Cell type-specific circular RNA expression in human glial cells.

The circular transcriptome of human glial cells is an area of neuroscience that has not been thoroughly elucidated. Circular RNAs (circRNAs) have the potential to facilitate the understanding of vast, complex and unknown mechanisms derived from the human transcriptome, including elements of the human brain that are not known and the evolution of the human brain, the complexities of which are not well understood. Moreover, the glial cells have been determined to contribute to human brain evolution. This study presents the first comprehensive analysis of the human brain glia circRNA transcriptome, that is, astrocytes, microglia and oligodendrocytes. After stringent criteria applied to the detection of circRNAs, it was found that the circular transcriptomes of these glia are unique from one another, and hence might be indicative of distinct roles for circRNAs within the brain. This study found 265, 239 and 442 circRNAs comprising the unique circular transcriptome of astrocytes, microglia and oligodendrocytes, respectively. The most abundant circRNAs in these glial cell types are expressed by parent genes co-expressing linear RNAs in low abundance, suggesting spliceosome activity favorable to the back-splicing mechanism instead of canonical splicing activity.

Early transcriptome changes in response to chemical long-term potentiation induced via activation of synaptic NMDA receptors in mouse hippocampal neurons.

Long term potentiation (LTP) is a form of synaptic plasticity. In the present study LTP was induced via activation of synaptic NMDA receptors in primary hippocampal neuron cultures from neonate mice and RNA was isolated for RNA sequencing at 20 min following LTP induction. RNA sequencing and differential expression testing was performed to determine the identity and abundance of protein-coding and non-coding RNAs in control and LTP induced neuron cultures. We show that expression levels of a small group of transcripts encoding proteins involved in negative regulation of gene expression (Adcyap1, Id3), protein translation (Rpl22L1), extracellular structure organization (Bgn), intracellular signalling (Ppm1H, Ntsr2, Cldn10) and protein citrullination (PAD2) are downregulated in the stimulated neurons. Our results suggest that the early stages of LTP are accompanied by the remodelling of the biosynthetic machinery, interactions with the extracellular matrix and intracellular signalling pathways at the transcriptional level.

HIV integration and the establishment of latency in CCL19-treated resting CD4(+) T cells require activation of NF-κB.

BACKGROUND: Eradication of HIV cannot be achieved with combination antiretroviral therapy (cART) because of the persistence of long-lived latently infected resting memory CD4(+) T cells. We previously reported that HIV latency could be established in resting CD4(+) T cells in the presence of the chemokine CCL19. To define how CCL19 facilitated the establishment of latent HIV infection, the role of chemokine receptor signalling was explored. RESULTS: In resting CD4(+) T cells, CCL19 induced phosphorylation of RAC-alpha serine/threonine-protein kinase (Akt), nuclear factor kappa B (NF-κB), extracellular-signal-regulated kinase (ERK) and p38. Inhibition of the phosphoinositol-3-kinase (PI3K) and Ras/Raf/Mitogen-activated protein kinase/ERK kinase (MEK)/ERK signalling pathways inhibited HIV integration, without significant reduction in HIV nuclear entry (measured by Alu-LTR and 2-LTR circle qPCR respectively). Inhibiting activation of MEK1/ERK1/2, c-Jun N-terminal kinase (JNK), activating protein-1 (AP-1) and NF-κB, but not p38, also inhibited HIV integration. We also show that HIV integrases interact with Pin1 in CCL19-treated CD4(+) T cells and inhibition of JNK markedly reduced this interaction, suggesting that CCL19 treatment provided sufficient signals to protect HIV integrase from degradation via the proteasome pathway. Infection of CCL19-treated resting CD4(+) T cells with mutant strains of HIV, lacking NF-κB binding sites in the HIV long terminal repeat (LTR) compared to infection with wild type virus, led to a significant reduction in integration by up to 40-fold (range 1-115.4, p = 0.03). This was in contrast to only a modest reduction of 5-fold (range 1.7-11, p > 0.05) in fully activated CD4(+) T cells infected with the same mutants. Finally, we demonstrated significant differences in integration sites following HIV infection of unactivated, CCL19-treated, and fully activated CD4(+) T cells. CONCLUSIONS: HIV integration in CCL19-treated resting CD4(+) T cells depends on NF-κB signalling and increases the stability of HIV integrase, which allow subsequent integration and establishment of latency. These findings have implications for strategies needed to prevent the establishment, and potentially reverse, latent infection.

Toxicity and in vitro activity of HIV-1 latency-reversing agents in primary CNS cells.

Despite the success of combination antiretroviral therapy (cART), HIV persists in long lived latently infected cells in the blood and tissue, and treatment is required lifelong. Recent clinical studies have trialed latency-reversing agents (LRA) as a method to eliminate latently infected cells; however, the effects of LRA on the central nervous system (CNS), a well-known site of virus persistence on cART, are unknown. In this study, we evaluated the toxicity and potency of a panel of commonly used and well-known LRA (panobinostat, romidepsin, vorinostat, chaetocin, disulfiram, hexamethylene bisacetamide [HMBA], and JQ-1) in primary fetal astrocytes (PFA) as well as monocyte-derived macrophages as a cellular model for brain perivascular macrophages. We show that most LRA are non-toxic in these cells at therapeutic concentrations. Additionally, romidepsin, JQ-1, and panobinostat were the most potent at inducing viral transcription, with greater magnitude observed in PFA. In contrast, vorinostat, chaetocin, disulfiram, and HMBA all demonstrated little or no induction of viral transcription. Together, these data suggest that some LRA could potentially activate transcription in latently infected cells in the CNS. We recommend that future trials of LRA also examine the effects of these agents on the CNS via examination of cerebrospinal fluid.

HIV-1 transcriptional regulation in the central nervous system and implications for HIV cure research.

Human immunodeficiency virus type-1 (HIV-1) invades the central nervous system (CNS) during acute infection which can result in HIV-associated neurocognitive disorders in up to 50% of patients, even in the presence of combination antiretroviral therapy (cART). Within the CNS, productive HIV-1 infection occurs in the perivascular macrophages and microglia. Astrocytes also become infected, although their infection is restricted and does not give rise to new viral particles. The major barrier to the elimination of HIV-1 is the establishment of viral reservoirs in different anatomical sites throughout the body and viral persistence during long-term treatment with cART. While the predominant viral reservoir is believed to be resting CD4(+) T cells in the blood, other anatomical compartments including the CNS, gut-associated lymphoid tissue, bone marrow, and genital tract can also harbour persistently infected cellular reservoirs of HIV-1. Viral latency is predominantly responsible for HIV-1 persistence and is most likely governed at the transcriptional level. Current clinical trials are testing transcriptional activators, in the background of cART, in an attempt to purge these viral reservoirs and reverse viral latency. These strategies aim to activate viral transcription in cells constituting the viral reservoir, so they can be recognised and cleared by the immune system, while new rounds of infection are blocked by co-administration of cART. The CNS has several unique characteristics that may result in differences in viral transcription and in the way latency is established. These include CNS-specific cell types, different transcription factors, altered immune surveillance, and reduced antiretroviral drug bioavailability. A comprehensive understanding of viral transcription and latency in the CNS is required in order to determine treatment outcomes when using transcriptional activators within the CNS.

Inhibition of catechol-O-methyl transferase (COMT) by tolcapone restores reductions in microtubule-associated protein 2 (MAP2) and synaptophysin (SYP) following exposure of neuronal cells to neurotropic HIV.

This investigation aimed to assess whether inhibition of cathecol-O-methyl transferase (COMT) by tolcapone could provide neuroprotection against HIV-associated neurodegenerative effects. This study was conducted based on a previous work, which showed that a single nucleotide polymorphism (SNP) at position 158 (val158met) in COMT, resulted in 40 % lower COMT activity. Importantly, this reduction confers a protective effect against HIV-associated neurocognitive disorders (HAND), which have been linked to HIV-associated brain changes. SH-SY5Y-differentiated neurons were exposed to macrophage-propagated HIV (neurotropic MACS2-Br strain) in the presence or absence of tolcapone for 6 days. RNA was extracted, and qPCR was performed using Qiagen RT2 custom array consisting of genes for neuronal and synaptic integrity, COMT and pro-inflammatory markers. Immunofluorescence was conducted to validate the gene expression changes at the protein level. Our findings demonstrated that HIV significantly increased the messenger RNA (mRNA) expression of COMT while reducing the expression of microtubule-associated protein 2 (MAP2) (p = 0.0015) and synaptophysin (SYP) (p = 0.012) compared to control. A concomitant exposure of tolcapone ameliorated the perturbed expression of MAP2 (p = 0.009) and COMT (p = 0.024) associated with HIV. Immunofluorescence revealed a trend reduction of SYP and MAP2 with exposure to HIV and that concomitant exposure of tolcapone increased SYP (p = 0.016) compared to HIV alone. Our findings demonstrated in vitro that inhibition of COMT can ameliorate HIV-associated neurodegenerative changes that resulted in the decreased expression of the structural and synaptic components MAP2 and SYP. As HIV-associated dendritic and synaptic damage are contributors to HAND, inhibition of COMT may represent a potential strategy for attenuating or preventing some of the symptoms of HAND.

A new way of measuring apoptosis by absolute quantitation of inter-nucleosomally fragmented genomic DNA.

Several critical events of apoptosis occur in the cell nucleus, including inter-nucleosomal DNA fragmentation (apoptotic DNA) and eventual chromatin condensation. The generation of apoptotic DNA has become a biochemical hallmark of apoptosis because it is a late 'point of no return' step in both the extrinsic (cell-death receptor) and intrinsic (mitochondrial) apoptotic pathways. Despite investigators observing apoptotic DNA and understanding its decisive role as a marker of apoptosis for over 20 years, measuring it has proved elusive. We have integrated ligation-mediated PCR and qPCR to design a new way of measuring apoptosis, termed ApoqPCR, which generates an absolute value for the amount (picogram) of apoptotic DNA per cell population. ApoqPCR's advances over current methods include a 1000-fold linear dynamic range yet sensitivity to distinguish subtle low-level changes, measurement with a 3- to 4-log improvement in sample economy, and capacity for archival or longitudinal studies combined with high-throughput capability. We demonstrate ApoqPCR's utility in both in vitro and in vivo contexts. Considering the fundamental role apoptosis has in vertebrate and invertebrate health, growth and disease, the reliable measurement of apoptotic nucleic acid by ApoqPCR will be of value in cell biology studies in basic and applied science.

CoRSeqV3-C: a novel HIV-1 subtype C specific V3 sequence based coreceptor usage prediction algorithm.

BACKGROUND: The majority of HIV-1 subjects worldwide are infected with HIV-1 subtype C (C-HIV). Although C-HIV predominates in developing regions of the world such as Southern Africa and Central Asia, C-HIV is also spreading rapidly in countries with more developed economies and health care systems, whose populations are more likely to have access to wider treatment options, including the CCR5 antagonist maraviroc (MVC). The ability to reliably determine C-HIV coreceptor usage is therefore becoming increasingly more important. In silico V3 sequence based coreceptor usage prediction algorithms are a relatively rapid and cost effective method for determining HIV-1 coreceptor specificity. In this study, we elucidated the V3 sequence determinants of C-HIV coreceptor usage, and used this knowledge to develop and validate a novel, user friendly, and highly sensitive C-HIV specific coreceptor usage prediction algorithm. RESULTS: We characterized every phenotypically-verified C-HIV gp120 V3 sequence available in the Los Alamos HIV Database. Sequence analyses revealed that compared to R5 C-HIV V3 sequences, CXCR4-using C-HIV V3 sequences have significantly greater amino acid variability, increased net charge, increased amino acid length, increased frequency of insertions and substitutions within the GPGQ crown motif, and reduced frequency of glycosylation sites. Based on these findings, we developed a novel C-HIV specific coreceptor usage prediction algorithm (CoRSeqV3-C), which we show has superior sensitivity for determining CXCR4 usage by C-HIV strains compared to all other available algorithms and prediction rules, including Geno2pheno[coreceptor] and WebPSSMSINSI-C, which has been designed specifically for C-HIV. CONCLUSIONS: CoRSeqV3-C is now openly available for public use at http://www.burnet.edu.au/coreceptor. Our results show that CoRSeqV3-C is the most sensitive V3 sequence based algorithm presently available for predicting CXCR4 usage of C-HIV strains, without compromising specificity. CoRSeqV3-C may be potentially useful for assisting clinicians to decide the best treatment options for patients with C-HIV infection, and will be helpful for basic studies of C-HIV pathogenesis.

A common mechanism of clinical HIV-1 resistance to the CCR5 antagonist maraviroc despite divergent resistance levels and lack of common gp120 resistance mutations.

BACKGROUND: The CCR5 antagonist maraviroc (MVC) inhibits human immunodeficiency virus type 1 (HIV-1) entry by altering the CCR5 extracellular loops (ECL), such that the gp120 envelope glycoproteins (Env) no longer recognize CCR5. The mechanisms of HIV-1 resistance to MVC, the only CCR5 antagonist licensed for clinical use are poorly understood, with insights into MVC resistance almost exclusively limited to knowledge obtained from in vitro studies or from studies of resistance to other CCR5 antagonists. To more precisely understand mechanisms of resistance to MVC in vivo, we characterized Envs isolated from 2 subjects who experienced virologic failure on MVC. RESULTS: Envs were cloned from subjects 17 and 24 before commencement of MVC (17-Sens and 24-Sens) and after virologic failure (17-Res and 24-Res). The Envs cloned during virologic failure showed broad divergence in resistance levels, with 17-Res Env exhibiting a relatively high maximal percent inhibition (MPI) of ~90% in NP2-CD4/CCR5 cells and peripheral blood mononuclear cells (PBMC), and 24-Res Env exhibiting a very low MPI of ~0 to 12% in both cell types, indicating relatively "weak" and "strong" resistance, respectively. Resistance mutations were strain-specific and mapped to the gp120 V3 loop. Affinity profiling by the 293-Affinofile assay and mathematical modeling using VERSA (Viral Entry Receptor Sensitivity Analysis) metrics revealed that 17-Res and 24-Res Envs engaged MVC-bound CCR5 inefficiently or very efficiently, respectively. Despite highly divergent phenotypes, and a lack of common gp120 resistance mutations, both resistant Envs exhibited an almost superimposable pattern of dramatically increased reliance on sulfated tyrosine residues in the CCR5 N-terminus, and on histidine residues in the CCR5 ECLs. This altered mechanism of CCR5 engagement rendered both the resistant Envs susceptible to neutralization by a sulfated peptide fragment of the CCR5 N-terminus. CONCLUSIONS: Clinical resistance to MVC may involve divergent Env phenotypes and different genetic alterations in gp120, but the molecular mechanism of resistance of the Envs studied here appears to be related. The increased reliance on sulfated CCR5 N-terminus residues suggests a new avenue to block HIV-1 entry by CCR5 N-terminus sulfopeptidomimetic drugs.

HIV infection of dendritic cells subverts the IFN induction pathway via IRF-1 and inhibits type 1 IFN production.

Many viruses have developed mechanisms to evade the IFN response. Here, HIV-1 was shown to induce a distinct subset of IFN-stimulated genes (ISGs) in monocyte-derived dendritic cells (DCs), without detectable type I or II IFN. These ISGs all contained an IFN regulatory factor 1 (IRF-1) binding site in their promoters, and their expression was shown to be driven by IRF-1, indicating this subset was induced directly by viral infection by IRF-1. IRF-1 and -7 protein expression was enriched in HIV p24 antigen-positive DCs. A HIV deletion mutant with the IRF-1 binding site deleted from the long terminal repeat showed reduced growth kinetics. Early and persistent induction of IRF-1 was coupled with sequential transient up-regulation of its 2 inhibitors, IRF-8, followed by IRF-2, suggesting a mechanism for IFN inhibition. HIV-1 mutants with Vpr deleted induced IFN, showing that Vpr is inhibitory. However, HIV IFN inhibition was mediated by failure of IRF-3 activation rather than by its degradation, as in T cells. In contrast, herpes simplex virus type 2 markedly induced IFNß and a broader range of ISGs to higher levels, supporting the hypothesis that HIV-1 specifically manipulates the induction of IFN and ISGs to enhance its noncytopathic replication in DCs.

Alternative coreceptor requirements for efficient CCR5- and CXCR4-mediated HIV-1 entry into macrophages.

Macrophage tropism of human immunodeficiency virus type 1 (HIV-1) is distinct from coreceptor specificity of the viral envelope glycoproteins (Env), but the virus-cell interactions that contribute to efficient HIV-1 entry into macrophages, particularly via CXCR4, are not well understood. Here, we characterized a panel of HIV-1 Envs that use CCR5 (n = 14) or CXCR4 (n = 6) to enter monocyte-derived macrophages (MDM) with various degrees of efficiency. Our results show that efficient CCR5-mediated MDM entry by Env-pseudotyped reporter viruses is associated with increased tolerance of several mutations within the CCR5 N terminus. In contrast, efficient CXCR4-mediated MDM entry was associated with reduced tolerance of a large deletion within the CXCR4 N terminus. Env sequence analysis and structural modeling identified amino acid variants at positions 261 and 263 within the gp41-interactive region of gp120 and a variant at position 326 within the gp120 V3 loop that were associated with efficient CXCR4-mediated MDM entry. Mutagenesis studies showed that the gp41 interaction domain variants exert a significant but strain-specific influence on CXCR4-mediated MDM entry, suggesting that the structural integrity of the gp120-gp41 interface is important for efficient CXCR4-mediated MDM entry of certain HIV-1 strains. However, the presence of Ile326 in the gp120 V3 loop stem, which we show by molecular modeling is located at the gp120-coreceptor interface and predicted to interact with the CXCR4 N terminus, was found to be critical for efficient CXCR4-mediated MDM entry of divergent CXCR4-using Envs. Together, the results of our study provide novel insights into alternative mechanisms of Env-coreceptor engagement that are associated with efficient CCR5- and CXCR4-mediated HIV-1 entry into macrophages.

Conformational alterations in the CD4 binding cavity of HIV-1 gp120 influencing gp120-CD4 interactions and fusogenicity of HIV-1 envelopes derived from brain and other tissues.

BACKGROUND: CD4-binding site (CD4bs) alterations in gp120 contribute to HIV-1 envelope (Env) mediated fusogenicity and the ability of gp120 to utilize low levels of cell-surface CD4. In a recent study, we constructed three-dimensional models of gp120 to illustrate CD4bs conformations associated with enhanced fusogenicity and enhanced CD4-usage of a modestly-sized panel of blood-derived HIV-1 Envs (n = 16). These conformations were characterized by a wider aperture of the CD4bs cavity, as constrained by the inner-most atoms at the gp120 V1V2 stem and the V5 loop. Here, we sought to provide further validation of the utility of these models for understanding mechanisms that influence Env function, by characterizing the structure-function relationships of a larger panel of Envs derived from brain and other tissues (n = 81). FINDINGS: Three-dimensional models of gp120 were generated by our recently validated homology modelling protocol. Analysis of predicted CD4bs structures showed correlations between the aperture width of the CD4bs cavity and ability of the Envs to mediate cell-cell fusion, scavenge low-levels of cell-surface CD4, bind directly to soluble CD4, and bind to the Env mAb IgG1b12 whose epitope overlaps the gp120 CD4bs. These structural alterations in the CD4bs cavity were associated with repositioning of the V5 loop. CONCLUSIONS: Using a large, independent panel of Envs, we can confirm the utility of three-dimensional gp120 structural models for illustrating CD4bs alterations that can affect Env function. Furthermore, we now provide new evidence that these CD4bs alterations augment the ability of gp120 to interact with CD4 by increasing the exposure of the CD4bs.

Genetic and functional heterogeneity of CNS-derived tat alleles from patients with HIV-associated dementia.

Human immunodeficiency virus type 1 (HIV-1) demonstrates a high degree of viral diversity which has an impact on viral fitness. Genetic compartmentalization of HIV-1 proteins between central nervous system (CNS) and lymphoid tissues is well established and reflects altered requirements for HIV-1 replication in macrophages/microglia, brain-specific immune selection pressures and possibly the timing of virus invasion of the CNS. Tat-encoding mRNA has been detected in the CNS of HIV-1 infected individuals and its neurotoxic effects in the CNS are well documented. However, while CNS-derived tat sequences have demonstrated significant diversity, the effect of this molecular diversity on transcriptional regulation and its impact on the pathogenesis of HIV-associated dementia (HAD) remains unclear. In this study, we cloned and characterised 44 unique tat alleles from brain, cerebral spinal fluid, spinal cord and blood/lymphoid tissue-derived HIV-1 isolates from five subjects with HAD. While phylogenetic analyses revealed tissue-specific compartmentalization of Tat variants for two patients, broad compartmentalization across the panel of tissue-derived viruses was not observed. Despite the lack of consistent tissue-specific compartmentalization, sequence variations within patients segregated CNS and non-CNS tat alleles. These amino acid alterations predominated within the transactivation domain of Tat and could account for alterations in the ability of particular Tat proteins to transactivate the LTR. Although a subset of patients demonstrated reduced transactivation capacity among CNS-derived Tat proteins compared to those from matched lymphoid tissues, overall Tat proteins from the CNS to lymphoid compartments maintained similar levels of transactivation function. Together, these data suggest that despite the observed heterogeneity in tat alleles isolated from matched lymphoid to CNS compartments, Tat function is maintained, highlighting the importance of Tat function in HIV-1 neuropathogenesis.

CD4 and MHC class 1 down-modulation activities of nef alleles from brain- and lymphoid tissue-derived primary HIV-1 isolates.

Human immunodeficiency virus type 1 (HIV-1) nef undergoes adaptive evolution in the central nervous system (CNS), reflecting altered requirements for HIV-1 replication in macrophages/microglia and brain-specific immune selection pressures. The role of Nef in HIV-1 neurotropism and pathogenesis of HIV-associated dementia (HAD) is unclear. In this study, we characterized 82 nef alleles cloned from brain, cerebral spinal fluid, spinal cord, and blood/lymphoid tissue-derived HIV-1 isolates from seven subjects with HAD. CNS isolate-derived nef alleles were genetically compartmentalized and had reduced sequence diversity compared to those from lymphoid tissue isolates. Defective nef alleles predominated in a brain-derived isolate from one of the seven subjects (MACS2-br). The ability of Nef to down-modulate CD4 and MHC class 1 (MHC-1) was generally conserved among nef alleles from both CNS and lymphoid tissues. However, the potency of CD4 and MHC-1 down-modulation was variable, which was associated with sequence alterations known to influence these Nef functions. These results suggest that CD4 and MHC-1 down-modulations are highly conserved functions among nef alleles from CNS- and lymphoid tissue-derived HIV-1 isolates that may contribute to viral replication and escape from immune surveillance in the CNS.

Both CD31(+) and CD31⁻ naive CD4(+) T cells are persistent HIV type 1-infected reservoirs in individuals receiving antiretroviral therapy.

BACKGROUND: Naive T cell recovery is critical for successful immune reconstitution after antiretroviral therapy (ART), but the relative contribution of CD31(+) and CD31⁻ naive T cells to immune reconstitution and viral persistence is unknown. METHODS: In a cross-sectional (n = 94) and longitudinal (n = 10) study of human immunodeficiency virus (HIV)-infected patients before and after ART, we examined the ratio of CD31(+) to CD31⁻ naive CD4(+) T cells. In the longitudinal cohort we then quantified the concentration of HIV-1 DNA in each cell subset and performed single-genome amplification of virus from memory and naive T cells. RESULTS: Patients receiving ART had a higher proportion of CD31(+) CD4(+) T cells than HIV-1-infected individuals naive to ART and uninfected control subjects (P < .001 and .007, respectively). After 24 months of ART, the proportion of CD31(+) naive CD4(+) T cells did not change, the concentration of HIV-1 DNA in memory CD4(+) T cells significantly decreased over time (P < .001), and there was no change in the concentration of HIV-1 DNA in CD31(+) or CD31⁻ naive CD4(+) T cells (P = .751 and .251, respectively). Single-genome amplification showed no evidence of virus compartmentalization in memory and naive T cell subsets before or after ART. CONCLUSIONS: After ART, both CD31(+) and CD31⁻ naive CD4(+) T cells expand, and both subsets represent a stable, persistent reservoir of HIV-1.

Modular Lentiviral Vectors for Highly Efficient Transgene Expression in Resting Immune Cells.

Gene/cell therapies are promising strategies for the many presently incurable diseases. A key step in this process is the efficient delivery of genes and gene-editing enzymes to many cell types that may be resistant to lentiviral vector transduction. Herein we describe tuning of a lentiviral gene therapy platform to focus on genetic modifications of resting CD4+ T cells. The motivation for this was to find solutions for HIV gene therapy efforts. Through selection of the optimal viral envelope and further modification to its expression, lentiviral fusogenic delivery into resting CD4+ T cells exceeded 80%, yet Sterile Alpha Motif and HD domain 1 (SAMHD1) dependent and independent intracellular restriction factors within resting T cells then dominate delivery and integration of lentiviral cargo. Overcoming SAMHD1-imposed restrictions, only observed up to 6-fold increase in transduction, with maximal gene delivery and expression of 35%. To test if the biologically limiting steps of lentiviral delivery are reverse transcription and integration, we re-engineered lentiviral vectors to simply express biologically active mRNA to direct transgene expression in the cytoplasm. In this setting, we observed gene expression in up to 65% of resting CD4+ T cells using unconcentrated MS2 lentivirus-like particles (MS2-LVLPs). Taken together, our findings support a gene therapy platform that could be readily used in resting T cell gene editing.

Extremely prolonged HIV seroconversion associated with an MHC haplotype carrying disease susceptibility genes for antibody deficiency disorders.

Severe immunodeficiency during primary human immunodeficiency virus (HIV) infection is unusual. Here, we characterized viral and immunological parameters in a subject presenting with Pneumocystis jirovecii pneumonia in the setting of prolonged primary HIV illness and delayed seroconversion. HIV antibody was only detected by enzyme-linked immunosorbent assay 12 months after presentation, and Western blot profiles remain indeterminate. Isolated virus was of R5 phenotype, exhibited poor viral fitness, but was otherwise unremarkable. Analysis of HIV antibody isotypes showed failure to mount a detectable HIV IgG response over nearly 2 years of infection, in particular IgG(1)- and IgG(3)-specific responses, despite normal responses to common infections and vaccines. Genetic analysis demonstrated homozygosity for part of an MHC haplotype containing susceptibility genes for common variable immunodeficiency (CVID) syndrome and other antibody deficiency disorders. Thus, a primary disorder of specific antibody production may explain exceptionally slow antibody development in an otherwise severe seroconversion illness. This highlights the need for multiparameter testing, in particular use of a fourth generation HIV test, for confirming HIV infection and underscores the importance of host factors in HIV pathogenesis.

Tissue-specific sequence alterations in the human immunodeficiency virus type 1 envelope favoring CCR5 usage contribute to persistence of dual-tropic virus in the brain.

Most human immunodeficiency virus type 1 (HIV-1) strains isolated from the brain use CCR5 for entry into macrophages and microglia. Strains that use both CCR5 and CXCR4 for entry (R5X4 strains) have been identified in the brains of some individuals, but mechanisms underlying the persistence of R5X4 viruses compartmentalized between the brain and other tissue reservoirs are unknown. Here, we characterized changes in the HIV-1 envelope (Env) that enhance the tropism of R5X4 variants for brain or lymphoid tissue. R5X4 Envs derived from the brains of two individuals had enhanced CCR5 usage in fusion assays compared to R5X4 Envs derived from matched spleen or blood, which was associated with reduced dependence on specific residues in the CCR5 N terminus and extracellular loop 1 (ECL1) and ECL3 regions. In contrast, spleen/blood-derived Envs had enhanced CXCR4 usage compared to brain-derived Envs, which was associated with reduced dependence on residues in the CXCR4 N terminus and ECL2 region. Consequently, brain-derived Envs had preferential CCR5 usage for HIV-1 entry into the JC53 cell line, could use either CCR5 or CXCR4 for entry into monocyte-derived macrophages (MDM), and could use CCR5 (albeit inefficiently) for entry into peripheral blood mononuclear cells (PBMC), whereas the entry of spleen-derived Envs was CXCR4 dependent in all three cell types. Mutagenesis studies of Env amino acid variants influencing coreceptor usage showed that S306 in the gp120 V3 region of brain-derived Envs reduces dependence on the CCR5 N terminus and enhances CCR5 usage for HIV-1 entry into PBMC and MDM, whereas R306 in spleen-derived Envs reduces dependence on the CXCR4 N terminus and confers the CXCR4 restricted phenotype. These results identify mechanisms underlying R5X4 HIV-1 persistence in different tissue reservoirs. Tissue-specific changes in the gp120 V3 region that increase the efficiency of CCR5 or CXCR4 usage, and thereby influence coreceptor preference, may enhance the tropism of R5X4 strains for CCR5-expressing macrophage lineage cells in the brain and CXCR4-expressing T cells in lymphoid tissues, respectively.

Identification of Specific Circular RNA Expression Patterns and MicroRNA Interaction Networks in Mesial Temporal Lobe Epilepsy.

Circular RNAs (circRNAs) regulate mRNA translation by binding to microRNAs (miRNAs), and their expression is altered in diverse disorders, including cancer, cardiovascular disease, and Parkinson's disease. Here, we compare circRNA expression patterns in the temporal cortex and hippocampus of patients with pharmacoresistant mesial temporal lobe epilepsy (MTLE) and healthy controls. Nine circRNAs showed significant differential expression, including circRNA-HOMER1, which is expressed in synapses. Further, we identified miRNA binding sites within the sequences of differentially expressed (DE) circRNAs; expression levels of mRNAs correlated with changes in complementary miRNAs. Gene set enrichment analysis of mRNA targets revealed functions in heterocyclic compound binding, regulation of transcription, and signal transduction, which maintain the structure and function of hippocampal neurons. The circRNA-miRNA-mRNA interaction networks illuminate the molecular changes in MTLE, which may be pathogenic or an effect of the disease or treatments and suggests that DE circRNAs and associated miRNAs may be novel therapeutic targets.

HIV latency can be established in proliferating and nonproliferating resting CD4+ T cells in vitro: implications for latency reversal.

OBJECTIVE: To determine whether latency can be established and reversed in both proliferating and nonproliferating CD4+ T cells in the same model in vitro. METHODS: Activated CD4+ T cells were infected with either a nonreplication competent, luciferase reporter virus or wild-type full-length enhanced green fluorescent protein (EGFP) reporter virus and cultured for 12 days. The cells were then sorted by flow cytometry to obtain two distinct T-cell populations that did not express the T-cell activation markers, CD69, CD25 and human leukocyte antigen (HLA)-DR: CD69CD25HLA-DR small cells (nonblasts) that had not proliferated in vitro following mitogen stimulation and CD69CD25HLA-DR large cells (which we here call transitional blasts) that had proliferated. The cells were then reactivated with latency-reversing agents and either luciferase or EGFP quantified. RESULTS: Inducible luciferase expression, consistent with latent infection, was observed in nonblasts and transitional blasts following stimulation with either phorbol-myristate-acetate/phytohemagglutinin (3.8 ±â€Š1 and 2.9 ±â€Š0.5 fold above dimethyl sulfoxide, respectively) or romidepsin (2.1 ±â€Š0.6 and 1.8 ±â€Š0.2 fold above dimethyl sulfoxide, respectively). Constitutive expression of luciferase was higher in transitional blasts compared with nonblasts. Using wild-type full-length EGFP reporter virus, inducible virus was observed in nonblasts but not in transitional blasts. No significant difference was observed in the response to latency-reversing agents in either nonblasts or transitional blasts. CONCLUSION: HIV latency can be established in vitro in resting T cells that have not proliferated (nonblasts) and blasts that have proliferated (transitional blasts). This model could potentially be used to assess new strategies to eliminate latency.