CORD-19_Custom_license_subset  

CORD-19:0aa9c6e5d23acccfae9077750071e2768bdeefe6 JSONTXT 9 Projects

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Id Subject Object Predicate Lexical cue
T1 0-25 Sentence denotes Yeast and the AIDS Virus:
T2 26-40 Sentence denotes The Odd Couple
T3 42-50 Sentence denotes Abstract
T4 51-330 Sentence denotes 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.
T5 331-450 Sentence denotes The complete genome of S. cerevisiae was the first of eukaryotic origin to be sequenced and contains about 6,000 genes.
T6 451-587 Sentence denotes More than 75% of the genes have an assigned function, while more than 40% share conserved sequences with known or predicted human genes.
T7 588-709 Sentence denotes This strong homology has allowed the function of human orthologs to be unveiled starting from the data obtained in yeast.
T8 710-766 Sentence denotes RNA plant viruses were the first to be studied in yeast.
T9 767-947 Sentence denotes 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.
T10 948-991 Sentence denotes This human retrovirus is the cause of AIDS.
T11 992-1164 Sentence denotes 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.
T12 1165-1256 Sentence denotes Current therapy is able to control the disease but there is no permanent cure or a vaccine.
T13 1257-1499 Sentence denotes 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.
T14 1501-1788 Sentence denotes Since the genetics and cell biology of higher eukaryotes are extremely complex, scientists looking for a good model have turned to the use of yeast as a simpler system for the study of various pathologies including virus proliferation and assay new drugs against these pathogenic agents.
T15 1789-1887 Sentence denotes The yeast Saccharomyces cerevisiae is a simple eukaryotic organism with approximately 6,000 genes.
T16 1888-2017 Sentence denotes It is inexpensive to cultivate, can exist in either haploid or diploid states, and is extremely simple for genetic manipulations.
T17 2018-2196 Sentence denotes The complete sequence of this yeast genome, the fruit of a worldwide collaboration of more than 100 laboratories from 1989 to 1996, was the first determined for a eukaryote cell.
T18 2197-2382 Sentence denotes More than 75% of the genes have an assigned function, while more than 40% share conserved sequences with at least one known or predicted human gene ( [1] ; http://www.yeastgenome.org/).
T19 2383-2570 Sentence denotes Owing to the high conservation of fundamental biochemical pathways, yeast has been used as a model to unravel biological processes in many higher eukaryotes (for a short review see [2] ).
T20 2571-2717 Sentence denotes A very important tool facilitating the use of these cells is the availability of yeast libraries in which each nonessential gene has been deleted.
T21 2718-2874 Sentence denotes It has been used for multiple studies including genome wide screenings for human disease genes and host factors that support virus replication [3] [4] [5] .
T22 2875-2971 Sentence denotes The above collection is commercially available and covers more than 90% of all yeast genes [6] .
T23 2972-3175 Sentence denotes An exciting example of the beneficial using this model system is found in two very recent reports, although not related to virus research, on the use of the yeast model to study a severe human pathology.
T24 3176-3423 Sentence denotes Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's Disease, is a neurodegenerative disease in which the motor neurons of the central nervous system that control muscles die off leading to paralysis and death within 3-5 years of onset.
T25 3424-3506 Sentence denotes The cause of the disease is unknown, and there is no treatment to stop or slow it.
T26 3507-3689 Sentence denotes A gene related to ALS, called FUS, has been used to explore its biology in yeast and highlight the potential for modeling elements of complex diseases in this simple eukaryotic cell.
T27 3690-3856 Sentence denotes Results from both studies suggest 2 Journal of Biomedicine and Biotechnology that defects in RNA processing and transport may be a key element of ALS pathophysiology.
T28 3857-4051 Sentence denotes In the cytoplasm of motor neurons of ALS patients, proteins aggregate to form insoluble aggregations, called inclusions, that may involve FUS and another ALS causing protein, called TDP-43 [7] .
T29 4052-4152 Sentence denotes When the two research groups overexpressed human FUS in yeast, they observed cytoplasmic inclusions.
T30 4153-4395 Sentence denotes In humans, the majority of FUS is found in the nucleus, and some ALSassociated mutations reduce the nuclear/cytoplasmic ratio of the protein, suggesting that transfer to the cytoplasm, rather than mutation, may be important in this pathology.
T31 4396-4546 Sentence denotes This hypothesis was supported by the observation that decreasing the overexpression of wild-type FUS in the yeast nucleus diminished its toxic effect.
T32 4547-4592 Sentence denotes Both FUS and TDP-43 are RNA binding proteins.
T33 4593-4775 Sentence denotes Genome-wide screens to identify yeast genes that specifically decreased cell toxicity were performed and the identified genes included some coding for other DNA/RNA binding proteins.
T34 4776-4882 Sentence denotes One of these yeast genes, called ECM32, has a human homolog, hUPF1, which was found to eliminate toxicity.
T35 4883-5040 Sentence denotes Among other functions hUPF1 plays an important role in mRNA quality control, supporting the idea that pathways involving RNA are defective in FUSinduced ALS.
T36 5041-5382 Sentence denotes Interestingly, expression of hUPF1 was able to rescue FUS toxicity in yeast without driving FUS out of the inclusions or sending it back to the nucleus, suggesting that it may be possible to overcome therapeutically the effects of abnormally localized FUS without addressing the difficult problem of restoring it to its original compartment.
T37 5383-5440 Sentence denotes Two important consequences may result from this approach.
T38 5441-5574 Sentence denotes By identifying new genes that can diminish ALS-linked toxicity, they point to RNA processing as a therapeutic target in this disease.
T39 5575-5696 Sentence denotes It has also been shown that yeast has the potential to be a much simpler and exciting system for modeling aspects of ALS.
T40 5697-5936 Sentence denotes Since testing ideas about pathogenesis and treatment is much faster and cheaper in yeast, these results may open the way for more rapid progress in understanding the disease, its treatment, and the role of this new gene in ALS development.
T41 5937-6022 Sentence denotes Important details of other neurodegenerative diseases have been obtained using yeast.
T42 6023-6180 Sentence denotes For example, protein misfolding associated with many human diseases, including Alzheimer's, Parkinson's, and Huntington's disease, has been studied in yeast.
T43 6181-6368 Sentence denotes As in the case of ALS mentioned above, protein misfolding often results in the formation of intracellular or extracellular inclusions whose role in protein misfolding diseases is unclear.
T44 6369-6620 Sentence denotes Studies on the implication of protein aggregation and toxicity in yeast provide an excellent experimental and conceptual paradigm that contributes to understanding the differences between the toxic and protective roles of protein conformation changes.
T45 6621-6824 Sentence denotes Results from these studies using yeast have the potential to transform basic concepts of protein folding in human diseases and may help in identifying new therapeutic strategies for their treatment [8] .
T46 6825-6884 Sentence denotes In virus research, S. cerevisiae is a very useful organism.
T47 6885-6979 Sentence denotes In terms of public health, the use of yeast to produce vaccines was a remarkable breakthrough.
T48 6980-7144 Sentence denotes For example, the first recombinant vaccine, the hepatitis B surface antigen expressed in yeast, has become a safe and efficient prophylactic vaccine worldwide [9] .
T49 7145-7474 Sentence denotes In addition, yeast has proved useful for drug discovery as illustrated by the power of applying genomic approaches using the yeast model to characterize the biological activity of small molecules and to identify their cellular targets, an important step towards understanding the mode of action of human therapeutic agents [10] .
T50 7475-7737 Sentence denotes This study and other recent reports show that the path from sequence and functional analysis to direct therapeutic applications is not necessarily long, at least using the yeast model, a simple organism, which could well be considered among "man's best friends."
T51 7738-7987 Sentence denotes In basic research in Virology, yeast has assisted the elucidation of the function of individual proteins from important pathogenic viruses such as Hepatitis C virus, and Epstein- Barr virus ([11, 12] ; for a review and further references see [13] ).
T52 7988-8197 Sentence denotes Furthermore, studies of viruses that infect yeast have provided many important contributions to the dissection of the life cycle of many other higher eukaryotes RNA viruses and the host factors involved [14] .
T53 8198-8296 Sentence denotes The study of the replication of RNA plant viruses was the first to be attempted using yeast cells.
T54 8297-8501 Sentence denotes The great majority of RNA+ viruses carry their own RNA polymerase and are replicated in the cytoplasm while entry to the nuclear compartment during the viral cycle is confined to retroviruses (see below).
T55 8502-8692 Sentence denotes The first higher eukaryotic virus reported to replicate in yeast was Brome mosaic virus, a positive-strand RNA ((+)RNA) virus that infects plants ( [15] , for an excellent review see [16] ).
T56 8693-8823 Sentence denotes Since the pioneering work of the Ahlquist laboratory, a growing list of viruses has been reported to undergo replication in yeast.
T57 8824-8949 Sentence denotes These include RNA and DNA viruses that infect plants, insects, mammals, and humans (Table 1 ) [17] [18] [19] [20] [21] [22] .
T58 8950-9034 Sentence denotes This wide range of viruses emphasizes the general applicability of the yeast system.
T59 9035-9205 Sentence denotes In this paper, we will focus on the contribution of the yeast model to the study of retroviruses with special emphasis on the human immunodeficiency virus type 1 (HIV-1).
T60 9206-9392 Sentence denotes The yeast two-hybrid system, also called double-hybrid, has been extensively used to search and characterize the potential partner(s) of a given protein ( [23] , for a review see [24] ).
T61 9393-9575 Sentence denotes The basis of this method allows searching and characterizing proteins capable of interacting with a known protein leading to obtaining the cloned genes of these interacting proteins.
T62 9576-9644 Sentence denotes Plasmids are constructed to encode two hybrid proteins ( Figure 1 ).
T63 9645-9925 Sentence denotes One hybrid consists of the DNA-binding domain of the yeast transcriptional activator protein GAL4 fused to the known protein; the other hybrid consists of the GAL4 activation domain fused to protein sequences encoded by a library of yeast (or other sources) genomic DNA fragments.
T64 9926-10122 Sentence denotes Interaction between the known protein and a protein encoded by one of the library plasmids leads to transcriptional activation of a reporter gene easy to detect containing a binding site for GAL4.
T65 10123-10600 Sentence denotes In addition of thousand of examples described in the literature using this system many cellular or viral partners of HIV-1 proteins have been Some examples are the interactions between the human and its ortholog yeast transcription factor SFN5/InI1 described below and that of the lens epithelium-derived growth factor (LEDGF/p75), the latter playing an important role by stimulating the integration step and facilitating the interaction with the host cell chromatin [25, 26] .
T66 10601-10740 Sentence denotes The interaction of HIV-1 IN with microtubules proteins [27] using the two-hybrid approach was confirmed later by using other methods [28] .
T67 10741-10844 Sentence denotes More recently were developed the three-hybrid and onehybrid systems derived from the two-hybrid system.
T68 10845-10942 Sentence denotes Even though retroviruses carry an RNA+ genome, their replicative cycle involves the cell nucleus.
T69 10943-11200 Sentence denotes These viruses are characterized by the fact that they carry an RNAdependent DNA polymerase (reverse transcriptase (RT)) that synthesizes a double-strand DNA called proviral DNA in the cytoplasm of the infected cell by using the viral RNA genome as template.
T70 11201-11437 Sentence denotes Proviral DNA and other viral and cellular factors forming the preintegration complex (PIC) enter the nuclear compartment and integrate the proviral double-stranded DNA in the host genome via an encoded retroviral enzyme, integrase (IN).
T71 11438-11664 Sentence denotes In this paper we will focus on the use of the yeast model system to study the following proteins encoded in the HIV-1: the enzymes reverse transcriptase, integrase and protease, Vpr, Rev and the translation machinery of HIV-1.
T72 11665-11687 Sentence denotes HIV-1 Encoded Enzymes.
T73 11688-11843 Sentence denotes All retroviruses carry three enzymes, an RNA-and DNA-dependent DNA polymerase or reverse transcriptase (RT), integrase (IN), and protease (PR) (see [29] ).
T74 11844-11971 Sentence denotes The organization of these proteins in several retroviruses is shown in Figure 2 while their 3D structure is shown in Figure 3 .
T75 11972-12107 Sentence denotes RT also contains an additional enzymatic activity, RNase H, which has been mapped to a separate, contiguous portion of the polypeptide.
T76 12108-12247 Sentence denotes First identified in the avian viruses, the retroviral enzymes are organized in domains on the Gag-Pro or Gag-Pro-Pol precursor polypeptide.
T77 12248-12315 Sentence denotes These domains are not always cleaved into separate mature proteins.
T78 12316-12457 Sentence denotes In most genera, all enzymes are translated together as a Gag-Pro-Pol precursor, which are processed to yield the mature forms of the enzymes.
T79 12458-12696 Sentence denotes Whether expression of pro and pol is due to frameshifting or termination suppression, they account for approximately 5% of RT and IN on a molecular basis but are synthesized and packaged in the virion at the same level as the Gag protein.
T80 12697-12742 Sentence denotes For most retroviruses, the same holds for PR.
T81 12743-12822 Sentence denotes A scheme describing the retroviral frameshifting process is shown in Figure 7 .
T82 12823-13062 Sentence denotes Since RT, IN, and PR are essential for viral replication and have characteristics that distinguish them from related cellular enzymes, they have all become privileged targets for drugs against the acquired immunodeficiency syndrome (AIDS).
T83 13063-13243 Sentence denotes At a time when the only assay to measure RT activity was in vitro assay, it seemed attractive to use the yeast S. cerevisiae as a model to test the in vivo activity of this enzyme.
T84 13244-13453 Sentence denotes For this purpose constructions of hybrid retrotransposons composed of the endogenous yeast Ty1 element and the reverse transcriptase (RT) of HIV-1 were shown to be active in the yeast Saccharomyces cerevisiae.
T85 13454-13554 Sentence denotes The RT activity of these hybrid Ty1/HIV-1 elements can be monitored by using a simple genetic assay.
T86 13555-13681 Sentence denotes The reverse transcription of yeast carrying this hybrid RT depends on both the DNA polymerase and RNase H domains of HIV-1 RT.
T87 13682-13821 Sentence denotes Most HIV-1 RT inhibitors can be divided into two classes, nucleoside analog RT inhibitors (NRTIs) and nonnucleoside RT inhibitors (NNRTIs).
T88 13822-14145 Sentence denotes NRTIs such as AZT (3-azido-3deoxythymidine) and ddI (dideoxyinosine) inhibit reverse transcription by a chain-termination mechanism; that is, when they are added to a growing DNA chain they block further synthesis while the NNRTIs act by blocking the synthesis by binding specifically an hydrophobic pocket of the HIV-1 RT.
T89 14146-14507 Sentence denotes The reverse transcriptase activity measured in yeast cells was shown to be sensitive to inhibitors of HIV-1 RT like 8Cl-TIBO, a well-characterized nonnucleoside RT inhibitor of HIV-1 RT while the hybrid constructions that express NNRTI-resistant RT variants of HIV-1 are insensitive to8Cl-TIBO demonstrating in yeast the specificity of inhibition in this assay.
T90 14508-14703 Sentence denotes These hybrid Ty1/HIV-1 (his3AI/AIDS RT), called HART, elements carrying NNRTI-resistant variants of HIV-1, RT were used to identify compounds that are active against drug-resistant viruses [30] .
T91 14704-14931 Sentence denotes The reverse transcriptase enzyme purified from the virus is a heterodimer with subunits of 66 and 51 kDa called p66 and p51 (Figure 3 (a)). p51 subunit is a shortened version of p66 after cleavage by the HIV-1 encoded protease.
T92 14932-15030 Sentence denotes As with many other recombinant proteins the yeast S. cerevisiae has been used to express HIV-1 RT.
T93 15031-15223 Sentence denotes Only the HIV-1 gene for the p66 subunit was carried by the expression vector used and surprisingly the recombinant enzyme purified after expression in yeast was the heterodimeric p66/p51 form.
T94 15224-15451 Sentence denotes Protein sequencing showed that the cleavage to produce the p51 subunit gave a product identical to the native viral enzyme showing that yeast possesses a protease with the same exquisite specificity as the HIV-1 protease [31] .
T95 15452-15576 Sentence denotes DNA into the host-cell genome (for general reviews on HIV-1 integrase and further references see [32] [33] [34] [35] [36] ).
T96 15577-15767 Sentence denotes In the first step of the integration reaction, termed 3 -end processing, two nucleotides are removed from each 3 -end of the double strand viral DNA to produce new "hydroxyl ends" (CA-3 OH).
T97 15768-15855 Sentence denotes This reaction occurs in the cytoplasm within a large viral nucleoprotein complex (PIC).
T98 15856-16160 Sentence denotes After entering the nucleus via the PIC multimeric complex, the 5 -ends of processed viral DNA are joined covalently to the host Journal of Biomedicine and Biotechnology Figure 3 (b)) is characterized by three highly conserved amino acid residues, D64, D116, and E152, forming the catalytic triad DD(35)E.
T99 16161-16221 Sentence denotes Each one of these residues is essential for enzyme activity.
T100 16222-16316 Sentence denotes The Cterminal region is the least conserved domain and is involved in nonspecific DNA binding.
T101 16317-16419 Sentence denotes The 3D structure of the three domains of HIV-1 IN has been determined by RMN or X-ray crystallography.
T102 16420-16586 Sentence denotes Despite considerable efforts, the low solubility of the native HIV-1 integrase has hampered the determination of the crystallographic structure of the entire protein.
T103 16587-16682 Sentence denotes The three HIV-1 IN domains are required for in vitro 3 -end processing and DNA strand transfer.
T104 16683-16866 Sentence denotes Mutation analyses of the viral integrase gene showed that this enzyme is required for retroviral replication and that it is a legitimate target for the design of antiretroviral drugs.
T105 16867-17094 Sentence denotes Recently, the 3D structure of the human foamy retrovirus integrase was determined, taking profit of the increased solubility and yield of this recombinant retroviral enzyme, and confirming a tetramer quaternary structure [37] .
T106 17095-17397 Sentence denotes Based on results dating from 1985 where the expression of the bacterial EcoR1 restriction endonuclease expressed in yeast cells led to the appearance of a lethal phenotype [38] the idea emerged that the expression of retroviral IN also carrying an endonuclease activity may produce a similar phenotype.
T107 17398-17561 Sentence denotes The idea behind this approach was that the feasibility of such a system may allow the setting up of an easy and rapid procedure for screening antiretroviral drugs.
T108 17562-17665 Sentence denotes However, the weak entrance of molecules through the yeast wall and cell membrane hampered this project.
T109 17666-17780 Sentence denotes The yeast model has been extremely useful to study many aspects of the integration step in retroviral replication.
T110 17781-17968 Sentence denotes An expression plasmid containing the retroviral integrase gene under the control of the inducible promoter ADH2/GAPDH regulated by the glucose concentration of the medium was constructed.
T111 17969-18080 Sentence denotes Haploid yeast strain W303-1A did not appear to be clearly sensitive to HIV-1 integrase expression ( Figure 4 ).
T112 18081-18271 Sentence denotes However, disruption of the RAD 52 gene, which is involved in the repair of double-strand DNA breaks, strongly increased the deleterious effects of the retroviral enzyme in this yeast strain.
T113 18272-18414 Sentence denotes The diploid strain constructed with W303-1A and an isogenic strain of the opposite mating type also showed strong sensitivity to the HIV-1 IN.
T114 18415-18581 Sentence denotes The lethal phenotype was suppressed by missense integrase mutations in the catalytic domain that are known to abolish HIV-1 IN activities in vitro ( Figure 4 ) [39] .
T115 18582-18745 Sentence denotes Subsequent studies were performed in order to determine the critical amino acid(s) and/or motif(s) required for the induction of the lethal phenotype in the [40] .
T116 18746-18945 Sentence denotes The question remained whether the lethal effect was related to the nonspecific endonuclease activity of the viral IN or whether the mechanism involved was due to a pleiotropic effect of this protein.
T117 18946-19130 Sentence denotes Lethality in yeast seems to be related to the mutagenic effect of the recombinant HIV-1 IN, most probably via the non-sequence-specific endonuclease activity carriedout by this enzyme.
T118 19131-19205 Sentence denotes This nonsequence-specific endonuclease activity was further characterized.
T119 19206-19425 Sentence denotes Although the enzyme was active on DNA substrates devoid of viral long terminal repeat (LTR) sequences characteristic of the retroviral proviral genome, the presence of LTR regions significantly stimulated this activity.
T120 19426-19668 Sentence denotes Genetic experiments showed that both the mutagenic effect and the level of recombination events were affected in cells expressing the active retroviral enzyme, while expression of the mutated inactive IN D116A had no significant effect [41] .
T121 19669-19933 Sentence denotes As mentioned above a nucleoprotein complex (PIC) comprising the proviral DNA, the IN, and other viral and cellular proteins is formed in the cytoplasm and enters the nucleus by an unknown mechanism where the retroviral DNA is inserted into the host nuclear genome.
T122 19934-20073 Sentence denotes The exact composition of the PIC is a controversial matter since different laboratories have described various viral and cellular proteins.
T123 20074-20273 Sentence denotes The yeast two-hybrid system was used to identify a human gene product that binds tightly to the human immunodeficiency virus type 1 (HIV-1) integrase in vitro and stimulates its DNA-joining activity.
T124 20274-20331 Sentence denotes This protein has been suggested as being part of the PIC.
T125 20332-20493 Sentence denotes The sequence of the gene suggests that the protein is a human homolog of yeast SNF5, a transcriptional activator required for highlevel expression of many genes.
T126 20494-20653 Sentence denotes The gene, termed INI1 (for integrase interactor 1), may encode a nuclear factor that promotes integration and targets incoming viral DNA to active genes [25] .
T127 20654-20801 Sentence denotes The analogy of the yeast and human orthologs prompted study of whether yeast SNF5 is involved in the lethal effect of HIV-1 IN expression in yeast.
T128 20802-20949 Sentence denotes The effect of the inactivation of the yeast gene encoding for SNF5 on the lethality induced by the yeast expression of HIV-1 IN has been described.
T129 20950-21165 Sentence denotes Results showed that the retroviral IN is unable to perform its lethal activity in cells where the SNF5 gene has been disrupted, suggesting that SNF5 may play a role in the lethal effect induced by IN in yeast [42] .
T130 21166-21250 Sentence denotes SNF5 inactivationdoes not affect neither yeast viability nor expression of HIV-1 IN.
T131 21251-21405 Sentence denotes Given the homology between SNF5 and its human counterpart INI1, these results suggest that this factor may be important for IN activity in infected cells.
T132 21406-21687 Sentence denotes Moreover, given the important role proposed for this transcription factor in the integration step and the fact that it is not essential for cell viability, the interaction between INI1/ySNF5 and HIV-1 IN should become a potential target in the search for new antiretroviral agents.
T133 21688-21921 Sentence denotes Given the proximity of many human and yeast proteins the expression of the human INI1 rescued the lethal phenotype in yeast cells where SNF5 was inactivated, again revealing the functional analogy of many human and yeast genes [42] .
T134 21922-22091 Sentence denotes A question that has not yet been fully answered concerning the HIV-1 cycle is the mechanism of entry of the PIC into the nucleus previous to integration in the host DNA.
T135 22092-22597 Sentence denotes This question about intracellular HIV-1 trafficking has been tackled using the yeast system.Based on previous results using the two-hybrid strategy and showing that HIV-1 IN interacts with two yeast microtubule-associated proteins, Dyn2p (dynein light chain protein) and Stu2p, a centrosomal protein [43] , the role of these proteins in intracellular trafficking and nuclear import in yeast was studied since it is thought that these biological mechanisms are evolutionarily conserved in eukaryotic cells.
T136 22598-22760 Sentence denotes IN was found to accumulate at the perinuclear microtubule organizing center (MTOC) called the yeast spindle pole body (SPB) via Stu2p colocalization ( Figure 5 ).
T137 22761-22974 Sentence denotes Disruption of the microtubule network by nocodazole or IN expression in a dynein2-deficient yeast strain prevented IN accumulation in the nuclear periphery and additionally inhibited IN transport into the nucleus.
T138 22975-23092 Sentence denotes By mutagenesis, the results indicated that trafficking of IN towards the SPB requires the C-terminus of the molecule.
T139 23093-23123 Sentence denotes Perinuclear Nuclear [28, 45] .
T140 23124-23338 Sentence denotes The strong similarities between human infected cells and budding yeast demonstrate the relevance of the yeast model to obtain a detailed picture of the molecular mechanisms involved in the HIV-1 cycle ( Figure 5 ).
T141 23339-23524 Sentence denotes 8 Journal of Biomedicine and Biotechnology As described above HIV-1 IN expressed in yeast in the absence of other viral proteins is able to enter the nucleus and cleave the host genome.
T142 23525-23656 Sentence denotes An exciting question remained whether it is able to integrate a proviral-like DNA in the nuclear yeast genome when expressed alone.
T143 23657-23850 Sentence denotes This was solved by setting up a system expressing only the IN and a short DNA mimicking the retroviral DNA by carrying the two LTRs flanking an antibiotic (zeocin) resistance gene ( Figure 6 ).
T144 23851-24029 Sentence denotes Multiple resistant yeast clones were observed under these conditions, while very few clones were obtained when using constructions carrying no IN or the inactive IN mutant D116A.
T145 24030-24204 Sentence denotes Zeocin-resistant clones were confirmed to represent real integration events by detecting the 800 bp proviral-like DNA in the yeast genomic DNA by PCR or Southern experiments.
T146 24205-24338 Sentence denotes Moreover, the sequence of the integration junctions showed that each integration event was disrupting a different open reading frame.
T147 24339-24514 Sentence denotes Since the sequence of these links between integrated LTRs and the target DNA constitutes a specific signature of the IN involved in this process, they were carefully analyzed.
T148 24515-24652 Sentence denotes The 5 bp repeats characterizing HIV integration were recovered for most clones, confirming that HIV-1 IN was responsible for the process.
T149 24653-24924 Sentence denotes Therefore, in this simple eukaryotic model, the retroviral integrase is the only viral protein necessary for the insertion of a DNA containing viral LTRs into the genome, thereby allowing the study of the isolated integration step independently of other viral mechanisms.
T150 24925-25037 Sentence denotes The identification of the yeast factors involved in the HIV-1 IN trafficking remains an interesting perspective.
T151 25038-25208 Sentence denotes Another issue is whether these cellular factors, which are analogs to those forming the PIC in human infected cells, may be used to reconstitute a PIC-like complex [46] .
T152 25209-25346 Sentence denotes Moreover these results may indicate that at least a fraction of the pool of integrase alone may enter the nucleus independent of the PIC.
T153 25347-25429 Sentence denotes The last step of the integration process involves the repair of the DNA junctions.
T154 25430-25550 Sentence denotes It has been widely reported that the cellular DNA repair system plays a crucial role in the retroviral integration step.
T155 25551-25627 Sentence denotes DNA repair factors like RAD18, RAD51, and RAD52 modulate this process [47] .
T156 25628-25843 Sentence denotes The effect of RAD51 and other DNA repair factors can be easily studied in yeast since, contrary to what has been observed in human cells, no deleterious effects are observed when RAD51 or RAD18 are deleted in yeast.
T157 25844-25984 Sentence denotes When RAD51 was deleted, a striking increase in integration events was observed after expression of IN and the viral-like DNA in yeast [46] .
T158 25985-26154 Sentence denotes Very recently, the close interaction between IN and RAD51 and the molecular mechanism played by this protein in HIV-1 integration was described in yeast and human cells.
T159 26155-26409 Sentence denotes In vitro integration assays performed under various conditions promoting or inhibiting hRAD51 activity demonstrated that the formation of an active hRAD51 nucleofilament is required for optimal inhibition involving the dissociation of the IN-DNA complex.
T160 26410-26571 Sentence denotes Furthermore, the availability of the RAD51 stimulatory agent, RAS-1, made it possible to show that this inhibition mechanism could be promoted in HIV- Figure 6 :
T161 26572-26599 Sentence denotes Integration assay in yeast.
T162 26600-26800 Sentence denotes Adapted from [46] . the restriction of viral proliferation by RAD51 pave the way for a new concept of antiretroviral therapy based on the enhancement of endogenous hRAD51 recombination activity [48] .
T163 26801-27040 Sentence denotes A lethal effect similar to that obtained by the expression of HIV-1 IN in yeast was observed more recently when the retrovirus-encoded protease (HIV-1 PR, Figure 3 (c)) was expressed both in Saccharomyces cerevisiae and in mammalian cells.
T164 27041-27127 Sentence denotes These findings contribute to a deeper understanding of HIV-1-induced cytopathogenesis.
T165 27128-27195 Sentence denotes Expression of HIV-1 PR stopped yeast growth followed by cell lysis.
T166 27196-27335 Sentence denotes The lytic phenotype included loss of plasma membrane integrity and cell wall breakage leading to the release of cell content to the medium.
T167 27336-27534 Sentence denotes Interestingly, this effect seems to be specific for HIV-1 PR since neither poliovirus 2A protease nor 2BC protein, which are both highly toxic for S. cerevisiae, was able to produce similar effects.
T168 27535-27632 Sentence denotes Drastic alterations in membrane permeability preceded the lysis in yeast expressing the HIV-1 PR.
T169 27633-27752 Sentence denotes The morphological changes after expression of HIV-1 PR in yeast and mammalian cells were similar in many aspects [49] .
T170 27753-27907 Sentence denotes During the replication of retroviruses, large numbers of Gag molecules must be generated to serve as precursors to the structural proteins of the virions.
T171 27908-28056 Sentence denotes However, the enzymes encoded by the pro and pol genes (PR, RT, and IN) are generally needed in lower amounts to carry out their catalytic functions.
T172 28057-28252 Sentence denotes Retroviruses have developed a mechanism leading to the synthesis of the Gag protein at higher levels relative to the pro and pol gene products, while retaining coregulated expression (Figure 7 ).
T173 28253-28361 Sentence denotes This is due to the use of the same initiation codon in the same mRNA to express the gag, pro, and pol genes.
T174 28362-28487 Sentence denotes Translation of this RNA leads occasionally to synthesis of a fusion protein that is usually called the Gag-Pro-Pol precursor.
T175 28488-28627 Sentence denotes Typically allows the same mechanism that targets the Gag precursor to the site of virion assembly also to direct the Gag-Pro-Pol precursor.
T176 28628-28744 Sentence denotes In all retroviruses, the gag gene is positioned at the 5 end of the viral genome, upstream of the pro and pol genes.
T177 28745-28976 Sentence denotes The Gag-Pro-Pol precursor is generated using a strategy in which the termination codon that defines the 3 terminus of the gag reading frame is bypassed, allowing translation to continue into the adjacent pro and pol reading frames.
T178 28977-29041 Sentence denotes Bypass of the termination codon occurs by one of two mechanisms.
T179 29042-29225 Sentence denotes The first mechanism (used by the mammalian type-C retroviruses) is read through (termination) suppression, in which the gag termination codon is occasionally misread as a sense codon.
T180 29226-29315 Sentence denotes Translation then continues past the termination codon and into the pro-pol reading frame.
T181 29316-29401 Sentence denotes The second mechanism, which is used by most retroviruses, is ribosomal frameshifting.
T182 29402-29526 Sentence denotes Here, occasional ribosomes slip backward one nucleotide (-1 frameshift, i.e., in the 5 direction) during translation of gag.
T183 29527-29683 Sentence denotes Thus, the ribosome leaves the gag reading frame (with its downstream termination codon) and shifts into an overlapping portion of the pro-pol reading frame.
T184 29684-29688 Sentence denotes Vpr.
T185 29689-29833 Sentence denotes The 14-Kd, 96-amino-acid HIV-1 encoded protein Vpr (viral protein R) [50, 51] , plays several roles in the replication cycle of this retrovirus.
T186 29834-30021 Sentence denotes Thus, it has been proposed that Vpr regulates the nuclear import of the preintegration complex (PIC) carrying the proviral DNA, the viral integrase, and other viral and cellular proteins.
T187 30022-30364 Sentence denotes It is also required in the replication of HIV-1 in nondividing cells like the macrophages, and much evidence indicates that it is able to induce the arrest of cell cycle at the G2 step in proliferating cells, the latter effect likely playing an important role in the immunosuppression process in AIDS patients (for a recent review see [52] ).
T188 30365-30504 Sentence denotes Several lines of evidence in yeast and human cells show that Vpr has an important effect on the G2 to M transition of the cell cycle [53] .
T189 30505-30687 Sentence denotes It has been reported that the induction of Vpr expression in the fission yeast Schizosaccharomyces pombe leads to several defects in the assembly and function of the mitotic spindle.
T190 30688-30791 Sentence denotes Some spindle pole body proteins were delocalized in Vprexpressing yeast cells perturbing its integrity.
T191 30792-30903 Sentence denotes In addition, nuclear envelope structure, contractile actin ring formation, and cytokinesis were also disrupted.
T192 30904-31099 Sentence denotes As similar problems in mitosis and cytokinesis were observed in human cells, it has been suggested that these defects account for some of the pathological effects associated with HIV-1 infection.
T193 31100-31336 Sentence denotes When VPR was expressed in the fission yeast Schizosaccharomyces pombe the induction of cell death observed was reminiscent of the effect of HIV-1 infection in mammalian cells, suggesting that VPR may affect a conserved cellular process.
T194 31337-31527 Sentence denotes In recent years proteins have been identified as Vpr suppressors that are able to overcome Vpr-induced cell death in fission yeast as well as arrest Vpr-induced apoptosis in mammalian cells.
T195 31528-31676 Sentence denotes Although not conclusive these results may suggest that Vpr-induced cell death in yeast resembles some of the apoptotic processes in mammalian cells.
T196 31677-31925 Sentence denotes Like Vpr-induced apoptosis in mammalian cells where mitochondria play an essential role, Vpr in yeast promotes phosphatidylserine externalization and induces hyperpolarization of mitochondria, leading to changes in mitochondrial membrane potential.
T197 31926-32097 Sentence denotes Moreover, Vpr triggers production of reactive oxygen species (ROS), indicating that cell death via an apoptotic-like mechanism might be mediated by these reactive species.
T198 32098-32248 Sentence denotes As mitochondria play a crucial role in apoptosis, it is intriguing that, in fission yeast, Vpr induces unique morphologic changes in these organelles.
T199 32249-32449 Sentence denotes The Vpr suppressor factors EF2, Hsp16, and Skp1 that suppress Vpr-induced apoptosis in mammalian abolished cell death mediated by Vpr and restored normal mitochondrial morphology in yeast cells [54] .
T200 32450-32704 Sentence denotes The similarity of Vpr-induced cell death in fission yeast with the mammalian apoptotic process reinforces the idea that fission yeast may be used as a simple model organism to study the apoptotic-like process induced by Vpr and other proapoptotic agents.
T201 32705-32914 Sentence denotes The 116-amino-acid HIV-1 Rev, an 18 kDa phosphoprotein discovered in 1986, is capable of being imported into the nucleus and binding specially to the Rev responsive element (RRE) a viral sequence-specific RNA.
T202 32915-33013 Sentence denotes Rev is able to form multimers and direct the nuclear export of large RRE-containing RNP complexes.
T203 33014-33108 Sentence denotes Rev activity is crucial in the nuclear export of intron-containing HIV-1 RNAs [55] [56] [57] .
T204 33109-33245 Sentence denotes Rev shuttles between the nucleus and the cytoplasm and has a nuclear localization signal (NLS) as well as a nuclear export signal (NES).
T205 33246-33394 Sentence denotes These essential peptide motifs have now been shown to function by accessing cellular signal-mediated pathways for nuclear import and nuclear export.
T206 33395-33552 Sentence denotes In addition to NLS and NES a nuclear import inhibitory signal (NIS) that inhibits the entry of low molecular weight proteins has been described in Rev [58] .
T207 33553-33660 Sentence denotes HIV-1 Rev is therefore an excellent system to study aspects of transport across the nuclear envelope [59] .
T208 33661-33770 Sentence denotes Human immunodeficiency virus type 1 (HIV-1) replication requires the expression of two classes of viral mRNA.
T209 33771-33868 Sentence denotes The early class of HIV-1 transcripts is fully spliced and encodes viral regulatory gene products.
T210 33869-34049 Sentence denotes The functional expression of Rev induces the cytoplasmic expression of the unspliced or incompletely spliced mRNAs that encode the viral structural proteins, including Gag and Env.
T211 34050-34215 Sentence denotes Based on experiments that indicate a similar function of Rev in the yeast S. cerevisiae, a yeast protein interacting with the effector domain of Rev was found [60] .
T212 34216-34332 Sentence denotes This protein called Rip1p is a novel small nucleoporin-like protein, some of which is associated with nuclear pores.
T213 34333-34452 Sentence denotes Its closest known yeast relative is a nuclear pore component also involved in mRNA transport from nucleus to cytoplasm.
T214 34453-34691 Sentence denotes Analysis of yeast strains that overexpress Rip1p or which are deleted for the RIP1 gene show that Rip1p is important for the effect of Rev on gene expression, indicating that the physical interaction is of functional significance in vivo.
T215 34692-34817 Sentence denotes These results suggest that Rev directly promotes the cytoplasmic transcripts transport by targeting them to the nuclear pore.
T216 34818-34958 Sentence denotes The NES domain of the Rev protein is required for Rev-mediated RNA export in mammals as well as in the yeast Saccharomyces cerevisiae [61] .
T217 34959-35104 Sentence denotes As mentioned above Rev NES has been shown to specifically interact with a human (hRIP/RAB1) and a yeast (yRip1p) protein in the two-hybrid assay.
T218 35105-35225 Sentence denotes Both of these interacting proteins are related to FG nucleoporins on the basis of the presence of typical repeat motifs.
T219 35226-35334 Sentence denotes Rev is able to interact with multiple FG repeat-containing nucleoporins from both S. cerevisiae and mammals.
T220 35335-35527 Sentence denotes Moreover, the ability of Rev NES mutants to interact with these FG nucleoporins parallels the ability of the mutants to promote RNA export in yeast, Xenopus oocytes, and mammalian cells [62] .
T221 35528-35648 Sentence denotes HIV-1 Rev-mediated nuclear export of viral RNAs involves the interaction of its leucine-rich NES with nuclear cofactors.
T222 35649-35895 Sentence denotes Using an extensive panel of nuclear export positive and negative mutants of the functionally homologous NES of the HIV-1 Rev, physiologically significant interactions of human Nup98 (hNup98) with NESs from various viral sources were demonstrated.
T223 35896-36088 Sentence denotes Recently, a cellular factor called CRM-1 was shown to be an essential nuclear export factor interacting directly with nuclear export signals including the Rev NES in a RanGTP-dependent manner.
T224 36089-36263 Sentence denotes It was shown that NLP-1, like the previously described Rev-interacting protein hRIP/Rab and several nucleoporins, also interacts with CRM-1 in both yeast and mammalian cells.
T225 36264-36407 Sentence denotes Missense mutations in the yeast nuclear export factor Crm1p abrogated Rev NES interaction with the XXFG repeat-containing nucleoporin Rab/hRIP.
T226 36408-36498 Sentence denotes These mutations had minimal effects on the interaction with GLFG repeat-containing hNup98.
T227 36499-36674 Sentence denotes Functional analysis of the Nup98 domains required for nuclear localization demonstrated that the entire ORF was required for efficient incorporation into the nuclear envelope.
T228 36675-36766 Sentence denotes A putative nuclear localization signal was identified downstream of the GLFG repeat region.
T229 36767-37072 Sentence denotes Whereas overexpression of both full-length Nup98 and the amino-terminal GLFG repeat region, but not the unique carboxy-terminal region, induced significant suppression of HIV unspliced RNA export, lower levels of exogenous Nup98 expression resulted in a relatively modest increase in unspliced RNA export.
T230 37073-37193 Sentence denotes These results suggest a physiological role for hNup98 in modulating Rev-dependent RNA export during HIV infection [63] .
T231 37194-37202 Sentence denotes Peptide.
T232 37203-37296 Sentence denotes The NIS signal encoded in Rev has been used as a therapeutic agent with anticandidal effects.
T233 37297-37438 Sentence denotes In addition this property was used to study the mechanism of action of this factor by studying the intracellular localization of the peptide.
T234 37439-37513 Sentence denotes The result showed that Rev rapidly accumulated on the fungal cell surface.
T235 37514-37662 Sentence denotes The cell wall regeneration test also indicated that Rev exerts its anticandidal activity on the fungal plasma membrane rather than on the cell wall.
T236 37663-37877 Sentence denotes By using fluorescent probes, the membrane-disruption mechanism of Rev was further confirmed, suggesting that it may be a potential therapeutic agent for treating fungal diseases caused by Candida species in humans.
T237 37878-38032 Sentence denotes Further studies showed that the antifungal effects of the nuclear entry inhibitory signal peptide of HIV-1 Rev protein had no hemolytic effects [64, 65] .
T238 38033-38266 Sentence denotes Retroviral frameshifting (see above) is a change in reading frame during gene expression and is a mechanism that allows to keep at a low level the synthesis rate of the functional proteins relative to that of the structural proteins.
T239 38267-38444 Sentence denotes Using S. cerevisiae to decipher viral frameshifting mechanisms Wilson et al. were the first to provide an in vivo demonstration of a frameshifting event, in S. cerevisiae [67] .
T240 38445-38640 Sentence denotes They inserted the Gag-Pol fragment containing the potential frameshifting site of HIV-1 (without the stimulatory element) into a yeast expression plasmid, upstream from the interferon (IFN) cDNA.
T241 38641-38715 Sentence denotes They monitored production of the frameshifted protein by Western blotting.
T242 38716-38899 Sentence denotes It is now clear that a stimulatory secondary structure is required for maximal frameshifting efficiency although the precise nature of this structure remained unclear for a long time.
T243 38900-39066 Sentence denotes Results using a dual reporter system in yeast showed that there is a direct correlation between HIV frameshifting efficiency and the stability of the stem loop [68] .
T244 39067-39178 Sentence denotes The stem loop analyzed in these studies was the upper part of the complete stimulatory element observed by NMR.
T245 39179-39281 Sentence denotes Under these conditions, the stability of this structure is clearly linked to frameshifting efficiency.
T246 39282-39458 Sentence denotes A structure was recently identified on the basis of the complete yeast genome sequence, and it seems interesting to explore frameshifting efficiency with the complete sequence.
T247 39459-39579 Sentence denotes The structure of the tetraloop is similar to the motif found in the RNase III recognition site from S. cerevisiae [69] .
T248 39580-39842 Sentence denotes As the ACAA motif in the tetraloop is poorly recognized by RNase III, the possibility of engineering the S. cerevisiae RNase III for selective targeting of the HIV-1 tetraloop followed by the expression of this protein in HIV-1-infected cells has been suggested.
T249 39843-40013 Sentence denotes This hypothetical approach remains to be confirmed but would provide an interesting therapeutic strategy, derived from experiments in yeast, to limit HIV-1 proliferation.
T250 40014-40201 Sentence denotes The similar slippage efficiencies of the HIV frameshifting site in vivo in yeast and in vitro in a mammalian system demonstrate the high level of conservation of frameshifting mechanisms.
T251 40202-40441 Sentence denotes Bidou et al. [66] suggested that the demonstration that frameshifting is conserved from yeast to humans paves the way for the use of yeast mutants to analyze retroviral frameshifting as already reported by several groups for other viruses.
T252 40442-40566 Sentence denotes For instance, SARS coronavirus (sometimes shortened to SARS-CoV) is the virus that causes severe acute respiratory syndrome.
T253 40567-40613 Sentence denotes SARS-CoV carries a frameshifting signal [70] .
T254 40614-40754 Sentence denotes The minimal frameshifting signal in this virus is a U UUA AAC slippery sequence and a stimulatory structure folding into a pseudoknot [71] .
T255 40755-40921 Sentence denotes This pseudoknot has several unusual features, including the third stem in loop 2 and the presence of two unpaired adenosine residues within the structure ( [70, 72] .
T256 40922-41017 Sentence denotes Plant and Dinman [70] demonstrated the ability of this new site to frameshift in S. cerevisiae.
T257 41018-41171 Sentence denotes The frequency of frameshifting in yeast was much lower (3%) than for other coronavirus sites tested in yeast (12% for infectious bronchitis virus (IBV)).
T258 41172-41263 Sentence denotes This may indicate the existence of subtle differences in terms of frameshifting mechanisms.
T259 41264-41408 Sentence denotes Indeed, the importance of the unpaired adenosine residues remains unclear as this part of the pseudoknot is thought to lie outside the ribosome.
T260 41409-41605 Sentence denotes It would be interesting to investigate the possible binding of a transacting factor, although the binding of such a factor has never been detected with the well-studied IBV coronavirus pseudoknot.
T261 41606-41688 Sentence denotes The yeast mak8-1 mutant is known to have a specific defect in frameshifting [73] .
T262 41689-41785 Sentence denotes It carries an altered form of ribosomal protein L3 in the ribosomal peptidyl-transferase center.
T263 41786-41981 Sentence denotes This strain was reported to have a slightly higher SARS-CoV frameshifting efficiency than wildtype strains [70] , in the first demonstration that this newly discovered frameshifting site is used.
T264 41982-42118 Sentence denotes In summary, although much of the potential promise of yeast is still to be revealed, it has proved extremely valuable in virus research.
T265 42119-42339 Sentence denotes In addition to techniques like the double-hybrid system, this simple eukaryotic cell has been very useful for producing recombinant viral proteins whose purification from native virions is difficult or simply impossible.
T266 42340-42534 Sentence denotes It also allows the mechanism of action of viral proteins to be studied thanks to the close analogy between human and yeast proteins, and this has led to the emergence of new therapeutic targets.
T267 42535-42839 Sentence denotes By associating the toxic phenotype induced by some viral proteins in yeast cells with the genetic manipulation facilitating the entry of drugs with potential therapeutic properties, it may become possible to establish a simple cheap system allowing faster screening of the antiviral agents of the future.
T268 42840-43040 Sentence denotes Moreover, future work should lead to the discovery of new cellular factors involved in virus proliferation, thus shortening the time necessary to develop new therapies against current and new viruses.
T269 43041-43303 Sentence denotes The ultimate information on the behavior of viruses or virus proteins inside the cell should be attained with plant, animal, and human cells or in vivo in whole organisms, although it may be difficult and costly for most laboratories to develop these approaches.
T270 43304-43563 Sentence denotes Since experiments with yeast will always be technically easier, more rapid, and cheaper than those with human and other complex eukaryote cells, yeast will remain a method of choice for studying virus infections mechanisms and the search for new drug targets.