Biology subjects

Campos, S. K.

Publications and source records attributed to Campos, S. K..

9 recordsLinked to original sources

bioRxiv2025

Human Papillomavirus Infection Blunts STING Signaling and Alters Downstream Response

The cGAS/STING pathway is an important innate immune pathway that senses and responds to foreign DNA or damaged host DNA. cGAS recognizes DNA and generates the second messenger cGAMP which activates STING. STING then creates a platform for IRF3 to be phosphorylated before pIRF3 induces a type I interferon response resulting in transcription of anti-viral genes. We examined how HPV18 infection modulates cGAS/STING activation following DNA stimulation in primary human foreskin keratinocytes from three different donors. Following exogenous activation, HPV(+) cells produced higher levels of cGAMP compared to patient-matched HPV(-) cells yet phosphorylation of STING and IRF3 was reduced in the HPV(+) cells. The reduced STING and IRF3 activation corresponded with a selective dampening of type I interferon driven antiviral genes and pro-inflammatory cytokines in HPV(+) cells. Simultaneously, HPV(+) cells had a baseline increase in genes associated with epithelial proliferation and skin development, which remained elevated following cGAMP treatment. We demonstrate that both E6 and E7 are required and sufficient to drive increased cGAMP levels and attenuate STING activation. Our results characterize cGAS/STING pathway activation across time in HPV positive and negative cells and demonstrate that the HPV oncogenes cooperate to suppress early STING activation despite higher levels of cGAMP in these HPV(+) cells. These findings indicate that HPV modulates cGAS/STING signaling to blunt antiviral defenses without impacting other arms of the cGAS/STING pathway response. ImportanceIn this study, we used primary human keratinocytes from multiple donors to track cGAS/STING activity over time following stimulation. We found that although HPV(+) cells produced more cGAMP, these cells showed reduced early activation of STING and IRF3. Downstream of cGAS/STING, many antiviral genes were suppressed by HPV however, epithelial proliferation-related genes had an HPV-mediated increase in baseline expression which remained higher than uninfected cells following cGAMP stimulation. We identified the HPV oncogenes E6 and E7 work together to enhance cGAMP levels and suppress parts of the downstream STING signaling response. These findings reveal that HPV attenuates cGAS/STING signaling and downstream events, selectively suppressing antiviral genes without inhibiting other parts of the pathway. Our results provide new insights into how HPV modulates innate immune responses.

microbiology↗

bioRxiv2024

Cell-based high-content approach for SARS-CoV-2 neutralization identifies unique monoclonal antibodies and PI3K pathway inhibitors.

The sudden rise of the SARS-CoV-2 virus and the delay in the development of effective therapeutics to mitigate it made evident a need for ways to screen for compounds that can block infection and prevent further pathogenesis and spread. Yet, identifying effective drugs efficacious against viral infection and replication with minimal toxicity for the patient can be difficult. Monoclonal antibodies were shown to be effective, yet as the SARS-CoV-2 mutated, these antibodies became ineffective. Small molecule antivirals were identified using pseudovirus constructs to recapitulate infection in non-human cells, such as Vero E6 cells. However, the impact was limited due to poor translation of these compounds in the clinical setting. This is partly due to the lack of similarity of screening platforms to the in vivo physiology of the patient and partly because drugs effective in vitro showed dose-limiting toxicities. In this study, we performed two high-throughput screens in human lung adenocarcinoma cells with authentic SARS-CoV-2 virus to identify both monoclonal antibodies that neutralize the virus and clinically useful kinase inhibitors to block the virus and prioritize minimal host toxicity. Using high-content imaging combined with single-cell and multidimensional analysis, we identified antibodies and kinase inhibitors that reduce virus infection without affecting the host. Our screening technique uncovered novel antibodies and overlooked kinase inhibitors (i.e. PIK3i, mTORi, multiple RTKi) that could be effective against SARS-CoV-2 virus. Further characterization of these molecules will streamline the repurposing of compounds for the treatment of future pandemics and uncover novel mechanisms viruses use to hijack and infect host cells.

pharmacology and toxicology↗

bioRxiv2024

A Peptide Derived from Sorting Nexin 1 Inhibits HPV16 Entry, Retrograde Trafficking, and L2 Membrane Spanning

High risk human papillomavirus (HPV) infection is responsible for 99% of cervical cancers and 5% of all human cancers worldwide. HPV infection requires the viral genome (vDNA) to gain access to nuclei of basal keratinocytes of epithelium. After virion endocytosis, the minor capsid protein L2 dictates the subcellular retrograde trafficking and nuclear localization of the vDNA during mitosis. Prior work identified a cell-permeable peptide termed SNX1.3, derived from the BAR domain of sorting nexin 1 (SNX1), that potently blocks the retrograde and nuclear trafficking of EGFR in triple negative breast cancer cells. Given the importance of EGFR and retrograde trafficking pathways in HPV16 infection, we set forth to study the effects of SNX1.3 within this context. SNX1.3 inhibited HPV16 infection by both delaying virion endocytosis, as well as potently blocking virion retrograde trafficking and Golgi localization. SNX1.3 had no effect on cell proliferation, nor did it affect post-Golgi trafficking of HPV16. Looking more directly at L2 function, SNX1.3 was found to impair membrane spanning of the minor capsid protein. Future work will focus on mechanistic studies of SNX1.3 inhibition, and the role of EGFR signaling and SNX1-mediated endosomal tubulation, cargo sorting, and retrograde trafficking in HPV infection.

microbiology↗

bioRxiv2021

Convergent antibody responses to the SARS-CoV-2 spike protein in convalescent and vaccinated individuals

Unrelated individuals can produce genetically similar clones of antibodies, known as public clonotypes, which have been seen in responses to different infectious diseases as well as healthy individuals. Here we identify 37 public clonotypes in memory B cells from convalescent survivors of SARS-CoV-2 infection or in plasmablasts from an individual after vaccination with mRNA-encoded spike protein. We identified 29 public clonotypes, including clones recognizing the receptor-binding domain (RBD) in the spike protein S1 subunit (including a neutralizing, ACE2-blocking clone that protects in vivo), and others recognizing non-RBD epitopes that bound the heptad repeat 1 region of the S2 domain. Germline-revertant forms of some public clonotypes bound efficiently to spike protein, suggesting these common germline-encoded antibodies are preconfigured for avid recognition. Identification of large numbers of public clonotypes provides insight into the molecular basis of efficacy of SARS-CoV-2 vaccines and sheds light on the immune pressures driving the selection of common viral escape mutants.

immunology↗

bioRxiv2020

Paradoxical effects of cigarette smoke and COPD on SARS-CoV2 infection and disease

IntroductionHow cigarette smoke (CS) and chronic obstructive pulmonary disease (COPD) affect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and severity is controversial. We investigated the protein and mRNA expression of SARS-CoV-2 entry receptor ACE2 and proteinase TMPRSS2 in lungs from COPD patients and controls, and lung tissue from mice exposed acutely and chronically to CS. Also, we investigated the effects of CS exposure on SARS-CoV-2 infection in human bronchial epithelial cells. MethodsIn Cohort 1, ACE2-positive cells were quantified by immunostaining in FFPE sections from both central and peripheral airways. In Cohort 2, we quantified pulmonary ACE2 protein levels by immunostaining and ELISA, and both ACE2 and TMPRSS2 mRNA levels by RT-qPCR. In C57BL/6 WT mice exposed to air or CS for up to 6 months, pulmonary ACE2 protein levels were quantified by triple immunofluorescence staining and ELISA. The effects of CS exposure on SARS-CoV-2 infection were evaluated after 72hr in vitro infection of Calu-3 cells. After SARS-CoV-2 infection, the cells were fixed for IF staining with dsRNA-specific J2 monoclonal Ab, and cell lysates were harvested for WB of viral nucleocapsid (N) protein. Supernatants (SN) and cytoplasmic lysates were obtained to measure ACE2 levels by ELISA. ResultsIn both human cohorts, ACE2 protein and mRNA levels were decreased in peripheral airways from COPD patients versus both smoker and NS controls, but similar in central airways. TMPRSS2 levels were similar across groups. Mice exposed to CS had decreased ACE2 protein levels in their bronchial and alveolar epithelia versus air-exposed mice exposed to 3 and 6 months of CS. In Calu3 cells in vitro, CS-treatment abrogated infection to levels below the limit of detection. Similar results were seen with WB for viral N protein, showing peak viral protein synthesis at 72hr. ConclusionsACE2 levels were decreased in both bronchial and alveolar epithelial cells from uninfected COPD patients versus controls, and from CS-exposed versus air-exposed mice. CS-pre-treatment did not affect ACE2 levels but potently inhibited SARS-CoV-2 replication in this in vitro model. These findings urge to further investigate the controversial effects of CS and COPD on SARS-CoV2 infection.

immunology↗

bioRxiv2020

In silico identification and validation of inhibitors of the interaction between neuropilin receptor 1 and SARS-CoV-2 Spike protein

Neuropilin-1 (NRP-1) is a multifunctional transmembrane receptor for ligands that affect developmental axonal growth and angiogenesis. In addition to a role in cancer, NRP-1 is a reported entry point for several viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal agent of coronavirus disease 2019 (COVID-19). The furin cleavage product of SARS-CoV-2 Spike protein takes advantage of the vascular endothelial growth factor A (VEGF-A) binding site on NRP-1 which accommodates a polybasic stretch ending in a C-terminal arginine. This site has long been a focus of drug discovery efforts for cancer therapeutics. We recently showed that interruption of the VEGF-A/NRP-1 signaling pathway ameliorates neuropathic pain and hypothesize that interference of this pathway by SARS-CoV-2 spike protein interferes with pain signaling. Here, we report hits from a small molecule and natural product screen of nearly 0.5 million compounds targeting the VEGF-A binding site on NRP-1. We identified nine chemical series with lead- or drug-like physico-chemical properties. Using an ELISA, we demonstrate that six compounds disrupt VEGF-A-NRP-1 binding more effectively than EG00229, a known NRP-1 inhibitor. Secondary validation in cells revealed that almost all tested compounds inhibited VEGF-A triggered VEGFR2 phosphorylation. Two compounds displayed robust inhibition of a recombinant vesicular stomatitis virus protein that utilizes the SARS-CoV-2 Spike for entry and fusion. These compounds represent a first step in a renewed effort to develop small molecule inhibitors of the VEGF-A/NRP-1 signaling for the treatment of neuropathic pain and cancer with the added potential of inhibiting SARS-CoV-2 virus entry.

neuroscience↗

bioRxiv2020

Vesicular Trafficking Permits Evasion of cGAS/STING Surveillance During Initial Human Papillomavirus Infection

Oncogenic human papillomaviruses (HPVs) replicate in differentiating epithelium, causing 5% of cancers worldwide. Like most other DNA viruses, HPV infection initiates after trafficking viral genome (vDNA) to host cell nuclei. Cells possess innate surveillance pathways to detect microbial components or physiological stresses often associated with microbial infections. One of these pathways, cGAS/STING, induces IRF3-dependent antiviral interferon (IFN) responses upon detection of cytosolic DNA. Virion-associated vDNA can activate cGAS/STING during initial viral entry and uncoating/trafficking, and thus cGAS/STING is an obstacle to many DNA viruses. HPV has a unique vesicular trafficking pathway compared to many other DNA viruses. As the capsid uncoats within acidic endosomal compartments, minor capsid protein L2 protrudes across vesicular membranes to facilitate transport of vDNA to the Golgi. L2/vDNA resides within the Golgi lumen until G2/M, whereupon vesicular L2/vDNA traffics along spindle microtubules, tethering to chromosomes to access daughter cell nuclei. L2/vDNA-containing vesicles likely remain intact until G1, following nuclear envelope reformation. We hypothesize that this unique vesicular trafficking protects HPV from cGAS/STING surveillance. Here, we investigate cGAS/STING responses to HPV infection. DNA transfection resulted in acute cGAS/STING activation and downstream IFN responses. In contrast, HPV infection elicited minimal cGAS/STING and IFN responses. To determine the role of vesicular trafficking in cGAS/STING evasion, we forced premature viral penetration of vesicular membranes with membrane-perturbing cationic lipids. Such treatment renders a non-infectious trafficking-defective mutant HPV infectious, yet susceptible to cGAS/STING detection. Overall, HPV evades cGAS/STING by its unique subcellular trafficking, a property that may contribute to establishment of infection. ImportancePersistent infection is the main risk factor for all HPV-associated cancers. However, cellular innate immune pathways exist to detect and limit viral infections. The cGAS/STING pathway senses cytosolic DNA to initiate antiviral IFN responses. Such responses would likely be detrimental towards the establishment of persistent HPV infections. We therefore hypothesize that HPV evades cGAS/STING detection via its unique vesicular trafficking mechanism. Here, we show that indeed HPV is a stealthy virus, capable of infecting keratinocytes with minimal activation of the cGAS/STING pathway. Such evasion is dependent on HPVs vesicular trafficking, as perturbation of vesicular integrity during infection results in sensing of virions.

microbiology↗

bioRxiv2019

Attenuation of cGAS/STING Activity During Mitosis

The innate immune system recognizes cytosolic DNA associated with microbial infections and cellular stress via the cGAS/STING pathway, leading to activation of phospho-IRF3 and downstream IFN-I and senescence responses. To prevent hyperactivation, cGAS/STING is presumed to be non-responsive to chromosomal self DNA during open mitosis, though specific regulatory mechanisms are lacking. Given a role for the Golgi in STING activation, we investigated the state of the cGAS/STING pathway in interphase cells with artificially vesiculated Golgi and in cells arrested in mitosis. We find that while cGAS activity is impaired through interaction with mitotic chromosomes, Golgi integrity has little effect on the enzymes production of cGAMP. In contrast, STING activation in response to either foreign DNA (cGAS-dependent) or exogenous cGAMP is impaired by a vesiculated Golgi. Overall our data suggest a secondary means for cells to limit potentially harmful cGAS/STING responses during open mitosis via natural Golgi vesiculation.

immunology↗

bioRxiv2019

Glutathione is Required for Efficient Post-Golgi Trafficking of Incoming HPV16 Genome

Human papillomavirus (HPV) is the most common sexually transmitted pathogen in the United States, causing 99% of cervical cancers and 5% of all human cancers worldwide. HPV infection requires transport of the viral genome (vDNA) into the nucleus of basal keratinocytes. During this process, minor capsid protein L2 facilitates subcellular retrograde trafficking of the vDNA from endosomes to the Golgi, and accumulation at host chromosomes during mitosis for nuclear retention and localization during interphase. Here we investigated the relationship between cytosolic GSH and HPV16 infection. siRNA knockdown of GSH biosynthetic enzymes results in a partial decrease of HPV16 infection. Likewise, infection of HPV16 in GSH depleted keratinocytes is inefficient, an effect that was not seen with adenoviral vectors. Analysis of trafficking revealed no defects in cellular binding, entry, furin cleavage of L2, or retrograde trafficking of HPV16, but GSH depletion hindered post-Golgi trafficking and translocation, decreasing nuclear accumulation of vDNA. Although precise mechanisms have yet to be defined, this work suggests that GSH is required for a specific post-Golgi trafficking step in HPV16 infection.

microbiology↗