Biology subjects

Gummuluru, S.

Publications and source records attributed to Gummuluru, S..

12 recordsLinked to original sources

bioRxiv2026

Bloom syndrome helicase is required for efficient HIV-1 reverse transcription in macrophages

The induction of DNA damage by HIV-1 prior to integration suggests a function for DNA damage responses (DDR) during early infection, however what this role is remains incompletely understood. Initial experiments, using specific inhibitors for DDR pathways demonstrate that both ATM and ATR are necessary for efficient HIV-1 infection of macrophages with ATM acting at the late reverse transcription step. To identify DDR factors associated with ATM/ATR pathways that influence HIV-1 infection, a CRISPR knockout screen using a DDR-focused sgRNA library was performed. Approximately 30 DDR genes that impacted HIV-1 infection were identified with 13 factors that facilitated HIV-1 infection and 17 DDR factors that restrict HIV-1 infection. Several hits were factors associated with the Fanconi anemia pathway, such as BTR complex proteins, including the RecQ helicase Bloom syndrome helicase. BLM was specifically demonstrated to enhance HIV-1 infection and replication with knockdown of BLM expression diminishing integration and the establishment of intact HIV-1 proviruses in macrophages by 50%. BLM is associated with HIV-1 late reverse transcription intermediates, the step that proceeds HIV-1 integration. These findings identify BLM as a DDR host factor that promotes early HIV-1 infection by facilitating completion of reverse transcription and subsequent integration. Significance StatementHIV-1 infection elicits cellular DNA damage responses although the role of DNA damage in HIV-1 infection has not been fully characterized. Our study identifies specific DNA Damage factors that facilitate or restrict HIV infection. In particular, we show the RecQ helicase Bloom syndrome helicase (BLM) is a mediator of HIV-1 reverse transcription and integration in macrophages. This work highlights a functional interface between DNA damage repair pathways and HIV-1 integration suggesting that targeting select host DNA damage response factors can limit HIV-1 infection and persistence.

microbiology↗

bioRxiv2026

Intragenic Transcription from Defective HIV Proviruses Triggers Interferon Responses in Myeloid Cells

The persistent HIV-1 reservoir includes a subset of cells harboring transcriptionally repressed latent HIV-1 that contributes to rebound upon antiretroviral treatment (ART) interruption. However, the majority of the reservoir consists of defective proviral genomes with mutations that prevent production of HIV -1 virions. People with HIV (PWH), even with suppression of viremia, demonstrate comorbidities of the central nervous system, heart, gut, and general aging-associated inflammation. Previously, we identified a transcriptionally active element within the envelope gene (env) of HIV-1 which mediates the expression of aberrant HIV-1 RNAs. We hypothesize that spurious expression of defective proviruses contributes to the general inflammation that drives these comorbidities. We observed correlations between levels of pro-inflammatory cytokines in serum of PWH and levels of HIV-1 transcripts from this intragenic promoter. To investigate the impact of defective proviruses, we employed CRISPR-Cas9 to render the 5 Long Terminal Repeat (LTR), which acts as the enhancer and promoter for proviral transcription, non-functional. HIV-1 infected cells harboring this deletion produce significantly higher levels of IP-10 in vitro in both monocytic cell lines and primary monocyte-derived macrophages. Transcripts generated from the env promoter, include a 5 cap and polyA tail and the induction of IP-10 expression was dependent on the cytosolic innate immune sensing pathway components MDA5 and MAVS. We propose that defective HIV proviruses contribute to chronic inflammation in PWH via an MDA5-dependent induction of type I interferon pathways. ImportancePeople with HIV-1 are at higher risk of developing age-associated comorbidities and immune exhaustion even when receiving antiviral treatments and having no detectable viremia. Transcription and translation have been documented from latent and defective proviruses, but their impact on inflammation associated with chronic HIV-1 infection remains poorly understood. The significance of this work is in identifying a role for defective HIV-1 proviruses and correlating their transcription in triggering a type I interferon response. These results highlight the importance of the persistent defective HIV-1 proviruses and understanding their impact on driving chronic inflammation to inform future strategies to assure people with HIV-1 healthy living and aging.

microbiology↗

bioRxiv2025

5'' cap sequestration is required for sensing of unspliced HIV-1 RNA by MDA5

Heterogenous transcription start site (TSS) usage dictates the structure and function of unspliced HIV-1 RNAs (usRNA). We and others have previously reported that expression and Rev/CRM1-mediated nuclear export of HIV-1 usRNA in macrophages activates MDA5, MAVS, and innate immune signaling cascades. In this study, we reveal that MDA5 sensing of viral usRNA is strictly determined by TSS, 5 leader structure, and RNA function. We show that cap-sequestered HIV-1 usRNAs (cap1G) destined for viral genome packaging are specifically targeted by MDA5, while translation-destined (cap3G) usRNAs are remarkably immunologically silent. Using mutant viruses which express usRNA with altered 5 cap-exposed leader structure, or inclusion of a retroviral constitutive transport element which drives mRNA-like NXF1-dependent nuclear export of viral usRNA, we show that cap exposure and nuclear export pathway choice are major determinants of both lentiviral RNA immunogenicity and function. In total, we identify innate immune system evasion as a possible rationale for the universal conservation of heterogenous TSS usage among ancestral and extant HIV-1 isolates and shed light on how MDA5 fundamentally discriminates between self and non-self RNAs. SIGNIFICANCEInnate immune activation is critical to both the process of initial infection establishment and ongoing chronic inflammation in HIV-1 infection. While MDA5 has been identified as the sensor which detects unspliced HIV-1 RNA produced in infected cells, it remains unclear how HIV-1 unspliced RNAs, which are generated by cellular transcriptional processes, are recognized as non-self. Here, we reveal that HIV-1 RNA function determines MDA5-driven immunogenicity. We show that only unspliced RNAs which traffic to membrane-associated viral assembly sites are immunogenic, while unspliced RNAs which are ribosomally translated to produce viral proteins are immunologically silent. These findings not only advance our knowledge of how the human innate immune system recognizes HIV-1 unspliced RNAs as foreign but also provide a rationale for the selective advantage to generate two pools of unspliced RNAs during HIV-1 replication.

microbiology↗

bioRxiv2025

Evasion of CARD8 Activation During HIV-1 Assembly

As intracellular parasites, viruses must devise sophisticated mechanisms to produce and assemble viral components while suppressing activation of innate immune effectors. Here, we report that coordination of HIV-1 assembly by the viral polyprotein Gag suppresses inappropriately-timed protease (PR) activity to evade the PR activity sensor, CARD8. Employing mutants of Gag, we show that disruption of domains controlling viral assembly site (MA) or virus particle release (NC and p6) lead to premature activation of PR and the CARD8 inflammasome, resulting in IL-1{beta} secretion and pyroptotic cell death. Further, we demonstrate that previously-observed host-adaptive mutations in HIV-1 MA (M30K) and p6 (PTAP duplication) associated with greater fitness in humans differentially modulate the process of viral assembly and budding to evade CARD8-mediated cell death. Altogether, this work reveals adaptation to human CARD8 by HIV-1 Gag upon zoonotic transmission from chimpanzees and suggests that assembly-regulated CARD8 activation influences the trajectory of HIV-1 evolution and fitness in humans.

microbiology↗

bioRxiv2024

Expression of Intron-containing HIV-1 RNA Induces NLRP1 Inflammasome Activation in Myeloid Cells

Despite the success of antiretroviral therapy (ART) in suppressing plasma viremia in people living with human immunodeficiency virus type-1 (HIV-1), persistent viral RNA expression in tissue reservoirs is observed and can contribute to HIV-1-induced immunopathology and comorbidities. Infection of long-lived innate immune cells, such as tissue-resident macrophages and microglia may contribute to persistent viral RNA production and chronic inflammation. We recently reported that de-novo cytoplasmic expression of HIV-1 intron-containing RNA (icRNA) in macrophages and microglia leads to MDA5 and MAVS-dependent innate immune sensing and induction of type I IFN responses, demonstrating that HIV icRNA is a pathogen-associated molecular pattern (PAMP). In this report, we show that cytoplasmic expression of HIV-1 icRNA also induces NLRP1 inflammasome activation and IL-1{beta} secretion in macrophages and microglia in a RLR- and endosomal TLR-independent manner. Infection of both macrophages and microglia with either replication-competent or single-cycle HIV-1 induced IL-1{beta} secretion, which was attenuated when cytoplasmic expression of viral icRNA was prevented. While IL-1{beta} secretion was blocked by treatment with caspase-1 inhibitors or knockdown of NLRP1 or caspase-1 expression in HIV- infected macrophages, overexpression of NLRP1 significantly enhanced IL-1{beta} secretion in an HIV-icRNA-dependent manner. Immunoprecipitation analysis revealed interaction of HIV-1 icRNA, but not multiply-spliced HIV-1 RNA, with NLRP1, suggesting that HIV-1 icRNA sensing by NLRP1 is sufficient to trigger inflammasome activation. Together, these findings reveal a pathway of NLRP1 inflammasome activation induced by de novo expressed HIV icRNA in HIV- infected myeloid cells.

microbiology↗

bioRxiv2024

Macrophage-intrinsic MDA5-IRF5 axis drives HIV-1 icRNA-induced inflammatory responses

Despite effective antiretroviral therapy (ART), transcriptionally competent HIV-1 reservoirs persist and contribute to persistent immune activation in people living with HIV (PWH). HIV-1-infected macrophages are important mediators of chronic innate immune activation, though mechanisms remain unclear. We previously reported that nuclear export and cytoplasmic expression of HIV-1 intron-containing RNA (icRNA) activates mitochondrial antiviral signaling protein (MAVS)-mediated type I interferon (IFN) responses in macrophages. In this study, we demonstrate an essential role of melanoma differentiation-associated protein 5 (MDA5) in sensing HIV-1 icRNA and promoting MAVS-dependent IRF5 activation in macrophages. Suppression of MDA5, but not RIG-I expression nor disruption of endosomal TLR pathway, abrogated HIV-1 icRNA-induced type I IFN responses and IP-10 expression in macrophages. Furthermore, induction of IP-10 in macrophages upon HIV-1 icRNA sensing by MDA5 was uniquely dependent on IRF5. Additionally, monocytes and MDMs from older (>50 years) individuals exhibit constitutively higher levels of IRF5 expression compared to younger (<35 years) individuals, and HIV-1 icRNA induced IP-10 expression was significantly enhanced in older macrophages, which was attenuated upon ablation of IRF5 expression suggesting that IRF5 functions as a major mediator of pro-inflammatory response downstream of MDA5-dependent HIV-1 icRNA sensing, dysregulation of which might contribute to chronic inflammation in older PWH.

microbiology↗

bioRxiv2023

Incorporation of 5 methylcytidine alleviates innate immune response to self-amplifying RNA vaccine

In order to improve vaccine effectiveness and safety profile of existing synthetic RNA-based vaccines, we have developed a self-amplifying RNA (saRNA)-based vaccine expressing membrane-anchored receptor binding domain (RBD) of SARS-CoV-2 S protein (S-RBD) and have demonstrated that a minimal dose of this saRNA vaccine elicits robust immune responses. Results from a recent clinical trial with 5-methylcytidine (5mC) incorporating saRNA vaccine demonstrated reduced vaccine-induced adverse effects while maintaining robust humoral responses. In this study, we investigate the mechanisms accounting for induction of efficient innate and adaptive immune responses and attenuated adverse effects induced by the 5mC-incorporated saRNA. We show that the 5mC-incorporating saRNA platform leads to prolonged and robust expression of antigen, while induction of type-I interferon (IFN-I), a key driver of reactogenicity, is attenuated in peripheral blood mononuclear cells (PBMCs), but not in macrophages and dendritic cells. Interestingly, we find that the major cellular source of IFN-I production in PBMCs is plasmacytoid dendritic cells (pDCs), which is attenuated upon 5mC incorporation in saRNA. In addition, we demonstrate that monocytes also play an important role in amplifying proinflammatory responses. Furthermore, we show that the detection of saRNA is mediated by a host cytosolic RNA sensor, RIG-I. Importantly, 5mC-incorporating saRNA vaccine candidate produced robust IgG responses against S-RBD upon injection in mice, thus providing strong support for the potential clinical use of 5mC-incorporating saRNA vaccines.

immunology↗

bioRxiv2022

CD169-mediated restrictive SARS-CoV-2 infection of macrophages induces pro-inflammatory responses

Exacerbated and persistent innate immune response marked by pro-inflammatory cytokine expression is thought to be a major driver of chronic COVID-19 pathology. Although macrophages are not the primary target cells of SARS-CoV-2 infection in humans, viral RNA and antigens in activated monocytes and macrophages have been detected in post-mortem samples, and dysfunctional monocytes and macrophages have been hypothesized to contribute to a protracted hyper-inflammatory state in COVID-19 patients. In this study, we demonstrate that CD169, a myeloid cell specific I-type lectin, facilitated ACE2-independent SARS-CoV-2 fusion and entry in macrophages. CD169- mediated SARS-CoV-2 entry in macrophages resulted in expression of viral genomic and sub-genomic (sg) RNAs with minimal viral protein expression and no infectious viral particle release, suggesting a post-entry restriction of the SARS-CoV-2 replication cycle. Intriguingly this post-entry replication block was alleviated by exogenous ACE2 expression in macrophages. Restricted expression of viral gRNA and sgRNA in CD169+ macrophages elicited a pro-inflammatory cytokine expression (TNF, IL-6 and IL-1{beta}) in a RIG-I, MDA-5 and MAVS-dependent manner, which was suppressed by remdesivir pre- treatment. These findings suggest that de novo expression of SARS-CoV-2 RNA in macrophages contributes to the pro-inflammatory cytokine signature and that blocking CD169-mediated ACE2 independent infection and subsequent activation of macrophages by viral RNA might alleviate COVID-19-associated hyperinflammatory response. Author SummaryOver-exuberant production of pro-inflammatory cytokine expression by macrophages has been hypothesized to contribute to severity of COVID-19 disease. Molecular mechanisms that contribute to macrophage-intrinsic immune activation during SARS- CoV-2 infection are not fully understood. Here we show that CD169, a macrophage- specific sialic-acid binding lectin, facilitates abortive SARS-CoV-2 infection of macrophages that results in innate immune sensing of viral replication intermediates and production of proinflammatory responses. We identify an ACE2-independent, CD169- mediated endosomal viral entry mechanism that results in cytoplasmic delivery of viral capsids and initiation of virus replication, but absence of infectious viral production. Restricted viral replication in CD169+ macrophages and detection of viral genomic and sub-genomic RNAs by cytoplasmic RIG-I-like receptor family members, RIG-I and MDA5, and initiation of downstream signaling via the adaptor protein MAVS, was required for innate immune activation. These studies uncover mechanisms important for initiation of innate immune sensing of SARS-CoV-2 infection in macrophages, persistent activation of which might contribute to severe COVID-19 pathophysiology.

immunology↗

bioRxiv2021

Multidose evaluation of 6,710 drug repurposing library identifies potent SARS-CoV-2 infection inhibitors In Vitro and In Vivo.

Identification of host factors contributing to replication of viruses and resulting disease progression remains a promising approach for development of new therapeutics. Here, we evaluated 6710 clinical and preclinical compounds targeting 2183 host proteins by immunocytofluorescence-based screening to identify SARS-CoV-2 infection inhibitors. Computationally integrating relationships between small molecule structure, dose-response antiviral activity, host target and cell interactome networking produced cellular networks important for infection. This analysis revealed 389 small molecules, >12 scaffold classes and 813 host targets with micromolar to low nanomolar activities. From these classes, representatives were extensively evaluated for mechanism of action in stable and primary human cell models, and additionally against Beta and Delta SARS-CoV-2 variants and MERS-CoV. One promising candidate, obatoclax, significantly reduced SARS-CoV-2 viral lung load in mice. Ultimately, this work establishes a rigorous approach for future pharmacological and computational identification of novel host factor dependencies and treatments for viral diseases.

microbiology↗

bioRxiv2020

CD209L/L-SIGN and CD209/DC-SIGN act as receptors for SARS-CoV-2 and are differentially expressed in lung and kidney epithelial and endothelial cells

As the COVID-19 pandemic continues to spread, investigating the processes underlying the interactions between SARS-CoV-2 and its hosts is of high importance. Here, we report the identification of CD209L/L-SIGN and the related protein CD209/DC-SIGN as receptors capable of mediating SARS-CoV-2 entry into human cells. Immunofluorescence staining of human tissues revealed prominent expression of CD209L in the lung and kidney epithelium and endothelium. Multiple biochemical assays using a purified recombinant SARS-CoV-2 spike receptor binding domain (S-RBD) or S1 encompassing both NTB and RBD and ectopically expressed CD209L and CD209 revealed that CD209L and CD209 interact with S-RBD. CD209L contains two N-glycosylation sequons, at sites N92 and N361, but we determined that only site N92 is occupied. Removal of the N-glycosylation at this site enhances the binding of S-RBD with CD209L. CD209L also interacts with ACE2, suggesting a role for heterodimerization of CD209L and ACE2 in SARS-CoV-2 entry and infection in cell types where both are present. Furthermore, we demonstrate that human endothelial cells are permissive to SARS-CoV-2 infection and interference with CD209L activity by knockdown strategy or with soluble CD209L inhibits virus entry. Our observations demonstrate that CD209L and CD209 serve as alternative receptors for SARS-CoV-2 in disease-relevant cell types, including the vascular system. This property is particularly important in tissues where ACE2 has low expression or is absent, and may have implications for antiviral drug development.

cell biology↗

bioRxiv2018

Strength of T cell signaling regulates HIV-1 replication and establishment of latency

A major barrier to curing HIV is the long-lived latent reservoir that supports re-emergence of HIV upon treatment interruption. Targeting this reservoir will require mechanistic insights into the establishment and maintenance of HIV latency. Whether T cell signaling at the time of HIV-1 infection influences productive replication or latency is not fully understood. We used a panel of chimeric antigen receptors (CARs) with different ligand binding affinities to induce a range of signaling strengths to model differential T cell receptor signaling at the time of HIV-1 infection. Stimulation of T cell lines or primary CD4+ T cells expressing chimeric antigen receptors supported HIV-1 infection regardless of affinity for ligand; however, only signaling by the highest affinity receptor facilitated HIV-1 expression. Activation of chimeric antigen receptors that had intermediate and low binding affinities did not support provirus transcription, suggesting that a minimal signal is required for optimal HIV-1 expression. In addition, strong signaling at the time of infection produced a latent population that was readily inducible, whereas latent cells generated in response to weaker signals were not easily reversed. Chromatin immunoprecipitation showed HIV-1 transcription was limited by transcriptional elongation and that robust signaling decreased the presence of negative elongation factor, a pausing factor, by more than 80%. These studies demonstrate that T cell signaling influences HIV-1 infection and the establishment of different subsets of latently infected cells, which may have implications for targeting the HIV reservoir.\n\nAuthor SummaryActivation of CD4+ T cells facilitates HIV-1 infection; however, whether there are minimal signals required for the establishment of infection, replication, and latency has not been explored. To determine how T cell signaling influences HIV-1 infection and the generation of latently infected cells, we used chimeric antigen receptors to create a tunable model. Stronger signals result in robust HIV-1 expression and an inducible latent population. Minimal signals predispose cells towards latent infections that are refractory to reversal. We discovered that repression of HIV-1 transcription immediately after infection is due to RNA polymerase II pausing and inefficient transcription elongation. These studies demonstrate that signaling events influence the course of HIV-1 infection and have implications for cure strategies. They also provide a mechanistic explanation for why a significant portion of the HIV latent reservoir is not responsive to latency reversing agents which function by modifiying chromatin.

microbiology↗

bioRxiv2017

HIV-1 Unspliced RNA Expression Induces Innate Immune Activation in Macrophages

Low-levels of type I interferons (IFN-I) are thought to be a driving force for immune activation and T cell exhaustion in HIV-1 infected individuals on highly active antiretroviral therapy (HAART), though the causative mechanisms for persistent IFN-I signaling have remained unclear. Here, we show Rev-CRM1 dependent nuclear export and peripheral membrane association of intron-containing HIV-1 unspliced RNA (usRNA), independent of primary viral sequence or viral protein expression, is subject to sensing, and results in IFN-I-dependent pro-inflammatory responses in macrophages. Our findings suggest that persistent expression of HIV-1 usRNA in macrophages contributes to chronic immune activation and that use of HIV RNA expression inhibitors as adjunct therapy might abrogate aberrant inflammation and restore immune function in HIV-infected individuals on HAART.

microbiology↗