Intracellular Reassociation of RNA-DNA Hybrids that Activates RNAi in HIV-Infected Cells.

Human immunodeficiency virus Type 1 (HIV-1) is the major cause of acquired immune deficiency syndrome (AIDS). In 2014, it was estimated that 1.2 million people died from AIDS-related illnesses. RNA interference-based therapy to block HIV replication is a field that, as of now, is without any FDA-approved drugs available for clinical use. In this chapter we describe a protocol for testing and utilizing a new approach that relies on reassociation of RNA-DNA hybrids activating RNAi and blocking HIV replication in human cells.

Versatile RNA tetra-U helix linking motif as a toolkit for nucleic acid nanotechnology.

RNA nanotechnology employs synthetically modified ribonucleic acid (RNA) to engineer highly stable nanostructures in one, two, and three dimensions for medical applications. Despite the tremendous advantages in RNA nanotechnology, unmodified RNA itself is fragile and prone to enzymatic degradation. In contrast to use traditionally modified RNA strands e.g. 2'-fluorine, 2'-amine, 2'-methyl, we studied the effect of RNA/DNA hybrid approach utilizing a computer-assisted RNA tetra-uracil (tetra-U) motif as a toolkit to address questions related to assembly efficiency, versatility, stability, and the production costs of hybrid RNA/DNA nanoparticles. The tetra-U RNA motif was implemented to construct four functional triangles using RNA, DNA and RNA/DNA mixtures, resulting in fine-tunable enzymatic and thermodynamic stabilities, immunostimulatory activity and RNAi capability. Moreover, the tetra-U toolkit has great potential in the fabrication of rectangular, pentagonal, and hexagonal NPs, representing the power of simplicity of RNA/DNA approach for RNA nanotechnology and nanomedicine community.

Elucidation of the Molecular Mechanism Driving Duplication of the HIV-1 PTAP Late Domain.

UNLABELLED: HIV-1 uses cellular machinery to bud from infected cells. This cellular machinery is comprised of several multiprotein complexes known as endosomal sorting complexes required for transport (ESCRTs). A conserved late domain motif, Pro-Thr-Ala-Pro (PTAP), located in the p6 region of Gag (p6(Gag)), plays a central role in ESCRT recruitment to the site of virus budding. Previous studies have demonstrated that PTAP duplications are selected in HIV-1-infected patients during antiretroviral therapy; however, the consequences of these duplications for HIV-1 biology and drug resistance are unclear. To address these questions, we constructed viruses carrying a patient-derived PTAP duplication with and without drug resistance mutations in the viral protease. We evaluated the effect of the PTAP duplication on viral release efficiency, viral infectivity, replication capacity, drug susceptibility, and Gag processing. In the presence of protease inhibitors, we observed that the PTAP duplication in p6(Gag) significantly increased the infectivity and replication capacity of the virus compared to those of viruses bearing only resistance mutations in protease. Our biochemical analysis showed that the PTAP duplication, in combination with mutations in protease, enhances processing between the nucleocapsid and p6 domains of Gag, resulting in more complete Gag cleavage in the presence of protease inhibitors. These results demonstrate that duplication of the PTAP motif in p6(Gag) confers a selective advantage in viral replication by increasing Gag processing efficiency in the context of protease inhibitor treatment, thereby enhancing the drug resistance of the virus. These findings highlight the interconnected role of PTAP duplications and protease mutations in the development of resistance to antiretroviral therapy. IMPORTANCE: Resistance to current drug therapy limits treatment options in many HIV-1-infected patients. Duplications in a Pro-Thr-Ala-Pro (PTAP) motif in the p6 domain of Gag are frequently observed in viruses derived from patients on protease inhibitor (PI) therapy. However, the reason that these duplications arise and their consequences for virus replication remain to be established. In this study, we examined the effect of PTAP duplication on PI resistance in the context of wild-type protease or protease bearing PI resistance mutations. We observe that PTAP duplication markedly enhances resistance to a panel of PIs. Biochemical analysis reveals that the PTAP duplication reverses a Gag processing defect imposed by the PI resistance mutations in the context of PI treatment. The results provide a long-sought explanation for why PTAP duplications arise in PI-treated patients.

Triggering RNAi with multifunctional RNA nanoparticles and their delivery.

Proteins are considered to be the key players in structure, function, and metabolic regulation of our bodies. The mechanisms used in conventional therapies often rely on inhibition of proteins with small molecules, but another promising method to treat disease is by targeting the corresponding mRNAs. In 1998, Craig Mellow and Andrew Fire discovered dsRNA-mediated gene silencing via RNA interference or RNAi. This discovery introduced almost unlimited possibilities for new gene silencing methods, thus opening new doors to clinical medicine. RNAi is a biological process that inhibits gene expression by targeting the mRNA. RNAi-based therapeutics have several potential advantages (i) a priori ability to target any gene, (ii) relatively simple design process, (iii) site-specificity, (iv) potency, and (v) a potentially safe and selective knockdown of the targeted cells. However, the problem lies within the formulation and delivery of RNAi therapeutics including rapid excretion, instability in the bloodstream, poor cellular uptake, and inefficient intracellular release. In an attempt to solve these issues, different types of RNAi therapeutic delivery strategies including multifunctional RNA nanoparticles are being developed. In this mini-review, we will briefly describe some of the current approaches.

Multifunctional RNA nanoparticles.

Our recent advancements in RNA nanotechnology introduced novel nanoscaffolds (nanorings); however, the potential of their use for biomedical applications was never fully revealed. As presented here, besides functionalization with multiple different short interfering RNAs for combinatorial RNA interference (e.g., against multiple HIV-1 genes), nanorings also allow simultaneous embedment of assorted RNA aptamers, fluorescent dyes, proteins, as well as recently developed RNA-DNA hybrids aimed to conditionally activate multiple split functionalities inside cells.

Activation of different split functionalities on re-association of RNA-DNA hybrids.

Split-protein systems, an approach that relies on fragmentation of proteins with their further conditional re-association to form functional complexes, are increasingly used for various biomedical applications. This approach offers tight control of protein functions and improved detection sensitivity. Here we report a similar technique based on a pair of RNA-DNA hybrids that can be used generally for triggering different split functionalities. Individually, each hybrid is inactive but when two cognate hybrids re-associate, different functionalities are triggered inside mammalian cells. As a proof of concept, this work mainly focuses on the activation of RNA interference. However, the release of other functionalities (such as resonance energy transfer and RNA aptamer) is also shown. Furthermore, in vivo studies demonstrate a significant uptake of the hybrids by tumours together with specific gene silencing. This split-functionality approach presents a new route in the development of 'smart' nucleic acid-based nanoparticles and switches for various biomedical applications.

Prevalence of etravirine-associated mutations in clinical samples with genotypic resistance to nevirapine and efavirenz in Brazilian clinics.

Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are potent and well tolerated. In Brazil, the first-generation NNRTI efavirenz is included in the majority of first-line antiretroviral treatment regimens. In this study, we evaluated the effectiveness of etravirine, a new second-generation NNRTI, among patients failing antiretroviral regimens containing first-generation NNRTIs. We assessed single resistance mutations to etravirine as well as complex resistance mutations profile and discuss the potential of introducing etravirine as salvage therapy.

Accumulation of P(T/S)AP late domain duplications in HIV type 1 subtypes B, C, and F derived from individuals failing ARV therapy and ARV drug-naive patients.

HIV-1 budding requires short peptide motifs in p6(Gag), known as late domains, that promote the release of infectious virions. The primary late domain of HIV-1 is a Pro-(Thr/Ser)-Ala-Pro (hereafter referred to as a PTAP) motif. This motif may be completely or partially duplicated. In this work we analyzed p6(Gag) sequences from 547 isolates from drug-naive patients and 213 isolates from patients failing HAART therapy. Complete duplications within PTAP were selected during HAART therapy in all HIV-1 subtypes analyzed: B (p = 0.0338), F1 (p = 0.0294), and C (p = 0.0001). Nevertheless, the patterns of these duplications were different; subtype C isolates accumulated longer duplications and displayed a higher frequency of duplications in both treated (54%) and drug-naive isolates (23%). Accumulation of PTAP duplications within subtypes B, F1, and C during therapy suggests a potential role of the duplications in antiretroviral drug resistance.

Genotypic and phenotypic characterization of human immunodeficiency virus type 1 isolates circulating in pregnant women from Mozambique.

This work evaluated HIV-1 subtypes from different geographic regions and phenotypic data from drug-naïve HIV-positive pregnant women from Mozambique. We analyzed 75 pol sequences from patients and the distribution of the subtypes in three regions of Mozambique and found that the majority of samples analyzed clustered with subtype C. In the northern region, multiple variants were found 5 (approximately 18%) subtype A, 3 (approximately 11%) subtype D and 2 (approximately 7.1%) mosaics (A/C/D and C/D), whereas 18 (64.3%) isolates were subtype C, from a total of 28 samples. Already in the southern region, only one (5%) isolate of 20 samples was subtype D, and the other 19 (95%) isolates were subtype C. All 27 (100%) isolates from the central region grouped within clade C. No primary resistance mutations to IP, NNRTI or NRTI were found. There was no evidence of phenotypic resistance in any of the isolates tested, suggesting that neither the polymorphism in the protease, nor the one found at codon 215 of the RT gene caused an increase in phenotypic resistance. This finding suggests that HAART regimens indicated by WHO will probably be successful in Mozambique.

Phylogenetic and genetic analysis of feline immunodeficiency virus gag, pol, and env genes from domestic cats undergoing nucleoside reverse transcriptase inhibitor treatment or treatment-naïve cats in Rio de Janeiro, Brazil.

Feline immunodeficiency virus (FIV) is the Lentivirus responsible for an immunodeficiency-like disease in domestic cats (Felis catus). FIV is divided into five phylogenetic subtypes (A, B, C, D, and E), based on genetic diversity. Knowledge of the geographical distribution of subtypes is relevant for understanding different disease progressions and for vaccine development. In this study, viral sequences of 26 infected cats from Rio de Janeiro, 8 undergoing treatment with zidovudine (AZT) for at least 5 years, were successfully amplified from blood specimens. gag capsid (CA), pol reverse transcriptase (RT), and env gp120 (V3-V4) regions were analyzed to determine subtypes and to evaluate potential mutations related to antiretroviral drug resistance among treated cats. Subtyping based on phylogenetic analysis was performed by the neighbor-joining and maximum likelihood methods. All of the sequences clustered with subtype B in the three regions, exhibiting low genetic variability. Additionally, we found evidence that the same virus is circulating in animals in close contact. The analysis of FIV RT sequences identified two new putative mutations related to drug resistance located in the RT "finger" domain, which has 60% identity to human immunodeficiency virus (HIV) sequence. Amino acid change K-->R at codons 64 and 69 was found in 25% and 37.5% of the treated animals, respectively. These signatures were comparable to K65R and K70R thymidine-associated mutations found in the HIV-1 HXB2 counterpart. This finding strongly suggests a position correlation between the mutations found in FIV and the K65R and K70R substitutions from drug-resistant HIV-1 strains.