Inhibition of HIV-1 replication by cell-penetrating peptides binding Rev

New therapeutic agents able to block HIV-1 replication are eagerly sought after to increase the possibilities of treatment of resistant viral strains. In this report, we describe a rational strategy to identify small peptide sequences owning the dual property of penetrating within lymphocytes and of binding to a protein target. Such sequences were identified for two important HIV-1 regulatory proteins, Tat and Rev. Their association to a stabilizing domain consisting of human small ubiquitin-related modifier-1 (SUMO-1) allowed the generation of small proteins named SUMO-1 heptapeptide protein transduction domain for binding Tat (SHPT) and SUMO-1 heptapeptide protein transduction domain for binding Rev (SHPR), which are stable and efficiently penetrate within primary lymphocytes. Analysis of the antiviral activity of these proteins showed that one SHPR is active in both primary lymphocytes and macrophages, whereas one SHPT is active only in the latter cells. These proteins may represent prototypes of new therapeutic agents targeting the crucial functions exerted by both viral regulatory factors.     

Sam68 is absolutely required for Rev function and HIV-1 production

Sam68 functionally complements for, as well as synergizes with, HIV-1 Rev in Rev response element (RRE)-mediated gene expression and virus production. Furthermore, C-terminal deletion/point mutants of Sam68 (Sam68ΔC/Sam68-P21) exert a transdominant negative phenotype for Rev function and HIV-1 production. However, the relevance of Sam68 in Rev/RRE function is not well defined. To gain more insight into the mechanism of Sam68 in Rev function, we used an RNAi (RNA interference) strategy to create stable Sam68 knockdown HeLa (SSKH) cells. In SSKH cells, Rev failed to activate both RRE-mediated reporter gene [chloramphenicol acetyltransferase (CAT) and/or gag] expressions. Importantly, reduction of Sam68 expression led to a dramatic inhibition of HIV-1 production. Inhibition of the reporter gene expression and HIV production correlated with the failure to export RRE-containing CAT mRNA and unspliced viral mRNAs to the cytoplasm, confirming that SSKH cells are defective for Rev-mediated RNA export. Taken together, these results suggest that Sam68 is involved in Rev-mediated RNA export and is absolutely required for HIV production.     

Rev-derived peptides inhibit HIV-1 replication by antagonism of Rev and a co-receptor,CXCR4

Rev, a viral regulatory protein of HIV-1, binds through its arginine-rich domain to the Rev-responsive element (RRE), a secondary structure in transcribed HIV-1 RNA. Binding of Rev to RRE mediates export of singly spliced or unspliced mRNAs from the nucleus to the cytoplasm. It has been previously shown that a certain arginine-rich peptide exhibits not only RRE-binding ability but also cell permeability and antagonism of CXCR4, one of the major coreceptors of HIV-1. Here we designed and synthesized arginine-rich peptides derived from the RNA-binding domain of Rev (Rev_34-50) and evaluated their anti-HIV-1 activities. Rev_34-50-A₄C, comprising Rev_34-50 with AAAAC at the C-terminus to increase the α-helicity, inhibited HIV-1 entry by CXCR4 antagonism and virus production in persistently HIV-1-infected PM1-CCR5 cells. Interestingly, similar motif of human lymphotropic virus type I Rex (Rex_1-21) also exerted moderate anti-HIV-1 activity. These results indicate that arginine-rich peptide, Rev_34-50-A₄C exerts dual antagonism against CXCR4 and Rev.     

Inhibition of chloroplast DNA recombination and repair by dominant negative mutants of Escherichia coli RecA.

The occurrence of homologous DNA recombination in chloroplasts is well documented, but little is known about the molecular mechanisms involved or their biological significance. The endosymbiotic origin of plastids and the recent finding of an Arabidopsis nuclear gene, encoding a chloroplast-localized protein homologous to Escherichia coli RecA, suggest that the plastid recombination system is related to its eubacterial counterpart. Therefore, we examined whether dominant negative mutants of the E. coli RecA protein can interfere with the activity of their putative homolog in the chloroplast of the unicellular green alga Chlamydomonas reinhardtii. Transformants expressing these mutant RecA proteins showed reduced survival rates when exposed to DNA-damaging agents, deficient repair of chloroplast DNA, and diminished plastid DNA recombination. These results strongly support the existence of a RecA-mediated recombination system in chloroplasts. We also found that the wild-type E. coli RecA protein enhances the frequency of plastid DNA recombination over 15-fold, although it has no effect on DNA repair or cell survival. Thus, chloroplast DNA recombination appears to be limited by the availability of enzymes involved in strand exchange rather than by the level of initiating DNA substrates. Our observations suggest that a primary biological role of the recombination system in plastids is in the repair of their DNA, most likely needed to cope with damage due to photooxidation and other environmental stresses. This hypothesis could explain the evolutionary conservation of DNA recombination in chloroplasts despite the predominantly uniparental inheritance of their genomes.     

Cell-dependent replication potentials of HIV-1 gag mutants

An infectious molecular clone of human immunodeficiency virus type 1 (HIV-1), designated pNLaiKH, which is tropic for both lymphocytic and monocytic cells, was constructed. To study the early function of HIV-1 Gag proteins in two types of cells, the mutations known to give host cell-dependent early defects were introduced into pNLaiKH, and the replication potentials and defective replication sites in the cells of the resultant mutants were monitored. All mutants grew in some lymphocytic cells, but not at all in monocytic cells. A nucleocapsid mutant was found to be defective at an early replication phase in all the cell lines to various extent, as expected. In contrast, a matrix mutant and a capsid mutant displayed a replication defect in a producer-cell-dependent manner. These results demonstrated that complex interactions of cell factors and Gag proteins are involved in an early process of HIV-1 replication.     

Apparent lack of trans-dominant negative effects of various vif mutants on the replication of HIV-1

The vif gene of human immunodeficiency virus type 1 (HIV-1) is essential for virus growth in non-permissive cells such as H9. To elucidate the mechanism of action of the Vif protein, vif mutants, which show trans-dominant negative effects on the replication of HIV-1, would be useful tools. In this study, a new assay system to identify the mutants of this category was established. For this new system, various reporter clones carrying both mutant and authentic vif sequences were generated. By determining the growth ability of the viruses derived from the reporter constructs, the potential negative effect of the mutant vif sequence was readily and sensitively monitored. Ten vif mutant sequences tested were found not to exert the trans-dominant negative effect on the replication of HIV-1.     

Inhibition of HIV-1 replication by ribozymes that show poor activity in vitro.

Self-cleaving RNAs (ribozymes) can be engineered to cleave target RNAs of choice in a sequence-specific manner (1). Consequently, they could be used to inhibit virus replication or to analyse host gene function in vivo. However, ribozymes that are catalytic in vitro are generally disappointing when analysed in cells unless expressed at high levels relative to their target RNAs (2, 3). Here we provide evidence that this can be overcome by optimizing ribozyme structure using cellular rather than cell-free assays. We show that ribozymes of relatively long flanking complementary regions (FCRs), while poor catalysts in vitro, can produce profound inhibition of HIV replication in cells. By examining a series of ribozymes in which the FCRs vary from 9 to 564 nucleotides, we establish that the optimum length for activity in the cell is > or = 33 nucleotides.     

Inhibition of HIV-1 multiplication by a modified U7 snRNA inducing Tat and Rev exon skipping

The HIV-1 regulatory proteins Tat and Rev are encoded by multiply spliced mRNAs that differ by the use of alternative 3' splice sites at the beginning of the internal exon. If these internal exons are skipped, the expression of these genes, and hence HIV-1 multiplication, should be inhibited. We have previously developed a strategy, based on antisense derivatives of U7 small nuclear RNA, that allows us to induce the skipping of an internal exon in virtually any gene. Here, we have successfully applied this approach to induce a partial skipping of the Tat, Rev (and Nef) internal exons. Three functional U7 constructs were subcloned into a lentiviral vector. Two of them strongly reduced the efficiency of lentiviral particle production compared to vectors carrying either no U7 insert or unrelated U7 cassettes. This defect could be partly or fully compensated by coexpressing Rev from an unspliced mRNA in the producing cell line. Upon stable transduction into CEM-SS or CEM T-lymphocytes, the most efficient of these constructs inhibits HIV-1 multiplication. Although the inhibition is not complete, it is more efficient in combination with another mechanism inhibiting HIV multiplication. Therefore, this new approach targeting HIV-1 regulatory genes at the level of pre-mRNA splicing, in combination with other antiviral strategies, may be a useful new tool in the fight against HIV/AIDS.     

Inhibition of human immunodeficiency virus type 1 replication is enhanced by a combination of transdominant Tat and Rev proteins.

Mutation of either of two critical human immunodeficiency virus type 1 (HIV-1) regulatory proteins, Tat and Rev, results in marked defects in viral replication. Thus, inhibition of the function of one or both of these proteins can significantly inhibit viral growth. In the present study, we constructed a novel transdominant Tat mutant protein and compared its efficiency in inhibiting HIV-1 replication with that of transdominant mutant Rev M10 when these proteins were stably expressed either alone or in combination in T-lymphocyte cell lines. The transdominant Tat mutant protein alone resulted in a modest inhibition of HIV replication, but it was able to enhance the ability of the M10 Rev mutant protein to inhibit HIV-1 replication. These results suggest a possible synergistic effect of these transdominant mutant proteins in inhibiting HIV-1 replication.     

Selective translational repression of HIV-1 RNA by Sam68DeltaC occurs by altering PABP1 binding to unspliced viral RNA

Background

The formation of new infectious human immunodeficiency type 1 virus (HIV-1) mainly relies on the homo-multimerization of the viral structural polyprotein Pr55^Gag and on the recruitment of host factors. We have previously shown that the double-stranded RNA-binding protein Staufen 1 (Stau1), likely through an interaction between its third double-stranded RNA-binding domain (dsRBD3) and the nucleocapsid (NC) domain of Pr55^Gag, participates in HIV-1 assembly by influencing Pr55^Gag multimerization.

Results

We now report the fine mapping of Stau1/Pr55^Gag association using co-immunoprecipitation and live cell bioluminescence resonance energy transfer (BRET) assays. On the one hand, our results show that the Stau1-Pr55^Gag interaction requires the integrity of at least one of the two zinc fingers in the NC domain of Pr55^Gag but not that of the NC N-terminal basic region. Disruption of both zinc fingers dramatically impeded Pr55^Gag multimerization and virus particle release. In parallel, we tested several Stau1 deletion mutants for their capacity to influence Pr55^Gag multimerization using the Pr55^Gag/Pr55^Gag BRET assay in live cells. Our results revealed that a molecular determinant of 12 amino acids at the N-terminal end of Stau1 is necessary to increase Pr55^Gag multimerization and particle release. However, this region is not required for Stau1 interaction with the viral polyprotein Pr55^Gag.

Conclusion

These data highlight that Stau1 is a modular protein and that Stau1 influences Pr55^Gag multimerization via 1) an interaction between its dsRBD3 and Pr55^Gag zinc fingers and 2) a regulatory domain within the N-terminus that could recruit host machineries that are critical for the completion of new HIV-1 capsids.     

Inhibition of HIV replication by amino-sugar derivatives

The plant alkaloids castanospermine, dihydroxymethyldihydroxypyrrolidine and deoxynojirimycin have recently been shown to have potential anti-HIV activity [(1987) Proc. Natl. Acad. Sci. USA 84, 8120-8124; (1987) Nature 330, 74-77; (1987) Lancet i, 1025-1026]. They are thought to act by inhibiting alpha-glucosidase I, an enzyme involved in the processing of N-linked oligosaccharides on glycoproteins. We report here the relative efficacy of a spectrum of amino-sugar derivatives as inhibition of HIV cytopathicity. Several alpha-glucosidase inhibitors and alpha-fucosidase inhibitors were found to be active at concentrations which were non-cytotoxic.     

Inhibition of human immunodeficiency virus (HIV-1) replication by synthetic oligo-RNA derivatives.

Several synthetic 2'-O-methyl-RNA oligomers and their derivatives have been evaluated for inhibitory effect against HIV-induced cytopathic effect and expression of the virus specific antigen in cultured MT-4 cells. In this study, oligo(2'-O-methyl)ribonucleoside phosphorothioates showed a potent inhibitory activity with size dependency (25-mer showed it at 1 microM), but by contrast both 2'-O-methylribo- and deoxy-oligomers with normal phosphate linkages failed to inhibit. However, it should be noted that the patched oligo(2'-O-methyl)ribonucleotide (20-mer), in which five linkages at 5'- and three linkages at 3'-ends of normal phosphates were replaced with thiophosphates, has recovered the substantial inhibitory effect. These results show that the size of oligomer and phosphorothioate linkages, probably resistant to exolytic nucleases, are essential for exhibiting antiviral activity.     

Expression of exogenous Sam68, the 68-kilodalton SRC-associated protein in mitosis, is able to alleviate impaired Rev function in astrocytes

Human immunodeficiency virus type 1 (HIV-1) gene expression in astrocytes is restricted, resulting in a brief and limited synthesis of HIV-1 viral structural proteins. Impaired Rev function has been documented in these cells. However, the molecular mechanisms underlying the impaired Rev function are not fully understood. Using the astroglial cell line U87.MG as a model, we report here that HIV-1 gene expression down-regulated expression of Sam68, the 68-kDa Src-associated protein in mitosis, which was constitutively expressed at a lower level in astrocytes. Elevating the endogenous level of Sam68 expression considerably restored HIV-1 Rev function in astrocytes, as determined by a Rev-dependent reporter gene assay. However, elevation of Sam68 expression achieved only a modest increase in HIV-1 production, further supporting the notion that there are multiple cellular restrictions of HIV-1 gene expression in astrocytes. Mutagenesis analysis identified the region between amino acids 321 and 410 of Sam68 as being directly involved in the binding of Sam68 to Rev, while a double mutation in Rev, L78D and E79L, like those in the dominant-negative Rev mutant M10, eliminated Rev binding to Sam68. Moreover, subcellular fractionation and digital fluorescence microscopic imaging revealed that Sam68 expression promoted Rev nuclear export. Taken together, our studies demonstrate that a lower level of constitutive Sam68 expression, followed by further down-regulation by HIV-1 infection, contributes to impaired Rev function in astrocytes, and they suggest that Sam68 may play an important role in Rev nuclear export.