Human otic progenitor cell models of congenital hearing loss reveal potential pathophysiologic mechanisms of Zika virus and cytomegalovirus infections.

Congenital hearing loss is a common chronic condition affecting children in both developed and developing nations. Viruses correlated with congenital hearing loss include human cytomegalovirus (HCMV) and Zika virus (ZIKV), which causes congenital Zika syndrome. The mechanisms by which HCMV and ZIKV infections cause hearing loss are poorly understood. It is challenging to study human inner ear cells because they are encased in bone and also scarce as autopsy samples. Recent advances in culturing human stem cell-derived otic progenitor cells (OPCs) have allowed us herein to describe successful in vitro infection of OPCs with HCMV and ZIKV, and also to propose potential mechanisms by which each viral infection could affect hearing. We find that ZIKV infection rapidly and significantly induces the expression of type I interferon and interferon-stimulated genes, while OPC viability declines, at least in part, from apoptosis. In contrast, HCMV infection did not appear to upregulate interferons or cause a reduction in cell viability, and instead disrupted expression of key genes and pathways associated with inner ear development and function, including Cochlin, nerve growth factor receptor, SRY-box transcription factor 11, and transforming growth factor-beta signaling. These findings suggest that ZIKV and HCMV infections cause congenital hearing loss through distinct pathways, that is, by inducing progenitor cell death in the case of ZIKV infection, and by disruption of critical developmental pathways in the case of HCMV infection. IMPORTANCE: Congenital virus infections inflict substantial morbidity and devastating disease in neonates worldwide, and hearing loss is a common outcome. It has been difficult to study viral infections of the human hearing apparatus because it is embedded in the temporal bone of the skull. Recent technological advances permit the differentiation of otic progenitor cells (OPCs) from human-induced pluripotent stem cells. This paper is important for demonstrating that inner ear virus infections can be modeled in vitro using OPCs. We infected OPCs with two viruses associated with congenital hearing loss: human cytomegalovirus (HCMV), a DNA virus, or Zika virus (ZIKV), an RNA virus. An important result is that the gene expression and cytokine production profiles of HCMV/ZIKV-infected OPCs are markedly dissimilar, suggesting that mechanisms of hearing loss are also distinct. The specific molecular regulatory pathways identified in this work could suggest important targets for therapeutics.

Direct SARS-CoV-2 infection of the human inner ear may underlie COVID-19-associated audiovestibular dysfunction.

BACKGROUND: COVID-19 is a pandemic respiratory and vascular disease caused by SARS-CoV-2 virus. There is a growing number of sensory deficits associated with COVID-19 and molecular mechanisms underlying these deficits are incompletely understood. METHODS: We report a series of ten COVID-19 patients with audiovestibular symptoms such as hearing loss, vestibular dysfunction and tinnitus. To investigate the causal relationship between SARS-CoV-2 and audiovestibular dysfunction, we examine human inner ear tissue, human inner ear in vitro cellular models, and mouse inner ear tissue. RESULTS: We demonstrate that adult human inner ear tissue co-expresses the angiotensin-converting enzyme 2 (ACE2) receptor for SARS-CoV-2 virus, and the transmembrane protease serine 2 (TMPRSS2) and FURIN cofactors required for virus entry. Furthermore, hair cells and Schwann cells in explanted human vestibular tissue can be infected by SARS-CoV-2, as demonstrated by confocal microscopy. We establish three human induced pluripotent stem cell (hiPSC)-derived in vitro models of the inner ear for infection: two-dimensional otic prosensory cells (OPCs) and Schwann cell precursors (SCPs), and three-dimensional inner ear organoids. Both OPCs and SCPs express ACE2, TMPRSS2, and FURIN, with lower ACE2 and FURIN expression in SCPs. OPCs are permissive to SARS-CoV-2 infection; lower infection rates exist in isogenic SCPs. The inner ear organoids show that hair cells express ACE2 and are targets for SARS-CoV-2. CONCLUSIONS: Our results provide mechanistic explanations of audiovestibular dysfunction in COVID-19 patients and introduce hiPSC-derived systems for studying infectious human otologic disease.

Progressive multifocal leukoencephalopathy presenting with acute sensorineural hearing loss in an intestinal transplant recipient.

A 20-year-old male presented 3.5 years after intestinal transplantation with rapidly progressive sensorineural hearing loss. Initial brain imaging was consistent with inflammation and/or demyelination. Lumbar puncture was initially non-diagnostic and a broad infectious workup was unrevealing. Three months after presentation, a repeat LP detected JC virus for which tests had not earlier been conducted. He continued to deteriorate despite withdrawal of prior immunosuppression and addition of mirtazapine, maraviroc, and steroids. He died of progressive neurologic decompensation 5 months after his initial presentation. This case highlights progressive multifocal leukoencephalopathy (PML) as a rare complication after solid organ transplantation and acute sensorineural hearing loss as an unusual first presenting symptom of PML. JC virus should be considered in the differential diagnosis of acute sensorineural hearing loss in any immunocompromised patient.

Pediatric post-transplant hepatic kaposi sarcoma due to donor-derived human herpesvirus 8.

In areas of the world where human herpesvirus 8 (HHV-8) is endemic, Kaposi sarcoma (KS) is a common SOT-associated cancer. In the United States, where the virus is not prevalent, PTKS is rare, and there is little literature on pediatric PTKS. We present a North American female who underwent deceased donor, left lateral segment liver transplant for biliary atresia at age 11 months. The donor was a male with no known history of KS, originally from an HHV-8-endemic country. Three months after transplantation, the patient developed liver nodules and portal vein thrombosis. Analysis of needle biopsy established the diagnosis of KS and confirmed that the transformed cells were donor-derived. HHV-8 viremia was detected, and ganciclovir dosing (which had been started prophylactically) was increased. Immunosuppression was changed from tacrolimus to sirolimus. After further disease progression, 8 cycles of paclitaxel were administered. Under this treatment, her nodules regressed, HHV-8 viremia resolved, and she had marked clinic improvement. Notably, the adult recipient of the right liver lobe from the same donor also developed PTKS. This is one of few pediatric PTKS cases described in the literature. It contributes to the mechanistic understanding of PTKS development, illustrating the risk posed by donors from HHV-8-endemic countries, as well as the potential for strong PTKS correlation between multiple recipients of organs from a single shared donor.

Gene activity in primary T cells infected with HIV89.6: intron retention and induction of genomic repeats.

BACKGROUND: HIV infection has been reported to alter cellular gene activity, but published studies have commonly assayed transformed cell lines and lab-adapted HIV strains, yielding inconsistent results. Here we carried out a deep RNA-Seq analysis of primary human T cells infected with the low passage HIV isolate HIV89.6. RESULTS: Seventeen percent of cellular genes showed altered activity 48 h after infection. In a meta-analysis including four other studies, our data differed from studies of HIV infection in cell lines but showed more parallels with infections of primary cells. We found a global trend toward retention of introns after infection, suggestive of a novel cellular response to infection. HIV89.6 infection was also associated with activation of several human endogenous retroviruses (HERVs) and retrotransposons, of interest as possible novel antigens that could serve as vaccine targets. The most highly activated group of HERVs was a subset of the ERV-9. Analysis showed that activation was associated with a particular variant of ERV-9 long terminal repeats that contains an indel near the U3-R border. These data also allowed quantification of >70 splice forms of the HIV89.6 RNA and specified the main types of chimeric HIV89.6-host RNAs. Comparison to over 100,000 integration site sequences from the same infected cell populations allowed quantification of authentic versus artifactual chimeric reads, showing that 5' read-in, splicing out of HIV89.6 from the D4 donor and 3' read-through were the most common HIV89.6-host cell chimeric RNA forms. CONCLUSIONS: Analysis of RNA abundance after infection of primary T cells with the low passage HIV89.6 isolate disclosed multiple novel features of HIV-host interactions, notably intron retention and induction of transcription of retrotransposons and endogenous retroviruses.

A reverse transcription loop-mediated isothermal amplification assay optimized to detect multiple HIV subtypes.

Diagnostic methods for detecting and quantifying HIV RNA have been improving, but efficient methods for point-of-care analysis are still needed, particularly for applications in resource-limited settings. Detection based on reverse-transcription loop-mediated isothermal amplification (RT-LAMP) is particularly useful for this, because when combined with fluorescence-based DNA detection, RT-LAMP can be implemented with minimal equipment and expense. Assays have been developed to detect HIV RNA with RT-LAMP, but existing methods detect only a limited subset of HIV subtypes. Here we report a bioinformatic study to develop optimized primers, followed by empirical testing of 44 new primer designs. One primer set (ACeIN-26), targeting the HIV integrase coding region, consistently detected subtypes A, B, C, D, and G. The assay was sensitive to at least 5000 copies per reaction for subtypes A, B, C, D, and G, with Z-factors of above 0.69 (detection of the minor subtype F was found to be unreliable). There are already rapid and efficient assays available for detecting HIV infection in a binary yes/no format, but the rapid RT-LAMP assay described here has additional uses, including 1) tracking response to medication by comparing longitudinal values for a subject, 2) detecting of infection in neonates unimpeded by the presence of maternal antibody, and 3) detecting infection prior to seroconversion.

Phylogeography and molecular epidemiology of an epidemic strain of dengue virus type 1 in Sri Lanka.

In 2009, a severe epidemic of dengue disease occurred in Sri Lanka, with higher mortality and morbidity than any previously recorded epidemic in the country. It corresponded to a shift to dengue virus 1 as the major disease-causing serotype in Sri Lanka. Dengue disease reached epidemic levels in the next 3 years. We report phylogenetic evidence that the 2009 epidemic DENV-1 strain continued to circulate within the population and caused severe disease in the epidemic of 2012. Bayesian phylogeographic analyses suggest that the 2009 Sri Lankan epidemic DENV-1 strain may have traveled directly or indirectly from Thailand through China to Sri Lanka, and after spreading within the Sri Lankan population, it traveled to Pakistan and Singapore. Our findings delineate the dissemination route of a virulent DENV-1 strain in Asia. Understanding such routes will be of particular importance to global control efforts.

Comparing DNA integration site clusters with scan statistics.

MOTIVATION: Gene therapy with retroviral vectors can induce adverse effects when those vectors integrate in sensitive genomic regions. Retroviral vectors are preferred that target sensitive regions less frequently, motivating the search for localized clusters of integration sites and comparison of the clusters formed by integration of different vectors. Scan statistics allow the discovery of spatial differences in clustering and calculation of false discovery rates providing statistical methods for comparing retroviral vectors. RESULTS: A scan statistic for comparing two vectors using multiple window widths is proposed with software to detect clustering differentials and compute false discovery rates. Application to several sets of experimentally determined HIV integration sites demonstrates the software. Simulated datasets of various sizes and signal strengths are used to determine the power to discover clusters and evaluate a convenient lower bound. This provides a toolkit for planning evaluations of new gene therapy vectors. AVAILABILITY AND IMPLEMENTATION: The geneRxCluster R package containing a simple tutorial and usage hints is available from http://www.bioconductor.org.

HIV latency and integration site placement in five cell-based models.

BACKGROUND: HIV infection can be treated effectively with antiretroviral agents, but the persistence of a latent reservoir of integrated proviruses prevents eradication of HIV from infected individuals. The chromosomal environment of integrated proviruses has been proposed to influence HIV latency, but the determinants of transcriptional repression have not been fully clarified, and it is unclear whether the same molecular mechanisms drive latency in different cell culture models. RESULTS: Here we compare data from five different in vitro models of latency based on primary human T cells or a T cell line. Cells were infected in vitro and separated into fractions containing proviruses that were either expressed or silent/inducible, and integration site populations sequenced from each. We compared the locations of 6,252 expressed proviruses to those of 6,184 silent/inducible proviruses with respect to 140 forms of genomic annotation, many analyzed over chromosomal intervals of multiple lengths. A regularized logistic regression model linking proviral expression status to genomic features revealed no predictors of latency that performed better than chance, though several genomic features were significantly associated with proviral expression in individual models. Proviruses in the same chromosomal region did tend to share the same expressed or silent/inducible status if they were from the same cell culture model, but not if they were from different models. CONCLUSIONS: The silent/inducible phenotype appears to be associated with chromosomal position, but the molecular basis is not fully clarified and may differ among in vitro models of latency.

Dynamic regulation of HIV-1 mRNA populations analyzed by single-molecule enrichment and long-read sequencing.

Alternative RNA splicing greatly expands the repertoire of proteins encoded by genomes. Next-generation sequencing (NGS) is attractive for studying alternative splicing because of the efficiency and low cost per base, but short reads typical of NGS only report mRNA fragments containing one or few splice junctions. Here, we used single-molecule amplification and long-read sequencing to study the HIV-1 provirus, which is only 9700 bp in length, but encodes nine major proteins via alternative splicing. Our data showed that the clinical isolate HIV-1(89.6) produces at least 109 different spliced RNAs, including a previously unappreciated ∼1 kb class of messages, two of which encode new proteins. HIV-1 message populations differed between cell types, longitudinally during infection, and among T cells from different human donors. These findings open a new window on a little studied aspect of HIV-1 replication, suggest therapeutic opportunities and provide advanced tools for the study of alternative splicing.

The effect of play type and collision closing distance on head impact biomechanics.

Football accounts for 55% of concussions to collegiate athletes. In the National Football League, players are at a greater risk for concussion during kickoffs and punts compared to rushing and passing plays. The two primary purposes of this study were to determine if game-related special teams head impacts were greater in magnitude than head impacts sustained during offensive and defensive plays, and to better understand the effect closing distance between players (short vs. long) had on head impact magnitude. Collegiate football players were enrolled in a prospective cohort study assessing head impact biomechanics during special teams, offensive, and defensive collisions; long closing distance (≥ 10 yards) and short closing distance (<10 yards) impacts were also studied. Data were analyzed using random intercepts general linear mixed models. Long closing distance collisions generated more severe head impacts than short closing distances. Collisions occurring on special teams plays over long closing distances were most severe while collisions occurring on special teams and defensive plays over short closing distances resulted in the least severe impacts. Decreasing the impact severity of collisions in collegiate football may be accomplished by reducing the closing distance prior to impact.

HIV-1 capsid-cyclophilin interactions determine nuclear import pathway, integration targeting and replication efficiency.

Lentiviruses such as HIV-1 traverse nuclear pore complexes (NPC) and infect terminally differentiated non-dividing cells, but how they do this is unclear. The cytoplasmic NPC protein Nup358/RanBP2 was identified as an HIV-1 co-factor in previous studies. Here we report that HIV-1 capsid (CA) binds directly to the cyclophilin domain of Nup358/RanBP2. Fusion of the Nup358/RanBP2 cyclophilin (Cyp) domain to the tripartite motif of TRIM5 created a novel inhibitor of HIV-1 replication, consistent with an interaction in vivo. In contrast to CypA binding to HIV-1 CA, Nup358 binding is insensitive to inhibition with cyclosporine, allowing contributions from CypA and Nup358 to be distinguished. Inhibition of CypA reduced dependence on Nup358 and the nuclear basket protein Nup153, suggesting that CypA regulates the choice of the nuclear import machinery that is engaged by the virus. HIV-1 cyclophilin-binding mutants CA G89V and P90A favored integration in genomic regions with a higher density of transcription units and associated features than wild type virus. Integration preference of wild type virus in the presence of cyclosporine was similarly altered to regions of higher transcription density. In contrast, HIV-1 CA alterations in another patch on the capsid surface that render the virus less sensitive to Nup358 or TRN-SR2 depletion (CA N74D, N57A) resulted in integration in genomic regions sparse in transcription units. Both groups of CA mutants are impaired in replication in HeLa cells and human monocyte derived macrophages. Our findings link HIV-1 engagement of cyclophilins with both integration targeting and replication efficiency and provide insight into the conservation of viral cyclophilin recruitment.

Role of the PWWP domain of lens epithelium-derived growth factor (LEDGF)/p75 cofactor in lentiviral integration targeting.

LEDGF/p75 is a chromatin-interacting, cellular cofactor of HIV integrase that dictates lentiviral integration site preference. In this study we determined the role of the PWWP domain of LEDGF/p75 in tethering and targeting of the lentiviral pre-integration complex, employing potent knockdown cell lines allowing analysis in the absence of endogenous LEDGF/p75. Deletion of the PWWP domain resulted in a diffuse subnuclear distribution pattern, loss of interaction with condensed chromatin, and failure to rescue proviral integration, integration site distribution, and productive virus replication. Substitution of the PWWP domain of LEDGF/p75 with that of hepatoma-derived growth factor or HDGF-related protein-2 rescued viral replication and lentiviral integration site distribution in LEDGF/p75-depleted cells. Replacing all chromatin binding elements of LEDGF/p75 with full-length hepatoma-derived growth factor resulted in more integration in genes combined with a preference for CpG islands. In addition, we showed that any PWWP domain targets SMYD1-like sequences. Analysis of integration preferences of lentiviral vectors for epigenetic marks indicates that the PWWP domain is critical for interactions specifying the relationship of integration sites to regions enriched in specific histone post-translational modifications.

HIV integration targeting: a pathway involving Transportin-3 and the nuclear pore protein RanBP2.

Genome-wide siRNA screens have identified host cell factors important for efficient HIV infection, among which are nuclear pore proteins such as RanBP2/Nup358 and the karyopherin Transportin-3/TNPO3. Analysis of the roles of these proteins in the HIV replication cycle suggested that correct trafficking through the pore may facilitate the subsequent integration step. Here we present data for coupling between these steps by demonstrating that depletion of Transportin-3 or RanBP2 altered the terminal step in early HIV replication, the selection of chromosomal sites for integration. We found that depletion of Transportin-3 and RanBP2 altered integration targeting for HIV. These knockdowns reduced HIV integration frequency in gene-dense regions and near gene-associated features, a pattern that differed from that reported for depletion of the HIV integrase binding cofactor Psip1/Ledgf/p75. MLV integration was not affected by the Transportin-3 knockdown. Using siRNA knockdowns and integration targeting analysis, we also implicated several additional nuclear proteins in proper target site selection. To map viral determinants of integration targeting, we analyzed a chimeric HIV derivative containing MLV gag, and found that the gag replacement phenocopied the Transportin-3 and RanBP2 knockdowns. Thus, our data support a model in which Gag-dependent engagement of the proper transport and nuclear pore machinery mediate trafficking of HIV complexes to sites of integration.

Metal cofactors in the structure and activity of the fowlpox resolvase.

Poxvirus DNA replication generates linear concatemers containing many copies of the viral genome with inverted repeat sequences at the junctions between monomers. The inverted repeats refold to generate Holliday junctions, which are cleaved by the virus-encoded resolvase enzyme to form unit-length genomes. Here we report studies of the influence of metal cofactors on the activity and structure of the resolvase of fowlpox virus, which provides a tractable model for in vitro studies. Small-molecule inhibitors of related enzymes bind simultaneously to metal cofactors and nearby surface amino acid residues, so understanding enzyme-cofactor interactions is important for the design of antiviral agents. Analysis of inferred active-site residues (D7, E60, K102, D132, and D135) by mutagenesis and metal rescue experiments specified residues that contribute to binding metal ions and that multiple binding sites are probably involved. Differential electrophoretic analysis was used to map the conformation of the DNA junction when bound by resolvase. For the wild-type complex in the presence of EDTA (ethylenediaminetetraacetic acid) or Ca(2+), migration was consistent with the DNA arms arranged in near-tetrahedral geometry. However, the D7N active-site mutant resolvase held the arms in a more planar arrangement in EDTA, Ca(2+), or Mg(2+) conditions, implicating metal-dependent contacts at the active site in the larger architecture of the complex. These data show how divalent metals dictate the conformation of FPV resolvase-DNA complexes and subsequent DNA cleavage.