (35-h) log-phase cultures. Specifically, NL4-3Vpr expression induced oxidative stress in the 14-h cultures leading, to decreased levels of superoxide anion (O(2)(·-)), hydroxyl radical (·OH) and glutathione (GSH), and significantly decreased activities of catalase, glutathione peroxidase, glutathione reductase, glucose-6-phosphate dehydrogenase and glutathione S-transferase. In the 35-h cultures, elevated levels of O(2)(·-) and peroxides were accompanied by increased activities of most antioxidant enzymes, suggesting that the Vpr-induced unbalanced redox state of the cells might contribute to the adverse effects in HIV-infected patients.

HIV-1 replication through hHR23A-mediated interaction of Vpr with 26S proteasome.

HIV-1 Vpr is a virion-associated protein. Its activities link to viral pathogenesis and disease progression of HIV-infected patients. In vitro, Vpr moderately activates HIV-1 replication in proliferating T cells, but it is required for efficient viral infection and replication in vivo in non-dividing cells such as macrophages. How exactly Vpr contributes to viral replication remains elusive. We show here that Vpr stimulates HIV-1 replication at least in part through its interaction with hHR23A, a protein that binds to 19S subunit of the 26S proteasome and shuttles ubiquitinated proteins to the proteasome for degradation. The Vpr-proteasome interaction was initially discovered in fission yeast, where Vpr was shown to associate with Mts4 and Mts2, two 19S-associated proteins. The interaction of Vpr with the 19S subunit of the proteasome was further confirmed in mammalian cells where Vpr associates with the mammalian orthologues of fission yeast Mts4 and S5a. Consistently, depletion of hHR23A interrupts interaction of Vpr with proteasome in mammalian cells. Furthermore, Vpr promotes hHR23A-mediated protein-ubiquitination, and down-regulation of hHR23A using RNAi significantly reduced viral replication in non-proliferating MAGI-CCR5 cells and primary macrophages. These findings suggest that Vpr-proteasome interaction might counteract certain host restriction factor(s) to stimulate viral replication in non-dividing cells.

HIV-1 Vpr-induced cell death in Schizosaccharomyces pombe is reminiscent of apoptosis.

Human immunodeficiency virus type 1 (HIV-1) Vpr induces cell death in mammalian and fission yeast cells, suggesting that Vpr may affect a conserved cellular process. It is unclear, however, whether Vpr-induced yeast cell death mimics Vpr-mediated apoptosis in mammalian cells. We have recently identified a number of Vpr suppressors that not only suppress Vpr-induced cell death in fission yeast, but also block Vpr-induced apoptosis in mammalian cells. These findings suggest that Vpr-induced cell death in yeast may resemble some of the apoptotic processes of mammalian cells. The goal of this study was to develop and validate a fission yeast model system for future studies of apoptosis. Similar to Vpr-induced apoptosis in mammalian cells, we show here that Vpr in fission yeast promotes phosphatidylserine externalization and induces hyperpolarization of mitochondria, leading to changes of mitochondrial membrane potential. Moreover, Vpr triggers production of reactive oxygen species (ROS), indicating that the apoptotic-like cell death might be mediated by ROS. Interestingly, Vpr induces unique morphologic changes in mitochondria that may provide a simple marker for measuring the apoptotic-like process in fission yeast. To verify this possibility, we tested two Vpr suppressors (EF2 and Hsp16) that suppress Vpr-induced apoptosis in mammalian cells in addition to a newly identified Vpr suppressor (Skp1). All three proteins abolished cell death mediated by Vpr and restored normal mitochondrial morphology in the yeast cells. In conclusion, Vpr-induced cell death in fission yeast resembles the mammalian apoptotic process. Fission yeast may thus potentially be used as a simple model organism for the future study of the apoptotic-like process induced by Vpr and other proapoptotic agents.

Human immunodeficiency virus type 1 Vpr induces cell cycle G2 arrest through Srk1/MK2-mediated phosphorylation of Cdc25.

Human immunodeficiency virus type 1 (HIV-1) Vpr induces cell cycle G(2) arrest in fission yeast (Schizosaccharomyces pombe) and mammalian cells, suggesting the cellular pathway(s) targeted by Vpr is conserved among eukaryotes. Our previous studies in fission yeast demonstrated that Vpr induces G(2) arrest in part through inhibition of Cdc25, a Cdc2-specific phosphatase that promotes G(2)/M transition. The goal of this study was to further elucidate molecular mechanism underlying the inhibitory effect of Vpr on Cdc25. We show here that, similar to the DNA checkpoint controls, expression of vpr promotes subcellular relocalization of Cdc25 from nuclear to cytoplasm and thereby prevents activation of Cdc2 by Cdc25. Vpr-induced nuclear exclusion of Cdc25 appears to depend on the serine/threonine phosphorylation of Cdc25 and the presence of Rad24/14-3-3 protein, since amino acid substitutions of the nine possible phosphorylation sites of Cdc25 with Ala (9A) or deletion of the rad24 gene abolished nuclear exclusion induced by Vpr. Interestingly, Vpr is still able to promote Cdc25 nuclear export in mutants defective in the checkpoints (rad3 and chk1/cds1), the kinases that are normally required for Cdc25 phosphorylation and nuclear exclusion of Cdc25, suggesting that others kinase(s) might modulate phosphorylation of Cdc25 for the Vpr-induced G(2) arrest. We report here that this kinase is Srk1. Deletion of the srk1 gene blocks the nuclear exclusion of Cdc25 caused by Vpr. Overexpression of srk1 induces cell elongation, an indication of cell cycle G(2) delay, in a similar fashion to Vpr; however, no additive effect of cell elongation was observed when srk1 and vpr were coexpressed, indicating Srk1 and Vpr are likely affecting the cell cycle G(2)/M transition through the same cellular pathway. Immunoprecipitation further shows that Vpr and Srk1 are part of the same protein complex. Consistent with our findings in fission yeast, depletion of the MK2 gene, a human homologue of Srk1, either by small interfering RNA or an MK2 inhibitor suppresses Vpr-induced cell cycle G(2) arrest in mammalian cells. Collectively, our data suggest that Vpr induces cell cycle G(2) arrest at least in part through a Srk1/MK2-mediated mechanism.

Phosphatase type 2A-dependent and -independent pathways for ATR phosphorylation of Chk1.

ATM and Rad3-related (ATR) is a regulatory kinase that, when activated by hydroxyurea, UV, or human immunodeficiency virus-1 Vpr, causes cell cycle arrest through Chk1-Ser(345) phosphorylation. We demonstrate here that of these three agents only Vpr requires protein phosphatase type 2A (PP2A) to activate ATR for Chk1-Ser(345) phosphorylation. A requirement for PP2A by Vpr was first shown with the PP2A-specific inhibitor okadaic acid, which reduced Vpr-induced G(2) arrest and Cdk1-Tyr(15) phosphorylation. Using small interference RNA to down-regulate specific subunits of PP2A indicated that the catalytic beta-isoform PP2A(Cbeta) and the A regulatory alpha-isoform PP2A(Aalpha) are involved in the G(2) induction, and these downregulations decreased the Vpr-induced, ATR-dependent phosphorylations of Cdk1-Tyr(15) and Chk1-Ser(345). In contrast, the same down-regulations had no effect on hydroxyurea- or UV-activated ATR-dependent Chk1-Ser(345) phosphorylation. Vpr and hydroxyurea/UV all induce ATR-mediated gammaH2AX-Ser(139) phosphorylation and foci formation, but down-regulation of PP2A(Aalpha) or PP2A(Cbeta) did not decrease gammaH2AX-Ser(139) phosphorylation by any of these agents or foci formation by Vpr. Conversely, H2AX down-regulation had little effect on PP2A(Aalpha/Cbeta)-mediated G(2) arrest and Chk1-Ser(345) phosphorylation by Vpr. The expression of vpr increases the amount and phosphorylation of Claspin, an activator of Chk1 phosphorylation. Down-regulation of either PP2A(Cbeta) or PP2A(Aalpha) had little effect on Claspin phosphorylation, but the amount of Claspin was reduced. Claspin may then be one of the phosphoproteins through which PP2A(Aalpha/Cbeta) affects Chk1 phosphorylation when ATR is activated by human immunodeficiency virus-1 Vpr.

Fission yeast homologue of Tip41-like proteins regulates type 2A phosphatases and responses to nitrogen sources.

A fission yeast (Schizosaccharomyces pombe) gene encoding a member of the TIP41-like protein family was identified and characterized. Deletion of the fission yeast tip41 gene leads to slower growth when ammonium chloride is the nitrogen source, but the growth rate is not affected when adenine is the nitrogen source. The tip41 mutant cells also enter the G1 phase of the cell cycle earlier than wild-type cells in response to nitrogen starvation. Overexpression of tip41(+) causes cell death, spherical cell morphology and blocks the shift to G1 phase upon nitrogen starvation. Overexpression of tip41(+) increases the activity of type 2A phosphatase. In a ppa2 deletion strain with reduced PP2A activity, overexpression of tip41(+) no longer blocks the shift to G1 upon nitrogen starvation. These results suggest that fission yeast Tip41 plays a role in cellular responses to nitrogen nutrient conditions at least partly through regulation of type 2A phosphatase activity.

HIV-1 viral protein R (Vpr) & host cellular responses.

During infection of host cells by HIV-1, active host-pathogen interactions take place. The final balance between these interactions determines the efficiency of viral infection and subsequent disease progression. HIV-infected cells respond to viral invasion with various antiviral strategies such as innate, cellular and humoral immune antiviral defense mechanisms. On the other hand, the virus has also developed tactics to suppress these host cellular responses. Among the many viral offensive strategies, viral protein R (Vpr) plays a particularly active role. Vpr involved in nuclear transport of the viral pre-integration complex, activation of viral transcription, induction of cell cycle G2/M arrest and apoptosis of the host cells. However, specific roles of these Vpr activities in viral pathogenesis and their contribution to disease progression are not fully understood. HIV-1 defective for some or all of these Vpr activities have been associated with slow disease progression in some patients. With regard to the host responses to vpr gene expression, studies show that Vpr is specifically targeted by CD8 T-lymphocytes during acute viral infection and that the host innate immune response may also play a crucial role in suppressing the effects of Vpr on various cellular activities. The effect of host cellular responses to vpr gene expression and its roles in nuclear transport, cell cycle G2/M regulation and induction of apoptosis are discussed in this review. Strategies with potential application for future antiviral therapies directed at suppressing Vpr activities are described.

Viral infections and cell cycle G2/M regulation.

Progression of cells from G2 phase of the cell cycle to mitosis is a tightly regulated cellular process that requires activation of the Cdc2 kinase, which determines onset of mitosis in all eukaryotic cells. In both human and fission yeast (Schizosaccharomyces pombe) cells, the activity of Cdc2 is regulated in part by the phosphorylation status of tyrosine 15 (Tyr15) on Cdc2, which is phosphorylated by Wee1 kinase during late G2 and is rapidly dephosphorylated by the Cdc25 tyrosine phosphatase to trigger entry into mitosis. These Cdc2 regulators are the downstream targets of two well-characterized G2/M checkpoint pathways which prevent cells from entering mitosis when cellular DNA is damaged or when DNA replication is inhibited. Increasing evidence suggests that Cdc2 is also commonly targeted by viral proteins, which modulate host cell cycle machinery to benefit viral survival or replication. In this review, we describe the effect of viral protein R (Vpr) encoded by human immunodeficiency virus type 1 (HIV-1) on cell cycle G2/M regulation. Based on our current knowledge about this viral effect, we hypothesize that Vpr induces cell cycle G2 arrest through a mechanism that is to some extent different from the classic G2/M checkpoints. One the unique features distinguishing Vpr-induced G2 arrest from the classic checkpoints is the role of phosphatase 2A (PP2A) in Vpr-induced G2 arrest. Interestingly, PP2A is targeted by a number of other viral proteins including SV40 small T antigen, polyomavirus T antigen, HTLV Tax and adenovirus E4orf4. Thus an in-depth understanding of the molecular mechanisms underlying Vpr-induced G2 arrest will provide additional insights into the basic biology of cell cycle G2/M regulation and into the biological significance of this effect during host-pathogen interactions.

Anti-Vpr activity of a yeast chaperone protein.

Human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) exerts multiple effects on viral and host cellular activities during viral infection, including nuclear transport of the proviral integration complex, induction of cell cycle G(2) arrest, and cell death. In this report, we show that a fission yeast chaperone protein Hsp16 inhibits HIV-1 by suppressing these Vpr activities. This protein was identified through three independent genome-wide screens for multicopy suppressors of each of the three Vpr activities. Consistent with the properties of a heat shock protein, heat shock-induced elevation or overproduction of Hsp16 suppressed Vpr activities through direct protein-protein interaction. Even though Hsp16 shows a stronger suppressive effect on Vpr in fission yeast than in mammalian cells, similar effects were also observed in human cells when fission yeast hsp16 was expressed either in vpr-expressing cells or during HIV-1 infection, indicating a possible highly conserved Vpr suppressing activity. Furthermore, stable expression of hsp16 prior to HIV-1 infection inhibits viral replication in a Vpr-dependent manner. Together, these data suggest that Hsp16 inhibits HIV-1 by suppressing Vpr-specific activities. This finding could potentially provide a new approach to studying the contribution of Vpr to viral pathogenesis and to reducing Vpr-mediated detrimental effects in HIV-infected patients.

A fission yeast homologue of the human uracil-DNA-glycosylase and their roles in causing DNA damage after overexpression.

A functional homologue (ung1) of the human uracil-DNA-glycosylase (UNG) gene was characterized from fission yeast (Schizosaccharomyces pombe). The ung1 gene is highly conserved and encodes a protein with uracil-DNA-glycosylase activity similar to human UNG. The Ung1 protein localizes predominantly to the nucleus, suggesting that it is more similar to the nuclear form (UNG2) than the mitochondrial form (UNG1) of human UNG. Even though deletion of ung1 does not cause any obvious defects, overexpression of ung1 increases the mutation frequency. Overexpression of ung1 or human UNG2 induces a DNA checkpoint-dependent cell cycle delay and causes cell death which is enhanced when the checkpoints are inactive. In addition, the steady-state level of AP (apurinic/apyrimidinic) sites increases after ung1 overexpression, indicating that AP sites are likely to be the DNA damage caused by overexpression. Analysis of mutant ung indicates that catalytic activity is not required for the effects of overexpression, but that binding of Ung1 or UNG2 to AP sites may be important.

Functional conservation of HIV-1 Vpr and variability in a mother-child pair of long-term non-progressors.

Increasing evidence suggests that HIV-1 Vpr is required in vivo for viral pathogenesis. Since Vpr displays multiple activities, little is known about which Vpr-specific activities are conserved in naturally occurring viruses or how natural mutations in Vpr might modulate viral pathogenesis in HIV-infected individuals. The goals of this study were to evaluate the functional variability of Vpr in naturally occurring viruses. The Vpr-specific activities of nuclear localization, induction of cell cycle G2 arrest and cell death were compared between viruses isolated from the fast progressing AIDS patients and a mother-child pair of long-term non-progressors (LTNPs). Wild-type Vpr activities were found in all of the viruses that were isolated from the fast progressing AIDS patients except for the truncated Vpr(IIIB) which lacked these activities. In contrast, defective Vpr were readily detected in viral populations isolated, over an 11-year period, from the mother-child pair. Sequence analyses indicated that these Vpr carried unique amino acid substitutions that frequently interrupted a highly conserved domain containing an N-terminal alpha-helix-turn-alpha-helix. Thus, Vpr activities are generally conserved in naturally occurring viruses. The functionally defective Vpr identified in the mother-child pair of LTNPs are likely to be unique and may possibly contribute to the slow disease progression.

Involvement of rhp23, a Schizosaccharomyces pombe homolog of the human HHR23A and Saccharomyces cerevisiae RAD23 nucleotide excision repair genes, in cell cycle control and protein ubiquitination.

A functional homolog (rhp23) of human HHR23A and Saccharomyces cerevisiae RAD23 was cloned from the fission yeast Schizosaccharomyces pombe and characterized. Consistent with the role of Rad23 homologs in nucleotide excision repair, rhp23 mutant cells are moderately sensitive to UV light but demonstrate wild-type resistance to gamma-rays and hydroxyurea. Expression of the rhp23, RAD23 or HHR23A cDNA restores UV resistance to the mutant, indicating that rhp23 is a functional homolog of the human and S.cerevisiae genes. The rhp23::ura4 mutation also causes a delay in the G2 phase of the cell cycle which is corrected when rhp23, RAD23 or HHR23A cDNA is expressed. Rhp23 is present throughout the cell but is located predominantly in the nucleus, and the nuclear levels of Rhp23 decrease around the time of S phase in the cell cycle. Rhp23 is ubiquitinated at low levels, but overexpression of the rhp23 cDNA induces a large increase in ubiquitination of other proteins. Consistent with a role in protein ubiquitination, Rhp23 binds ubiquitin, as determined by two-hybrid analysis. Thus, the rhp23 gene plays a role not only in nucleotide excision repair but also in cell cycle regulation and the ubiquitination pathways.

HIV-1 VPR modulates cell cycle G2/M transition through an alternative cellular mechanism other than the classic mitotic checkpoints.

HIV-1 Vpr induces cell cycle G2/M arrest in both human and fission yeast cells, suggesting a highly conserved activity of this viral protein. In this review, we summarize the current understanding of Vpr-induced G2 arrest based on studies from both mammalian cells and the fission yeast (Schizosaccharomyces pombe) model system. Fission yeast has proven to be an excellent model system to investigate cell cycle G2/M control of eukaryotic cells. Similarly, fission yeast has also been instrumental in defining the molecular mechanism underlying the G2 arrest induced by Vpr. We have compared the classic DNA-damage and DNA-replication checkpoint controls of the cell cycle G2/M transition to the G2 arrest conferred by Vpr. Based on the current findings, we hypothesize that Vpr induces cell cycle G2 arrest through an alternative novel cellular pathway(s) rather than through the classic mitotic checkpoint controls. A number of cellular proteins which may be involved in this new cellular pathway(s) have been identified and are discussed.