RESUMEN
Multiple omics analyzes of Vaccinia virus (VACV) infection have defined molecular characteristics of poxvirus biology. However, little is known about the monkeypox (mpox) virus (MPXV) in humans, which has a different disease manifestation despite its high sequence similarity to VACV. Here, we perform an in-depth multi-omics analysis of the transcriptome, proteome, and phosphoproteome signatures of MPXV-infected primary human fibroblasts to gain insights into the virus-host interplay. In addition to expected perturbations of immune-related pathways, we uncover regulation of the HIPPO and TGF-ß pathways. We identify dynamic phosphorylation of both host and viral proteins, which suggests that MAPKs are key regulators of differential phosphorylation in MPXV-infected cells. Among the viral proteins, we find dynamic phosphorylation of H5 that influenced the binding of H5 to dsDNA. Our extensive dataset highlights signaling events and hotspots perturbed by MPXV, extending the current knowledge on poxviruses. We use integrated pathway analysis and drug-target prediction approaches to identify potential drug targets that affect virus growth. Functionally, we exemplify the utility of this approach by identifying inhibitors of MTOR, CHUK/IKBKB, and splicing factor kinases with potent antiviral efficacy against MPXV and VACV.
Asunto(s)
Fibroblastos , Monkeypox virus , Mpox , Proteínas Virales , Humanos , Monkeypox virus/genética , Fosforilación , Mpox/virología , Mpox/metabolismo , Fibroblastos/virología , Fibroblastos/metabolismo , Proteínas Virales/metabolismo , Proteínas Virales/genética , Proteoma/metabolismo , Interacciones Huésped-Patógeno , Transducción de Señal , Proteómica/métodos , Transcriptoma , Antivirales/farmacología , MultiómicaRESUMEN
Inflammasomes integrate cytosolic evidence of infection or damage to mount inflammatory responses. The inflammasome sensor NLRP1 is expressed in human keratinocytes and coordinates inflammation in the skin. We found that diverse stress signals induce human NLRP1 inflammasome assembly by activating MAP kinase p38: While the ribotoxic stress response to UV and microbial molecules exclusively activates p38 through MAP3K ZAKα, infection with arthropod-borne alphaviruses, including Semliki Forest and Chikungunya virus, activates p38 through ZAKα and potentially other MAP3K. We demonstrate that p38 directly phosphorylates NLRP1 and that serine 107 in the linker region is critical for activation. NLRP1 phosphorylation is followed by ubiquitination of NLRP1PYD, N-terminal degradation of NLRP1, and nucleation of inflammasomes by NLRP1UPA-CARD. In contrast, activation of NLRP1 by nanobody-mediated ubiquitination, viral proteases, or inhibition of DPP9 was independent of p38 activity. Taken together, we define p38 activation as a unifying signaling hub that controls NLRP1 inflammasome activation by integrating a variety of cellular stress signals relevant to the skin.
Asunto(s)
Inflamasomas , Virosis , Proteínas Quinasas p38 Activadas por Mitógenos , Humanos , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Inflamasomas/metabolismo , Proteínas NLR/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Orthomyxo- and bunyaviruses steal the 5' cap portion of host RNAs to prime their own transcription in a process called "cap snatching." We report that RNA modification of the cap portion by host 2'-O-ribose methyltransferase 1 (MTr1) is essential for the initiation of influenza A and B virus replication, but not for other cap-snatching viruses. We identified with in silico compound screening and functional analysis a derivative of a natural product from Streptomyces, called trifluoromethyl-tubercidin (TFMT), that inhibits MTr1 through interaction at its S-adenosyl-l-methionine binding pocket to restrict influenza virus replication. Mechanistically, TFMT impairs the association of host cap RNAs with the viral polymerase basic protein 2 subunit in human lung explants and in vivo in mice. TFMT acts synergistically with approved anti-influenza drugs.
Asunto(s)
Alphainfluenzavirus , Antivirales , Betainfluenzavirus , Productos Biológicos , Inhibidores Enzimáticos , Metiltransferasas , Caperuzas de ARN , Tubercidina , Replicación Viral , Animales , Humanos , Ratones , Caperuzas de ARN/metabolismo , ARN Mensajero/metabolismo , ARN Viral/biosíntesis , Replicación Viral/efectos de los fármacos , Alphainfluenzavirus/efectos de los fármacos , Betainfluenzavirus/efectos de los fármacos , Productos Biológicos/química , Productos Biológicos/farmacología , Antivirales/química , Antivirales/farmacología , Tubercidina/análogos & derivados , Tubercidina/farmacología , Metiltransferasas/antagonistas & inhibidores , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Streptomyces/química , Simulación por Computador , Células A549RESUMEN
Inflammasomes sense intracellular clues of infection, damage, or metabolic imbalances. Activated inflammasome sensors polymerize the adaptor ASC into micron-sized "specks" to maximize caspase-1 activation and the maturation of IL-1 cytokines. Caspase-1 also drives pyroptosis, a lytic cell death characterized by leakage of intracellular content to the extracellular space. ASC specks are released among cytosolic content, and accumulate in tissues of patients with chronic inflammation. However, if extracellular ASC specks contribute to disease, or are merely inert remnants of cell death remains unknown. Here, we show that camelid-derived nanobodies against ASC (VHHASC ) target and disassemble post-pyroptotic inflammasomes, neutralizing their prionoid, and inflammatory functions. Notably, pyroptosis-driven membrane perforation and exposure of ASC specks to the extracellular environment allowed VHHASC to target inflammasomes while preserving pre-pyroptotic IL-1ß release, essential to host defense. Systemically administrated mouse-specific VHHASC attenuated inflammation and clinical gout, and antigen-induced arthritis disease. Hence, VHHASC neutralized post-pyroptotic inflammasomes revealing a previously unappreciated role for these complexes in disease. VHHASC are the first biologicals that disassemble pre-formed inflammasomes while preserving their functions in host defense.
Asunto(s)
Inflamasomas , Anticuerpos de Dominio Único , Animales , Proteínas Adaptadoras de Señalización CARD/metabolismo , Caspasa 1/metabolismo , Humanos , Inflamasomas/metabolismo , Inflamación/metabolismo , Ratones , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , PiroptosisRESUMEN
The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread, with devastating consequences. For passive immunization efforts, nanobodies have size and cost advantages over conventional antibodies. In this study, we generated four neutralizing nanobodies that target the receptor binding domain of the SARS-CoV-2 spike protein. We used x-ray crystallography and cryo-electron microscopy to define two distinct binding epitopes. On the basis of these structures, we engineered multivalent nanobodies with more than 100 times the neutralizing activity of monovalent nanobodies. Biparatopic nanobody fusions suppressed the emergence of escape mutants. Several nanobody constructs neutralized through receptor binding competition, whereas other monovalent and biparatopic nanobodies triggered aberrant activation of the spike fusion machinery. These premature conformational changes in the spike protein forestalled productive fusion and rendered the virions noninfectious.