Viral interference during influenza A-SARS-CoV-2 coinfection of the human airway epithelium and reversal by oseltamivir.

To gain insight into interactions among respiratory viruses, we modeled influenza A virus (IAV) - SARS-CoV-2 coinfections using differentiated human airway epithelial cultures. Replicating IAV induced a more robust interferon response than SARS-CoV-2 and suppressed SARS-CoV-2 replication in both sequential and simultaneous infections, whereas SARS-CoV-2 did not enhance host cell defense during influenza infection or suppress IAV replication. Oseltamivir, an antiviral targeting influenza, reduced IAV replication during coinfection but also reduced the host antiviral response and restored SARS-CoV-2 replication. These results demonstrate how perturbations in one viral infection can impact its effect on a coinfecting virus.

Loss of TRIM33 causes resistance to BET bromodomain inhibitors through MYC- and TGF-ß-dependent mechanisms.

Bromodomain and extraterminal domain protein inhibitors (BETi) hold great promise as a novel class of cancer therapeutics. Because acquired resistance typically limits durable responses to targeted therapies, it is important to understand mechanisms by which tumor cells adapt to BETi. Here, through pooled shRNA screening of colorectal cancer cells, we identified tripartite motif-containing protein 33 (TRIM33) as a factor promoting sensitivity to BETi. We demonstrate that loss of TRIM33 reprograms cancer cells to a more resistant state through at least two mechanisms. TRIM33 silencing attenuates down-regulation of MYC in response to BETi. Moreover, loss of TRIM33 enhances TGF-ß receptor expression and signaling, and blocking TGF-ß receptor activity potentiates the antiproliferative effect of BETi. These results describe a mechanism for BETi resistance and suggest that combining inhibition of TGF-ß signaling with BET bromodomain inhibition may offer new therapeutic benefits.

Counterintuitive effect of antiviral therapy on influenza A-SARS-CoV-2 coinfection due to viral interference.

The resurgence of influenza and continued circulation of SARS-CoV-2 raise the question of how these viruses interact in a co-exposed host. Here we studied virus-virus and host-virus interactions during influenza A virus (IAV) -SARS-CoV-2 coinfection using differentiated cultures of the human airway epithelium. Coexposure to IAV enhanced the tissue antiviral response during SARS-CoV-2 infection and suppressed SARS-CoV-2 replication. Oseltamivir, an antiviral targeting influenza, reduced IAV replication during coinfection but also reduced the antiviral response and paradoxically restored SARS-CoV-2 replication. These results highlight the importance of diagnosing coinfections and compel further study of how coinfections impact the outcome of antiviral therapy.

Magnitude and timing of the antiviral response determine SARS-CoV-2 replication early in infection.

The interferon response is a potent antiviral defense mechanism, but its effectiveness depends on its timing relative to viral replication. Here, we report viral replication and host response kinetics in patients at the start of SARS-CoV-2 infection and explore the impact of these kinetics experimentally. In both longitudinal patient nasopharyngeal samples and airway epithelial organoids, we found that SARS-CoV-2 initially replicated exponentially with a doubling time of ~6hr, and induced interferon stimulated genes (ISGs) with delayed timing relative to viral replication. Prior exposure to rhinovirus increased ISG levels and blocked SARS-CoV-2 replication. Conversely, inhibiting ISG induction abrogated interference by rhinovirus and enhanced SARS-CoV-2 replication rate. These results demonstrate the importance of initial interferon-mediated defenses in determining the extent to which SARS-CoV-2 can replicate at the start of infection and indicate that biological variables that alter the airway interferon response, including heterologous induction of innate immunity by other viruses, could profoundly impact SARS-CoV-2 susceptibility and transmission.

Dynamic innate immune response determines susceptibility to SARS-CoV-2 infection and early replication kinetics.

Initial replication of SARS-CoV-2 in the upper respiratory tract is required to establish infection, and the replication level correlates with the likelihood of viral transmission. Here, we examined the role of host innate immune defenses in restricting early SARS-CoV-2 infection using transcriptomics and biomarker-based tracking in serial patient nasopharyngeal samples and experiments with airway epithelial organoids. SARS-CoV-2 initially replicated exponentially, with a doubling time of ∼6 h, and induced interferon-stimulated genes (ISGs) in the upper respiratory tract, which rose with viral replication and peaked just as viral load began to decline. Rhinovirus infection before SARS-CoV-2 exposure accelerated ISG responses and prevented SARS-CoV-2 replication. Conversely, blocking ISG induction during SARS-CoV-2 infection enhanced viral replication from a low infectious dose. These results show that the activity of ISG-mediated defenses at the time of SARS-CoV-2 exposure impacts infection progression and that the heterologous antiviral response induced by a different virus can protect against SARS-CoV-2.

Interference between rhinovirus and influenza A virus: a clinical data analysis and experimental infection study.

BACKGROUND: During the 2009 pandemic of an emerging influenza A virus (IAV; H1N1pdm09), data from several European countries indicated that the spread of the virus might have been interrupted by the annual autumn rhinovirus epidemic. We aimed to investigate viral interference between rhinovirus and IAV with use of clinical data and an experimental model. METHODS: We did a clinical data analysis and experimental infection study to investigate the co-occurrence of rhinovirus and IAV in respiratory specimens from adults (≥21 years) tested with a multiplex PCR panel at Yale-New Haven Hospital (CT, USA) over three consecutive winter seasons (Nov 1 to March 1, 2016-17, 2017-18, and 2018-19). We compared observed versus expected co-detections using data extracted from the Epic Systems electronic medical record system. To assess how rhinovirus infection affects subsequent IAV infection, we inoculated differentiated primary human airway epithelial cultures with rhinovirus (HRV-01A; multiplicity of infection [MOI] 0·1) or did mock infection. On day 3 post-infection, we inoculated the same cultures with IAV (H1N1 green fluorescent protein [GFP] reporter virus or H1N1pdm09; MOI 0·1). We used reverse transcription quantitative PCR or microscopy to quantify host cell mRNAs for interferon-stimulated genes (ISGs) on day 3 after rhinovirus or mock infection and IAV RNA on days 4, 5, or 6 after rhinovirus or mock infection. We also did sequential infection studies in the presence of BX795 (6 µM), to inhibit the interferon response. We compared ISG expression and IAV RNA and expression of GFP by IAV reporter virus. FINDINGS: Between July 1, 2016, and June 30, 2019, examination of 8284 respiratory samples positive for either rhinovirus (n=3821) or IAV (n=4463) by any test method was used to establish Nov 1 to March 1 as the period of peak virus co-circulation. After filtering for samples within this time frame meeting the inclusion criteria (n=13 707), there were 989 (7·2%) rhinovirus and 922 (6·7%) IAV detections, with a significantly lower than expected odds of co-detection (odds ratio 0·16, 95% CI 0·09-0·28). Rhinovirus infection of cell cultures induced ISG expression and protected against IAV infection 3 days later, resulting in an approximate 50 000-fold decrease in IAV H1N1pdm09 viral RNA on day 5 post-rhinovirus inoculation. Blocking the interferon response restored IAV replication following rhinovirus infection. INTERPRETATION: These findings show that one respiratory virus can block infection with another through stimulation of antiviral defences in the airway mucosa, supporting the idea that interference from rhinovirus disrupted the 2009 IAV pandemic in Europe. These results indicate that viral interference can potentially affect the course of an epidemic, and this possibility should be considered when designing interventions for seasonal influenza epidemics and the ongoing COVID-19 pandemic. FUNDING: National Institutes of Health, National Institute of General Medical Sciences, and the Yale Department of Laboratory Medicine.

Regional Differences in Airway Epithelial Cells Reveal Tradeoff between Defense against Oxidative Stress and Defense against Rhinovirus.

Rhinovirus is a leading cause of acute respiratory infections and asthma attacks, but infections are also frequently cleared from the nasal mucosa without causing symptoms. We sought to better understand host defense against rhinovirus by investigating antiviral defense in primary human nasal and bronchial airway epithelial cells cultured ex vivo. Surprisingly, upon rhinovirus infection or RIG-I stimulation, nasal-derived epithelial cells exhibited much more robust antiviral responses than bronchial-derived cells. Conversely, RIG-I stimulation triggered more robust activation of the NRF2-dependent oxidative stress response in bronchial cells compared to nasal cells. NRF2 activation dampened epithelial antiviral responses, whereas NRF2 knockdown enhanced antiviral responses and was protective during rhinovirus infection. These findings demonstrate a tradeoff in epithelial defense against distinct types of airway damage, namely, viral versus oxidative, and reveal differential calibration of defense responses in cells derived from different airway microenvironments.

Decreased expression of the DNA mismatch repair gene Mlh1 under hypoxic stress in mammalian cells.

The hypoxic tumor microenvironment has been shown to contribute to genetic instability. As one possible mechanism for this effect, we report that expression of the DNA mismatch repair (MMR) gene Mlh1 is specifically reduced in mammalian cells under hypoxia, whereas expression of other MMR genes, including Msh2, Msh6, and Pms2, is not altered at the mRNA level. However, levels of the PMS2 protein are reduced, consistent with destabilization of PMS2 in the absence of its heterodimer partner, MLH1. The hypoxia-induced reduction in Mlh1 mRNA was prevented by the histone deacetylase inhibitor trichostatin A, suggesting that hypoxia causes decreased Mlh1 transcription via histone deacetylation. In addition, treatment of cells with the iron chelator desferrioxamine also reduced MLH1 and PMS2 levels, in keeping with low oxygen tension being the stress signal that provokes the altered MMR gene expression. Functional MMR deficiency under hypoxia was detected as induced instability of a (CA)(29) dinucleotide repeat and by increased mutagenesis in a chromosomal reporter gene. These results identify a potential new pathway of genetic instability in cancer: hypoxia-induced reduction in the expression of key MMR proteins. In addition, this stress-induced genetic instability may represent a conceptual parallel to the pathway of stationary-phase mutagenesis seen in bacteria.

Human T-cell leukemia virus I tax protein sensitizes p53-mutant cells to DNA damage.

Mutations in p53 are a common cause of resistance of cancers to standard chemotherapy and, thus, treatment failure. Reports have shown that Tax, a human T-cell leukemia virus type I encoded protein that has been associated with genomic instability and perturbation of transcription and cell cycle, sensitizes HeLa cells to UV treatment. The extent to which Tax can sensitize cells and the mechanism by which it exerts its effect are unknown. In this study, we show that Tax sensitizes p53-mutant cells to a broad range of DNA-damaging agents, including mitomycin C, a bifunctional alkylator, etoposide, a topoisomerase II drug, and UV light, but not ionizing radiation, a double-strand break agent, or vinblastine, a tubulin poison. Tax caused hypersensitivity in all p53-deleted cell lines and several, but not all, mutant-expressed p53-containing cell lines, while unexpectedly being protective in p53 wild-type (wt) cells. The effect observed in p53-deleted lines could be reversed for this by transfection of wt p53. We also show that Tax activates a p53-independent proapoptotic program through decreased expression of the retinoblastoma protein and subsequent increased E2F1 expression. The expression of several proapoptotic proteins was also induced by Tax, including Puma and Noxa, culminating in a substantial increase in Bax dimerization. Our results show that Tax can sensitize p53-mutant cells to DNA damage while protecting p53 wt cells, a side benefit that might result in reduced toxicity in normal cells. Such studies hold the promise of a novel adjunctive therapy that could make cancer chemotherapy more effective.

HTLV-1 Tax oncoprotein subverts the cellular DNA damage response via binding to DNA-dependent protein kinase.

Human T-cell leukemia virus type-1 is the causative agent for adult T-cell leukemia. Previous research has established that the viral oncoprotein Tax mediates the transformation process by impairing cell cycle control and cellular response to DNA damage. We showed previously that Tax sequesters huChk2 within chromatin and impairs the response to ionizing radiation. Here we demonstrate that DNA-dependent protein kinase (DNA-PK) is a member of the Tax.Chk2 nuclear complex. The catalytic subunit, DNA-PKcs, and the regulatory subunit, Ku70, were present. Tax-containing nuclear extracts showed increased DNA-PK activity, and specific inhibition of DNA-PK prevented Tax-induced activation of Chk2 kinase activity. Expression of Tax induced foci formation and phosphorylation of H2AX. However, Tax-induced constitutive signaling of the DNA-PK pathway impaired cellular response to new damage, as reflected in suppression of ionizing radiation-induced DNA-PK phosphorylation and gammaH2AX stabilization. Tax co-localized with phospho-DNA-PK into nuclear speckles and a nuclear excluded Tax mutant sequestered endogenous phospho-DNA-PK into the cytoplasm, suggesting that Tax interaction with DNA-PK is an initiating event. We also describe a novel interaction between DNA-PK and Chk2 that requires Tax. We propose that Tax binds to and stabilizes a protein complex with DNA-PK and Chk2, resulting in a saturation of DNA-PK-mediated damage repair response.