Modeling HIV-1 infection and NeuroHIV in hiPSCs-derived cerebral organoid cultures.

The human immunodeficiency virus (HIV) epidemic is an ongoing global health problem affecting 38 million people worldwide with nearly 1.6 million new infections every year. Despite the advent of combined antiretroviral therapy (cART), a large percentage of people with HIV (PWH) still develop neurological deficits, grouped into the term of HIV-associated neurocognitive disorders (HAND). Investigating the neuropathology of HIV is important for understanding mechanisms associated with cognitive impairment seen in PWH. The major obstacle for studying neuroHIV is the lack of suitable in vitro human culture models that could shed light into the HIV-CNS interactions. Recent advances in induced pluripotent stem cell (iPSC) culture and 3D brain organoid systems have allowed the generation of 2D and 3D culture methods that possess a potential to serve as a model of neurotropic viral diseases, including HIV. In this study, we first generated and characterized several hiPSC lines from healthy human donor skin fibroblast cells. hiPSCs were then used for the generation of microglia-containing human cerebral organoids (hCOs). Once fully characterized, hCOs were infected with HIV-1 in the presence and absence of cART regimens and viral infection was studied by cellular, molecular/biochemical, and virological assays. Our results revealed that hCOs were productively infected with HIV-1 as evident by viral p24-ELISA in culture media, RT-qPCR and RNAscope analysis of viral RNA, as well as ddPCR analysis of proviral HIV-1 in genomic DNA samples. More interestingly, replication and gene expression of HIV-1 were also greatly suppressed by cART in hCOs as early as 7 days post-infections. Our results suggest that hCOs derived from hiPSCs support HIV-1 replication and gene expression and may serve as a unique platform to better understand neuropathology of HIV infection in the brain.

Preclinical safety and biodistribution of CRISPR targeting SIV in non-human primates.

In this study, we demonstrate the safety and utility of CRISPR-Cas9 gene editing technology for in vivo editing of proviral DNA in ART-treated, virally controlled simian immunodeficiency virus (SIV) infected rhesus macaques, an established model for HIV infection. EBT-001 is an AAV9-based vector delivering SaCas9 and dual guide RNAs designed to target multiple regions of the SIV genome: the viral LTRs, and the Gag gene. The results presented here demonstrate that a single IV inoculation of EBT-001 at each of 3 dose levels (1.4 × 1012, 1.4 × 1013 and 1.4 × 1014 genome copies/kg) resulted in broad and functional biodistribution of AAV9-EBT-001 to known tissue reservoirs of SIV. No off-target effects or abnormal pathology were observed, and animals returned to their normal body weight after receiving EBT-001. Importantly, the macaques that received the 2 highest doses of EBT-001 showed improved absolute lymphocyte counts as compared to antiretroviral-treated controls. Taken together, these results demonstrate safety, biodistribution, and in vivo proviral DNA editing following IV administration of EBT-001, supporting the further development of CRISPR-based gene editing as a potential therapeutic approach for HIV in humans.

iPSC-derived three-dimensional brain organoid models and neurotropic viral infections.

Progress in stem cell research has revolutionized the medical field for more than two decades. More recently, the discovery of induced pluripotent stem cells (iPSCs) has allowed for the development of advanced disease modeling and tissue engineering platforms. iPSCs are generated from adult somatic cells by reprogramming them into an embryonic-like state via the expression of transcription factors required for establishing pluripotency. In the context of the central nervous system (CNS), iPSCs have the potential to differentiate into a wide variety of brain cell types including neurons, astrocytes, microglial cells, endothelial cells, and oligodendrocytes. iPSCs can be used to generate brain organoids by using a constructive approach in three-dimensional (3D) culture in vitro. Recent advances in 3D brain organoid modeling have provided access to a better understanding of cell-to-cell interactions in disease progression, particularly with neurotropic viral infections. Neurotropic viral infections have been difficult to study in two-dimensional culture systems in vitro due to the lack of a multicellular composition of CNS cell networks. In recent years, 3D brain organoids have been preferred for modeling neurotropic viral diseases and have provided invaluable information for better understanding the molecular regulation of viral infection and cellular responses. Here we provide a comprehensive review of the literature on recent advances in iPSC-derived 3D brain organoid culturing and their utilization in modeling major neurotropic viral infections including HIV-1, HSV-1, JCV, ZIKV, CMV, and SARS-CoV2.

Coronavirus infection in chemosensory cells.

Clinical manifestations of human coronavirus (HCoV)-related diseases are mostly related to the respiratory system, although secondary complications such as headache, anosmia, ageusia, and myalgia have been reported. HCoV infection and replication in chemosensory cells associated with ageusia and anosmia is poorly understood. Here, we characterized HCoV-OC43 and SARS-CoV-2 infection in two types of chemosensory cells, olfactory and taste cells, with their unique molecular and histological characteristics. We first assessed HCoV-OC43 infection in in vitro cultured human olfactory epithelial cells (hOECs) and fungiform taste papilla (HBO) cells. Interestingly, while both cell types were susceptible to HCoV-OC43 infection, viral replication rates were significantly reduced in HBO cells compared to hOECs. More interestingly, while culture media from hOECs was able to produce secondary infection in Vero cells, there was very limited secondary infection from HBO cells, suggesting that HBO cells may not be able to release infectious virus. On the other hand, unlike HCoV-OC43, SARS-CoV-2 showed comparable levels of viral infection rates in both hOECs and HBO cells. Furthermore, our RT-qPCR-based gene array studies revealed that several key genes involved in taste and olfactory functions were significantly altered by SARS-CoV-2 infection. These results may suggest a possible mechanism associated with chemosensory symptoms, such as anosmia and ageusia in patients infected with SARS-CoV-2.

Zika virus infection in chemosensory cells.

Zika virus (ZIKV) is an emerging virus belonging to the genus Flavivirus. ZIKV infection is a significant health concern, with increasing numbers of reports of microcephaly cases in fetuses and Guillain-Barré syndrome (GBS) in adults. Interestingly, chemosensory disturbances are also reported as one of the manifestations of GBS. ZIKV infects several human tissues and cell types in vitro and in vivo. However, there is no study demonstrating ZIKV infection and replication in chemosensory cells, including olfactory and taste cells. Taste papilla and olfactory cells are chemosensory receptor cells with unique histological, molecular, and physiological characteristics. Here we examined ZIKV infection (PRVABC59) in cultured human olfactory epithelial cells (hOECs) and fungiform taste papilla (HBO) cells in vitro, as well as in vivo mouse taste and olfactory epithelial and olfactory bulb tissues. Interestingly, while HBO cells showed resistance to ZIKV replication, hOECs were highly susceptible for ZIKV infection and replication. Further, we demonstrated the presence of ZIKV particles and expression of viral proteins in olfactory epithelium, as well as in olfactory bulb, but not in taste papillae, of immunocompromised mice (ifnar/-) infected with the PRVABC59 strain of ZIKV. These observations suggest that chemosensory cells in the olfactory neuroepithelium and olfactory bulb may be important tissues for ZIKV replication and dissemination.

Suppression of Zika Virus Infection in the Brain by the Antiretroviral Drug Rilpivirine.

Zika virus (ZIKV) infection is associated with microcephaly in neonates and Guillain-Barré syndrome in adults. ZIKV produces a class of nonstructural (NS) regulatory proteins that play a critical role in viral transcription and replication, including NS5, which possesses RNA-dependent RNA polymerase (RdRp) activity. Here we demonstrate that rilpivirine (RPV), a non-nucleoside reverse transcriptase inhibitor (NNRTI) used in the treatment of HIV-1 infection, inhibits the enzymatic activity of NS5 and suppresses ZIKV infection and replication in primary human astrocytes. Similarly, other members of the NNRTI family, including etravirine and efavirenz, showed inhibitory effects on viral infection of brain cells. Site-directed mutagenesis identified 14 amino acid residues within the NS5 RdRp domain (AA265-903), which are important for the RPV interaction and the inhibition of NS5 polymerase activity. Administration of RPV to ZIKV-infected interferon-alpha/beta receptor (IFN-A/R) knockout mice improved the clinical outcome and prevented ZIKV-induced mortality. Histopathological examination of the brains from infected animals revealed that RPV reduced ZIKV RNA levels in the hippocampus, frontal cortex, thalamus, and cerebellum. Repurposing of NNRTIs, such as RPV, for the inhibition of ZIKV replication offers a possible therapeutic strategy for the prevention and treatment of ZIKV-associated disease.

Alcohol-Mediated Missplicing of Mcl-1 Pre-mRNA is Involved in Neurotoxicity.

BACKGROUND: Heavy and chronic ethanol (EtOH) exposure can cause significant structural and functional damage to the adult brain. The most devastating consequence of EtOH exposure is the neurotoxicity associated with the depletion of neurons. Regulation of splice variants in the brain can modulate protein functions, which may ultimately affect behaviors associated with alcohol dependence and EtOH-mediated neurotoxicity. As alcohol consumption is associated with neurotoxicity, it is possible that altered splicing of survival and pro-survival factors during the development of alcoholism may contribute to the neurotoxicity. METHODS: Primary human neurons and a neuroblastoma cell line were exposed to different concentrations of EtOH for various time periods. Cell viability and neuronal marker expression were analyzed by MTT assay and immunoblotting, respectively. Effect of EtOH exposure on splicing regulatory protein expression and alternative splicing of candidate genes was analyzed by a biochemical approach. Transcriptional activity of serine/arginine-rich splicing factor 1 (SRSF1) gene was determined by reporter gene analysis. RESULTS: Our results suggest that EtOH exposure to neuronal cells at 25 mM and higher concentrations are detrimental. In addition, EtOH exposure caused a dramatic reduction in SRSF1 expression levels. Furthermore, EtOH exposure led to pre-mRNA missplicing of Mcl-1, a pro-survival member of the Bcl-2 family, by down-regulating the expression levels of SRSF1. Moreover, ectopic expression of both SRSF1 and Mcl-1L isoform was able to recover EtOH-mediated neurotoxicity. CONCLUSIONS: Our results suggest that EtOH exposure can lead to pre-mRNA missplicing of Mcl-1 in neuronal cells. Our results indicate that EtOH exposure of neurons leads to a decrease in the ratio of Mcl-1L/Mcl-1S by favoring pro-apoptotic Mcl-1S splicing over anti-apoptotic Mcl-1L isoform suggesting that Mcl-1S may play a crucial role in neurotoxicity associated with alcohol consumption.

Modulation of OPRM1 Alternative Splicing by Morphine and HIV-1 Nef.

Clinically used opioids, such as morphine, activate the mu opioid receptor (MOR) encoded by Opioid Receptor Mu 1 (OPRM1) gene. Examination of the opioid receptor genes showed that the human OPRM1 pre-mRNA undergoes extensive alternative splicing events and capable of expressing 21 isoforms. However, characterization of OPRM1 signaling is generalized, and only one isoform (MOR-1) has been extensively studied. Compounding this issue is the increasing significance of intravenous drug abuse in HIV neuropathogenesis. Here, we investigated the molecular impact of morphine and HIV-1 on regulation of OPRM1 pre-mRNA splicing in in vitro and in vivo models. Our results suggested that morphine treatment specifically induces the alternative splicing of MOR-1X isoform among the other isoforms analyzed in neuronal cells. Interestingly, alternative splicing and expression of MOR-1X isoform was also induced in postmortem brain tissues obtained from people with HIV (PWH). Additionally, treatment of control rats with morphine induced alternative splicing of MOR-1X in the brain regions involved in the reward pathways. More interestingly, HIV-1 transgenic (HIV-1Tg) rats, showed an additive induction of MOR-1X isoform with the exposure to morphine. To further assess the possible role of HIV secretory proteins in alternative splicing of OPRM1 gene, we analyzed the impact of HIV-1 Tat, gp120 and Nef proteins on alternative splicing of MOR-1X isoform. While the Tat and gp120 had no visible effects, treatment of neurons with Nef induced MOR-1X alternative splicing that was comparable to treatment with morphine. Altogether, our results suggest that HIV-1 may alter MOR isoform expression with Nef protein by amplifying the rate of MOR-1X alternative splicing induced by morphine.

Characterization of Nef expression in different brain regions of SIV-infected macaques.

OBJECTIVE: HIV-associated CNS dysfunction is a significant problem among people with HIV (PWH), who now live longer due to viral suppression from combined anti-retroviral therapy (ART). Over the course of infection, HIV generates toxic viral proteins and induces inflammatory cytokines that have toxic effects on neurons in the CNS. Among these viral proteins, HIV Nef has been found in neurons of postmortem brain specimens from PWH. However, the source of Nef and its impact on neuronal cell homeostasis are still elusive. METHODS AND RESULTS: Here, in using a simian immunodeficiency virus (SIV) infected rhesus macaque model of neuroHIV, we find SIV Nef reactivity in the frontal cortex, hippocampus and cerebellum of SIV-infected animals using immunohistochemistry (IHC). Interestingly, SIV-infected macaques treated with ART also showed frequent Nef positive cells in the cerebellum and hippocampus. Using dual quantitative RNAscope and IHC, we observed cells that were positive for Nef, but were not for SIV RNA, suggesting that Nef protein is present in cells that are not actively infected with SIV. Using cell specific markers, we observed Nef protein in microglia/macrophages and astrocytes. Importantly, we also identified a number of NeuN-positive neurons, which are not permissive to SIV infection, but contained Nef protein. Further characterization of Nef-positive neurons showed caspase 3 activation, indicating late stage apoptosis in the CNS neurons. CONCLUSIONS: Our results suggest that regardless of ART status, Nef is expressed in the brain of SIV infected macaques and may contribute to neurological complications seen in PWH.

Molecular and Cellular Impact of Inflammatory Extracellular Vesicles (EVs) Derived from M1 and M2 Macrophages on Neural Action Potentials.

Several factors can contribute to neuroinflammatory disorders, such as cytokine and chemokines that are produced and released from peripherally derived immune cells or from locally activated cells such as microglia and perivascular macrophages in the brain. The primary function of these cells is to clear inflammation; however, following inflammation, circulating monocytes are recruited to the central nervous system (CNS). Monocyte-derived macrophages in the CNS play pivotal roles in mediating neuroinflammatory responses. Macrophages are heterogeneous both in normal and in pathological conditions due to their plasticity, and they are classified in two main subsets, classically activated (M1) or alternatively activated (M2). There is accumulating evidence suggesting that extracellular vesicles (EVs) released from activated immune cells may play crucial roles in mediating inflammation. However, a possible role of EVs released from immune cells such as M1 and M2 macrophages on neuronal functions in the brain is not known. In order to investigate the molecular and cellular impacts of macrophages and EVs released from macrophage subtypes on neuronal functions, we used a recently established in vitro M1 and M2 macrophage culture model and isolated and characterized EVs from these macrophage subtypes, treated primary neurons with M1 or M2 EVs, and analyzed the extracellular action potentials of neurons with microelectrode array studies (MEA). Our results introduce evidence on the interfering role of inflammatory EVs released from macrophages in interneuronal signal transmission processes, with implications in the pathogenesis of neuroinflammatory diseases induced by a variety of inflammatory insults.

CRISPR based editing of SIV proviral DNA in ART treated non-human primates.

Elimination of HIV DNA from infected individuals remains a challenge in medicine. Here, we demonstrate that intravenous inoculation of SIV-infected macaques, a well-accepted non-human primate model of HIV infection, with adeno-associated virus 9 (AAV9)-CRISPR/Cas9 gene editing construct designed for eliminating proviral SIV DNA, leads to broad distribution of editing molecules and precise cleavage and removal of fragments of the integrated proviral DNA from the genome of infected blood cells and tissues known to be viral reservoirs including lymph nodes, spleen, bone marrow, and brain among others. Accordingly, AAV9-CRISPR treatment results in a reduction in the percent of proviral DNA in blood and tissues. These proof-of-concept observations offer a promising step toward the elimination of HIV reservoirs in the clinic.

Host-Immune Interactions in JC Virus Reactivation and Development of Progressive Multifocal Leukoencephalopathy (PML).

With the advent of immunomodulatory therapies and the HIV epidemic, the impact of JC Virus (JCV) on the public health system has grown significantly due to the increased incidence of Progressive Multifocal Leukoencephalopathy (PML). Currently, there are no pharmaceutical agents targeting JCV infection for the treatment and the prevention of viral reactivation leading to the development of PML. As JCV primarily reactivates in immunocompromised patients, it is proposed that the immune system (mainly the cellular-immunity component) plays a key role in the regulation of JCV to prevent productive infection and PML development. However, the exact mechanism of JCV immune regulation and reactivation is not well understood. Likewise, the impact of host factors on JCV regulation and reactivation is another understudied area. Here we discuss the current literature on host factor-mediated and immune factor-mediated regulation of JCV gene expression with the purpose of developing a model of the factors that are bypassed during JCV reactivation, and thus are potential targets for the development of therapeutic interventions to suppress PML initiation. Graphical Abstract.

Alcohol exposure alters pre-mRNA splicing of antiapoptotic Mcl-1L isoform and induces apoptosis in neural progenitors and immature neurons.

Alternative splicing and expression of splice variants of genes in the brain may lead to the modulation of protein functions, which may ultimately influence behaviors associated with alcohol dependence and neurotoxicity. We recently showed that ethanol exposure can lead to pre-mRNA missplicing of Mcl-1, a pro-survival member of the Bcl-2 family, by downregulating the expression levels of serine/arginine rich splicing factor 1 (SRSF1). Little is known about the physiological expression of these isoforms in neuronal cells and their role in toxicity induced by alcohol exposure during the developmental period. In order to investigate the impact of alcohol exposure on alternative splicing of Mcl-1 pre-mRNA and its role in neurotoxicity, we developed a unique primary human neuronal culture model where neurospheres (hNSPs), neural progenitors (hNPCs), immature neurons, and mature neurons were cultured from the matching donor fetal brain tissues. Our data suggest that neural progenitors and immature neurons are highly sensitive to the toxic effects of ethanol, while mature neuron cultures showed resistance to ethanol exposure. Further analysis of Mcl-1 pre-mRNA alternative splicing by semi-quantitative and quantitative analysis revealed that ethanol exposure causes a significant decrease in Mcl-1L/Mcl-1S ratio in a dose and time dependent manner in neural progenitors. Interestingly, ectopic expression of Mcl-1L isoform in neural progenitors was able to recover the viability loss and apoptosis induced by alcohol exposure. Altogether, these observations suggest that alternative splicing of Mcl-1 may play a crucial role in neurotoxicity associated with alcohol exposure in the developing fetal brain.

Sequential LASER ART and CRISPR Treatments Eliminate HIV-1 in a Subset of Infected Humanized Mice.

Elimination of HIV-1 requires clearance and removal of integrated proviral DNA from infected cells and tissues. Here, sequential long-acting slow-effective release antiviral therapy (LASER ART) and CRISPR-Cas9 demonstrate viral clearance in latent infectious reservoirs in HIV-1 infected humanized mice. HIV-1 subgenomic DNA fragments, spanning the long terminal repeats and the Gag gene, are excised in vivo, resulting in elimination of integrated proviral DNA; virus is not detected in blood, lymphoid tissue, bone marrow and brain by nested and digital-droplet PCR as well as RNAscope tests. No CRISPR-Cas9 mediated off-target effects are detected. Adoptive transfer of human immunocytes from dual treated, virus-free animals to uninfected humanized mice fails to produce infectious progeny virus. In contrast, HIV-1 is readily detected following sole LASER ART or CRISPR-Cas9 treatment. These data provide proof-of-concept that permanent viral elimination is possible.

Viral tumor antigen expression is no longer required in radiation-resistant subpopulation of JCV induced mouse medulloblastoma cells.

The human neurotropic polyomavirus JC, JC virus (JCV), infects the majority of human population during early childhood and establishes a latent/persistent infection for the rest of the life. JCV is the etiologic agent of the fatal demyelinating disease of the central nervous system, progressive multifocal leukoencephalopathy (PML) that is seen primarily in immunocompromised individuals. In addition to the PML, JCV has also been shown to transform cells in culture systems and cause a variety of tumors in experimental animals. Moreover, JCV genomic DNA and tumor antigen expression have been shown in a variety of human tumors with CNS origin. Similar to all polyomaviruses, JCV encodes for several tumor antigens from a single transcript of early coding region via alternative splicing. There is little known regarding the characteristics of JCV induced tumors and impact of DNA damage induced by radiation on viral tumor antigen expression and growth of these cells. Here we analyzed the possible impact of ionizing radiation on transformed phenotype and tumor antigen expression by utilizing a mouse medulloblastoma cell line (BSB8) obtained from a mouse transgenic for JCV tumor antigens. Our results suggest that a small subset of BSB8 cells survives and shows radiation resistance. Further analysis of the transformed phenotype of radiation resistant BSB8 cells (BSB8-RR) have revealed that they are capable of forming significantly higher numbers and sizes of colonies under anchorage dependent and independent conditions with reduced viral tumor antigen expression. Moreover, BSB8-RR cells show an increased rate of double-strand DNA break repair by homologous recombination (HR). More interestingly, knockout studies of JCV tumor antigens by utilizing CRISPR/Cas9 gene editing reveal that unlike parental BSB8 cells, BSB8-RR cells are no longer required the expression of viral tumor antigens in order to maintain transformed phenotype.