A Camptothetin Analog, Topotecan, Promotes HIV Latency via Interference with HIV Transcription and RNA Splicing.

Low level HIV transcription during modern antiretroviral therapy (ART) in persons with HIV is linked to residual inflammation and associated diseases, like cardiovascular disease and cancer. The "block and lock" approach to hold HIV in a state of deep latency may help decrease residual inflammation in a person with HIV on ART and thus improve health. A camptothecin analog topotecan (TPT) was previously implicated as an inhibitor of active HIV replication. Using an in vitro primary T cell model of HIV latency, we demonstrated that (i) TPT reduces HIV transcriptional activity in latently infected cells; (ii) downregulation of HIV RNA by TPT cannot be reversed by latency reversing agents; (iii) several primary and secondary mechanism of action of TPT may be involved in control of HIV replication; (iv) regulation of HIV RNA by TPT is dependent on splicing complexity; (v) increase in proportion of unspliced HIV transcripts was facilitated by intron retention and upregulation of splicing factors, specifically SRSF6, by TPT. Although high TPT dosing (10 µM) was needed to achieve the observed effects, viability of primary CD4+ T cells was not greatly affected. Because toxicity can be observed with TPT in persons with cancer, TPT is unlikely to be used as an anti-HIV agent in clinic, but our study provides proof that camptothetin has "block and lock" activity. Other camptothetin analogs, which are less toxic than TPT, should be designed and tested as HIV "block and lock" agents. IMPORTANCE HIV survives in a state of very low activity, called latency, for long periods in persons with HIV on antiretroviral therapy. This low activity of HIV is linked to residual inflammation and associated diseases, such as heart disease and cancer. New strategies are being explored to further silence the HIV provirus and suppress residual inflammation. This study provides strong evidence that the camptothetin analog, Topotecan, can reduce residual activity of HIV in an experimental model of HIV latency. While Topotecan itself is likely not suitable for use in the clinic due to its toxicity, other camptothetin analogs should be designed and investigated as "block and lock" agents.

Transcriptomic Analysis Implicates the p53 Signaling Pathway in the Establishment of HIV-1 Latency in Central Memory CD4 T Cells in an In Vitro Model.

The search for an HIV-1 cure has been greatly hindered by the presence of a viral reservoir that persists despite antiretroviral therapy (ART). Studies of HIV-1 latency in vivo are also complicated by the low proportion of latently infected cells in HIV-1 infected individuals. A number of models of HIV-1 latency have been developed to examine the signaling pathways and viral determinants of latency and reactivation. A primary cell model of HIV-1 latency, which incorporates the generation of primary central memory CD4 T cells (TCM), full-length virus infection (HIVNL4-3) and ART to suppress virus replication, was used to investigate the establishment of HIV latency using RNA-Seq. Initially, an investigation of host and viral gene expression in the resting and activated states of this model indicated that the resting condition was reflective of a latent state. Then, a comparison of the host transcriptome between the uninfected and latently infected conditions of this model identified 826 differentially expressed genes, many of which were related to p53 signaling. Inhibition of the transcriptional activity of p53 by pifithrin-α during HIV-1 infection reduced the ability of HIV-1 to be reactivated from its latent state by an unknown mechanism. In conclusion, this model may be used to screen latency reversing agents utilized in shock and kill approaches to cure HIV, to search for cellular markers of latency, and to understand the mechanisms by which HIV-1 establishes latency.

Mixed effects of suberoylanilide hydroxamic acid (SAHA) on the host transcriptome and proteome and their implications for HIV reactivation from latency.

Suberoylanilide hydroxamic acid (SAHA) has been assessed in clinical trials as part of a "shock and kill" strategy to cure HIV-infected patients. While it was effective at inducing expression of HIV RNA ("shock"), treatment with SAHA did not result in a reduction of reservoir size ("kill"). We therefore utilized a combined analysis of effects of SAHA on the host transcriptome and proteome to dissect its mechanisms of action that may explain its limited success in "shock and kill" strategies. CD4+ T cells from HIV seronegative donors were treated with 1µM SAHA or its solvent dimethyl sulfoxide (DMSO) for 24h. Protein expression and post-translational modifications were measured with iTRAQ proteomics using ultra high-precision two-dimensional liquid chromatography-tandem mass spectrometry. Gene expression was assessed by Illumina microarrays. Using limma package in the R computing environment, we identified 185 proteins, 18 phosphorylated forms, 4 acetylated forms and 2982 genes, whose expression was modulated by SAHA. A protein interaction network integrating these 4 data types identified the HIV transcriptional repressor HMGA1 to be upregulated by SAHA at the transcript, protein and acetylated protein levels. Further functional category assessment of proteins and genes modulated by SAHA identified gene ontology terms related to NFκB signaling, protein folding and autophagy, which are all relevant to HIV reactivation. In summary, SAHA modulated numerous host cell transcripts, proteins and post-translational modifications of proteins, which would be expected to have very mixed effects on the induction of HIV-specific transcription and protein function. Proteome profiling highlighted a number of potential counter-regulatory effects of SAHA with respect to viral induction, which transcriptome profiling alone would not have identified. These observations could lead to a more informed selection and design of other HDACi with a more refined targeting profile, and prioritization of latency reversing agents of other classes to be used in combination with SAHA to achieve more potent induction of HIV expression.

Maraviroc intensification in patients with suppressed HIV viremia has limited effects on CD4+ T cell recovery and gene expression.

Addition of the CCR5 inhibitor Maraviroc (MVC) to ongoing antiretroviral therapy increases CD4+ T cell counts in some virologically suppressed patients with suboptimal CD4+ T cell recovery. To understand the mechanisms by which MVC elicits increases in CD4+ T cell counts, the present study was undertaken to identify host factors (i.e. genes) that are modulated and are correlated with CD4+ T cell recovery during the 24weeks of MVC intensification in 32 subjects. Median changes of CD4+ T cell counts over 24weeks of MVC compared to baseline were 38cells/mm(3) (p<0.001). The median slope of CD4+ T cell recovery was 39cells/mm(3) per year before initiation of MVC and 76cells/mm(3) per year during MVC intensification, however, this increase was not statistically significant (p=0.33). Microarray analysis (N=31,426 genes) identified a single differentially expressed gene, tumor necrosis factor alpha (TNF), which was modestly (1.44-fold, p<0.001) downregulated by MVC at week 24 compared to baseline. TNF differential expression was evaluated using an independent method of droplet digital PCR, but the difference was not significant (p=0.6). Changes in gene expression did not correlate with CD4+ T cell recovery or any changes in the CD4+ T cell maturation, proliferation and activation phenotypes. In summary, our data suggest that modest improvements of CD4+ T cell counts during MVC intensification cannot be explained by changes in gene expression elicited by MVC. However, the modest changes in T cell composition, including reduction of the percentages of Tregs, proliferating CD4+ T cells and senescent CD8+ T cells, suggest immunologically favorable effects of MVC.

Differential gene expression in HIV-infected individuals following ART.

Previous studies of the effect of ART on gene expression in HIV-infected individuals have identified small numbers of modulated genes. Since these studies were underpowered or cross-sectional in design, a paired analysis of peripheral blood mononuclear cells (PBMCs), isolated before and after ART, from a robust number of HIV-infected patients (N=32) was performed. Gene expression was assayed by microarray and 4157 differentially expressed genes (DEGs) were identified following ART using multivariate permutation tests. Pathways and gene ontology (GO) terms over-represented for DEGs reflected the transition from a period of active virus replication before ART to one of viral suppression (e.g., repression of JAK-STAT signaling) and possible prolonged drug exposure (e.g., oxidative phosphorylation pathway) following ART. CMYC was the DEG whose product made the greatest number of interactions at the protein level in protein interaction networks (PINs), which has implications for the increased incidence of Hodgkin's lymphoma (HL) in HIV-infected patients. The differential expression of multiple genes was confirmed by RT-qPCR including well-known drug metabolism genes (e.g., ALOX12 and CYP2S1). Targets not confirmed by RT-qPCR (i.e., GSTM2 and RPL5) were significantly confirmed by droplet digital (ddPCR), which may represent a superior method when confirming DEGs with low fold changes. In conclusion, a paired design revealed that the number of genes modulated following ART was an order of magnitude higher than previously recognized.

HIV downregulates interferon-stimulated genes in primary macrophages.

HIV is able to outpace the innate immune response, including that mediated by interferon (IFN), to establish a productive infection. Primary macrophages, however, may be protected from HIV infection by treatment with type I IFN before virus exposure. The ability of HIV to modulate the type I IFN-mediated innate immune response when it encounters a cell that has already been exposed to IFN remains poorly defined. The optimal pretreatment time (12 h) and the most potent HIV-inhibitors (e.g., IFN-α2 and -ω) were identified to investigate the ability of HIV to modulate an established type I IFN response. Gene expression at the level of the entire transcriptome was then compared between primary macrophages treated with type I IFNs, as opposed to treated with IFNs and then infected with HIV. Although HIV was not able to establish a robust infection, the virus was able to downregulate a number of IFN-stimulated genes (ISGs) with a fold change greater than 1.5 (i.e., AXL, IFI27, IFI44, IFI44L, ISG15, OAS1, OAS3, and XAF1). The downregulation of OAS1 by the presence of HIV was confirmed by real-time quantitative polymerase chain reaction. In conclusion, even though HIV replication is significantly inhibited by IFN pretreatment, the virus is able to downregulate the transcription of known antiviral ISGs (e.g., IFI44, ISG15, and OAS1).

Sequence requirements for localization and packaging of Ty3 retroelement RNA.

Retroviruses and retrotransposons package genomic RNA into virus-like particles (VLPs) in a poorly understood process. Expression of the budding yeast retrotransposon Ty3 results in the formation of cytoplasmic Ty3 VLP assembly foci comprised of Ty3 RNA and proteins, and cellular factors associated with RNA processing body (PB) components, which modulate translation and effect nonsense-mediated decay (NMD). A series of Ty3 RNA variants were tested to understand the effects of read-through translation via programmed frameshifting on RNA localization and packaging into VLPs, and to identify the roles of coding and non-coding sequences in those processes. These experiments showed that a low level of read-through translation of the downstream open reading frame (as opposed to no translation or translation without frameshifting) is important for localization of full-length Ty3 RNA to foci. Ty3 RNA variants associated with PB components via independent determinants in the native Ty3 untranslated regions (UTRs) and in GAG3-POL3 sequences flanked by UTRs adapted from non-Ty3 transcripts. However, despite localization, RNAs containing GAG3-POL3 but lacking Ty3 UTRs were not packaged efficiently. Surprisingly, sequences within Ty3 UTRs, which bind the initiator tRNA(Met) proposed to provide the dimerization interface, were not required for packaging of full-length Ty3 RNA into VLPs. In summary, our results demonstrate that Gag3 is sufficient and required for localization and packaging of RNAs containing Ty3 UTRs and support a role for POL3 sequences, translation of which is attenuated by programmed frameshifting, in both localization and packaging of the Ty3 full-length gRNA.

Ty3 nuclear entry is initiated by viruslike particle docking on GLFG nucleoporins.

Yeast retrotransposons form intracellular particles within which replication occurs. Because fungal nuclear membranes do not break down during mitosis, similar to retroviruses infecting nondividing cells, the cDNA produced must be translocated through nuclear pore complexes. The Saccharomyces cerevisiae long terminal repeat retrotransposon Ty3 assembles its Gag3 and Gag3-Pol3 precursor polyproteins into viruslike particles in association with perinuclear P-body foci. These perinuclear clusters of Ty3 viruslike particles localized to sites of clustered nuclear pore complexes (NPCs) in a nup120Delta mutant, indicating that Ty3 particles and NPCs interact physically. The NPC channels are lined with nucleoporins (Nups) with extended FG (Phe-Gly) motif repeat domains, further classified as FG, FxFG, or GLFG repeat types. These domains mediate partitioning of proteins between the cytoplasm and the nucleus. Here we have systematically examined the requirements for FG repeat domains in Ty3 nuclear transport. The GLFG domains interacted in vitro with virus-like particle Gag3, and this interaction was disrupted by mutations in the amino-terminal domain of Gag3, which is predicted to lie on the external surface of the particles. Accordingly, Ty3 transposition was decreased in strains with the GLFG repeats deleted. The spacer-nucleocapsid domain of Gag3, which is predicted to be internal to the particle, interacted with GLFG repeats and nucleocapsid localized to the nucleus. We conclude that Ty3 particle docking on nuclear pores is facilitated by interactions between Gag3 and GLFG Nups and that nuclear entry of the preintegration complex is further promoted by nuclear localization signals within the nucleocapsid and integrase.

Ty3 capsid mutations reveal early and late functions of the amino-terminal domain.

The Ty3 retrotransposon assembles into 50-nm virus-like particles that occur in large intracellular clusters in the case of wild-type (wt) Ty3. Within these particles, maturation of the Gag3 and Gag3-Pol3 polyproteins by Ty3 protease produces the structural proteins capsid (CA), spacer, and nucleocapsid. Secondary and tertiary structure predictions showed that, like retroviral CA, Ty3 CA contains a large amount of helical structure arranged in amino-terminal and carboxyl-terminal bundles. Twenty-six mutants in which alanines were substituted for native residues were used to study CA subdomain functions. Transposition was measured, and particle morphogenesis and localization were characterized by analysis of protein processing, cDNA production, genomic RNA protection, and sedimentation and by fluorescence and electron microscopy. These measures defined five groups of mutants. Proteins from each group could be sedimented in a large complex. Mutations in the amino-terminal domain reduced the formation of fluorescent Ty3 protein foci. In at least one major homology region mutant, Ty3 protein concentrated in foci but no wt clusters of particles were observed. One mutation in the carboxyl-terminal domain shifted assembly from spherical particles to long filaments. Two mutants formed foci separate from P bodies, the proposed sites of assembly, and formed defective particles. P-body association was therefore found to be not necessary for assembly but correlated with the production of functional particles. One mutation in the amino terminus blocked transposition after cDNA synthesis. Our data suggest that Ty3 proteins are concentrated first, assembly associated with P bodies occurs, and particle morphogenesis concludes with a post-reverse transcription, CA-dependent step. Particle formation was generally resistant to localized substitutions, possibly indicating that multiple domains are involved.

Virus-like particles of the Ty3 retrotransposon assemble in association with P-body components.

Retroviruses and retrotransposons assemble intracellular immature core particles around a RNA genome, and nascent particles collect in association with membranes or as intracellular clusters. How and where genomic RNA are identified for retrovirus and retrotransposon assembly, and how translation and assembly processes are coordinated is poorly understood. To understand this process, the subcellular localization of Ty3 RNA and capsid proteins and virus-like particles was investigated. We demonstrate that mRNAs, proteins, and virus-like particles of the yeast Ty3 retrotransposon accumulate in association with cytoplasmic P-bodies, which are sites of mRNA translation repression, storage, and degradation. Deletions of genes encoding P-body proteins decreased Ty3 transposition and caused changes in the pattern of Ty3 foci, underscoring the biological significance of the association of Ty3 virus-like protein components and P-bodies. These results suggest the hypothesis that P-bodies may serve to segregate translation and assembly functions of the Ty3 genomic RNA to promote assembly of virus-like particles. Because Ty3 has features of a simple retrovirus and P-body functions are conserved between yeast and metazoan organisms, these findings may provide insights into host factors that facilitate retrovirus assembly.