RESUMEN
Human parvovirus B19 (B19V), like most parvoviruses, possesses phospholipase A2 (PLA2) activity, which is thought to mediate endosomal escape by membrane disruption. Here, we challenge this model and find evidence for a mechanism of B19V entry mediated by the glycosphingolipid globoside without endosome disruption and retrograde transport to the Golgi. We show that B19V PLA2 activity requires specific calcium levels and pH conditions that are not optimal in endosomes. Accordingly, endosomal membrane integrity was maintained during B19V entry. Furthermore, endosomes remained intact when loaded with MS2 bacteriophage particles pseudotyped with multiple B19V PLA2 subunits, providing superior enzymatic potential compared to native B19V. In globoside knockout cells, incoming viruses are arrested in the endosomal compartment and the infection is blocked. Infection can be rescued by promoting endosomal leakage with polyethyleneimine (PEI), demonstrating the essential role of globoside in facilitating endosomal escape. Incoming virus colocalizes with Golgi markers and interfering with Golgi function blocks infection, suggesting that globoside-mediated entry involves the Golgi compartment, which provides conditions favorable for the lipolytic PLA2. Our study challenges the current model of B19V entry and identifies globoside as an essential intracellular receptor required for endosomal escape.
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Endosomas , Globósidos , Aparato de Golgi , Parvovirus B19 Humano , Internalización del Virus , Endosomas/metabolismo , Endosomas/virología , Humanos , Aparato de Golgi/metabolismo , Aparato de Golgi/virología , Parvovirus B19 Humano/metabolismo , Parvovirus B19 Humano/fisiología , Parvovirus B19 Humano/genética , Globósidos/metabolismo , Fosfolipasas A2/metabolismo , Calcio/metabolismoRESUMEN
Fusion of enveloped viruses with endosomal membranes and subsequent release of the viral genome into the cytoplasm are crucial to the viral infection cycle. It is often modeled by performing fusion between virus particles and target lipid vesicles. We utilized fluorescence microscopy to characterize the kinetic aspects of the transfer of influenza viral ribonucleoprotein (vRNP) complexes to target vesicles and their spatial distribution within the fused volumes to gain deeper insight into the mechanistic aspects of endosomal escape. The fluorogenic RNA-binding dye QuantiFluor (Promega) was found to be well-suited for direct and sensitive microscopic observation of vRNPs which facilitated background-free detection and kinetic analysis of fusion events on a single particle level. To determine the extent to which the viral contents are transferred to the target vesicles through the fusion pore, we carried out virus-vesicle fusion in a side-by-side fashion. Measurement of the Euclidean distances between the centroids of superlocalized membrane and content dye signals within the fused volumes allowed determination of any symmetry (or the lack thereof) between them as expected in the event of transfer (or the lack thereof) of vRNPs, respectively. We found that, in the case of fusion between viruses and 100 nm target vesicles, â¼39% of the events led to transfer of viral contents to the target vesicles. This methodology provides a rapid, generic, and cell-free way to assess the inhibitory effects of antiviral drugs and therapeutics on the endosomal escape behavior of enveloped viruses.
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Colorantes Fluorescentes , Colorantes Fluorescentes/química , Humanos , Microscopía Fluorescente/métodos , Endosomas/metabolismo , Endosomas/virología , Animales , Ribonucleoproteínas/metabolismo , Orthomyxoviridae/aislamiento & purificación , Células de Riñón Canino Madin Darby , CinéticaRESUMEN
A multistep priming process involving furin and endosomal cathepsin B and L (CatB/L) has been described for the Orthoebolavirus zairense (EBOV) glycoprotein GP. Inhibition or knockdown of either furin or endosomal cathepsins, however, did not prevent virus multiplication in cell cultures. Moreover, an EBOV mutant lacking the furin cleavage motif (RRTRRâAGTAA) was able to replicate and cause fatal disease in nonhuman primates, indicating that furin cleavage may be dispensable for virus infectivity. Here, by using protease inhibitors and EBOV GP-carrying recombinant vesicular stomatitis virus (VSV) and transcription and replication-competent virus-like particles (trVLPs) we found that processing of EBOV GP is mediated by different proteases in different cell lines depending on the protease repertoire available. Endosomal cathepsins were essential for EBOV GP entry in Huh-7 but not in Vero cells, in which trypsin-like proteases and stably expressed trypsin-like transmembrane serine protease 2 (TMPRSS2) supported wild-type EBOV GP and EBOV GP_AGTAA mutant entry. Furthermore, we show that the EBOV GP_AGTAA mutant is cleaved into fusion-competent GP2 by TMPRSS2 and by CatL at a so far unknown site. Fluorescence microscopy co-localization studies indicate that EBOV GP cleavage by TMPRSS2 may occur in the TGN prior to virus release or in the late endosome at the stage of virus entry into a new cell. Our data show that EBOV GP must be proteolytically activated to support virus entry but has even greater flexibility in terms of proteases and the precise cleavage site than previously assumed.
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Catepsina L , Ebolavirus , Furina , Serina Endopeptidasas , Proteínas del Envoltorio Viral , Internalización del Virus , Catepsina L/metabolismo , Catepsina L/genética , Furina/metabolismo , Furina/genética , Ebolavirus/genética , Ebolavirus/fisiología , Ebolavirus/metabolismo , Animales , Humanos , Serina Endopeptidasas/metabolismo , Serina Endopeptidasas/genética , Chlorocebus aethiops , Proteínas del Envoltorio Viral/metabolismo , Proteínas del Envoltorio Viral/genética , Proteolisis , Células Vero , Línea Celular , Endosomas/metabolismo , Endosomas/virologíaRESUMEN
African swine fever (ASF) is a highly contagious, fatal disease of pigs caused by African swine fever virus (ASFV). The complexity of ASFV and our limited understanding of its interactions with the host have constrained the development of ASFV vaccines and antiviral strategies. To identify host factors required for ASFV replication, we developed a genome-wide CRISPR knockout (GeCKO) screen that contains 186,510 specific single guide RNAs (sgRNAs) targeting 20,580 pig genes and used genotype II ASFV to perform the GeCKO screen in wild boar lung (WSL) cells. We found that knockout of transmembrane protein 239 (TMEM239) significantly reduced ASFV replication. Further studies showed that TMEM239 interacted with the early endosomal marker Rab5A, and that TMEM239 deletion affected the co-localization of viral capsid p72 and Rab5A shortly after viral infection. An ex vivo study showed that ASFV replication was significantly reduced in TMEM239-/- peripheral blood mononuclear cells from TMEM239 knockout piglets. Our study identifies a novel host factor required for ASFV replication by facilitating ASFV entry into early endosomes and provides insights for the development of ASF-resistant breeding.
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Virus de la Fiebre Porcina Africana , Fiebre Porcina Africana , Sistemas CRISPR-Cas , Endosomas , Proteínas de la Membrana , Internalización del Virus , Replicación Viral , Animales , Porcinos , Virus de la Fiebre Porcina Africana/genética , Virus de la Fiebre Porcina Africana/fisiología , Fiebre Porcina Africana/virología , Fiebre Porcina Africana/metabolismo , Fiebre Porcina Africana/genética , Endosomas/metabolismo , Endosomas/virología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Técnicas de Inactivación de GenesRESUMEN
Viral diseases are among the main threats to public health. Understanding the factors affecting viral invasion is important for antiviral research. Until now, it was known that most viruses have very low plaque-forming unit (PFU)-to-particle ratios. However, further investigation is required to determine the underlying factors. Here, using quantitative single-particle analysis methods, the invasion of Semliki Forest virus (SFV), Japanese encephalitis virus (JEV), and influenza A virus (IAV) containing attachment to the cell surface, entry into the cell, transport towards the cell interior, and fusion with endosomes to release nucleocapsids were quantitatively analysed in parallel. It was found that for SFV with an PFU-to-particle ratio of approximately 1:2, an entry efficiency of approximately 31% limited infection. For JEV, whose PFU-to-particle ratio was approximately 1:310, an attachment efficiency of approximately 27% and an entry efficiency of 10% were the main factors limiting its infection. Meanwhile, for IAV with PFU-to-particle ratios of 1:8100, 5% attachment efficiency, 9% entry efficiency, and 53% fusion efficiency significantly limited its infection. These results suggest that viruses with different infectivities have different limited steps in the invasion process. Moreover, there are significant differences in attachment efficiencies among viruses, emphasizing the pivotal role of attachment in viral invasion. The influence of the virus purification method on virus invasion was also investigated. This study, for the first time, reports the efficiencies of different stages of virus invasion, leading to a better understanding of virus invasion and providing a protocol to quantitatively analyse the virus invasion efficiency.
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Virus de la Influenza A , Virus de los Bosques Semliki , Internalización del Virus , Virus de la Influenza A/fisiología , Animales , Virus de los Bosques Semliki/fisiología , Humanos , Virus de la Encefalitis Japonesa (Especie)/fisiología , Línea Celular , Acoplamiento Viral , Endosomas/virologíaRESUMEN
The cellular endosomal sorting complex required for transport (ESCRT) system comprises five distinct components and is involved in many different physiological processes. Recent studies have shown that different viruses rely upon the host ESCRT system for viral infection. However, whether this system is involved in white spot syndrome virus (WSSV) infection remains unclear. Here, we identified 24 homologs of ESCRT subunits in kuruma shrimp, Marsupenaeus japonicus, and found that some key components were strongly upregulated in shrimp after WSSV infection. Knockdown of key components of the ESCRT system using RNA interference inhibited virus replication, suggesting that the ESCRT system is beneficial for WSSV infection. We further focused on TSG101, a crucial member of the ESCRT-I family that plays a central role in recognizing cargo and activating the ESCRT-II and ESCRT-III complexes. TSG101 colocalized with WSSV in hemocytes. The addition of N16 (a TSG101 inhibitor) markedly decreased WSSV replication. TSG101 and ALIX of the ESCRT system interact with WSSV envelope proteins. The host proteins TSG101, RAB5, and RAB7, the viral protein VP28, and DNA were detected in endosomes isolated from hemocytes of WSSV-infected shrimp. Knockdown of Rab5 and Rab7 expression reduced viral replication. Taken together, these results suggest that the ESCRT system is hijacked by WSSV for transport through the early to late endosome pathway. Our work identified a novel requirement for the intracellular trafficking and infection of WSSV, and provided novel therapeutic targets for the prevention and control of WSSV in shrimp aquaculture. IMPORTANCE: Viruses utilize the ESCRT machinery in a variety of strategies for their replication and infection. This study revealed that the interaction of ESCRT complexes with WSSV envelope proteins plays a crucial role in WSSV infection in shrimp. The ESCRT system is conserved in the shrimp Marsupenaeus japonicus, and 24 homologs of the ESCRT system were identified in the shrimp. WSSV exploits the ESCRT system for transport and propagation via the interaction of envelope proteins with host TSG101 and ALIX in an endosome pathway-dependent manner. Understanding the underlying mechanisms of WSSV infection is important for disease control and breeding in shrimp aquaculture.
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Proteínas de Unión al ADN , Complejos de Clasificación Endosomal Requeridos para el Transporte , Penaeidae , Replicación Viral , Virus del Síndrome de la Mancha Blanca 1 , Animales , Virus del Síndrome de la Mancha Blanca 1/fisiología , Virus del Síndrome de la Mancha Blanca 1/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Penaeidae/virología , Penaeidae/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Endosomas/metabolismo , Endosomas/virología , Hemocitos/virología , Hemocitos/metabolismo , Interacciones Huésped-Patógeno , Proteínas del Envoltorio Viral/metabolismo , Proteínas del Envoltorio Viral/genética , Interferencia de ARNRESUMEN
Lymphocytic choriomeningitis virus (LCMV) is an enveloped and segmented negative-sense RNA virus classified within the Arenaviridae family of the Bunyavirales order. LCMV is associated with fatal disease in immunocompromised populations and, as the prototypical arenavirus member, acts as a model for the many highly pathogenic members of the Arenaviridae family, such as Junín, Lassa, and Lujo viruses, all of which are associated with devastating hemorrhagic fevers. To enter cells, the LCMV envelope fuses with late endosomal membranes, for which two established requirements are low pH and interaction between the LCMV glycoprotein (GP) spike and secondary receptor CD164. LCMV subsequently uncoats, where the RNA genome-associated nucleoprotein (NP) separates from the Z protein matrix layer, releasing the viral genome into the cytosol. To further examine LCMV endosome escape, we performed an siRNA screen which identified host cell potassium ion (K+) channels as important for LCMV infection, with pharmacological inhibition confirming K+ channel involvement during the LCMV entry phase completely abrogating productive infection. To better understand the K+-mediated block in infection, we tracked incoming virions along their entry pathway under physiological conditions, where uncoating was signified by separation of NP and Z proteins. In contrast, K+ channel blockade prevented uncoating, trapping virions within Rab7 and CD164-positive endosomes, identifying K+ as a third LCMV entry requirement. K+ did not increase GP-CD164 binding or alter GP-CD164-dependent fusion. Thus, we propose that K+ mediates uncoating by modulating NP-Z interactions within the virion interior. These results suggest K+ channels represent a potential anti-arenaviral target.IMPORTANCEArenaviruses can cause fatal human disease for which approved preventative or therapeutic options are not available. Here, using the prototypical LCMV, we identified K+ channels as critical for arenavirus infection, playing a vital role during the entry phase of the infection cycle. We showed that blocking K+ channel function resulted in entrapment of LCMV particles within late endosomal compartments, thus preventing productive replication. Our data suggest K+ is required for LCMV uncoating and genome release by modulating interactions between the viral nucleoprotein and the matrix protein layer inside the virus particle.
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Endosomas , Virus de la Coriomeningitis Linfocítica , Potasio , Internalización del Virus , Desencapsidación Viral , Endosomas/virología , Endosomas/metabolismo , Virus de la Coriomeningitis Linfocítica/fisiología , Virus de la Coriomeningitis Linfocítica/genética , Humanos , Potasio/metabolismo , Proteínas de Unión a GTP rab7 , Línea Celular , Animales , Canales de Potasio/metabolismo , Canales de Potasio/genéticaRESUMEN
During entry, human papillomavirus (HPV) traffics from the endosome to the trans Golgi network (TGN) and Golgi and then the nucleus to cause infection. Although dynein is thought to play a role in HPV infection, how this host motor recruits the virus to support infection and which entry step(s) requires dynein are unclear. Here we show that the dynein cargo adaptor BICD2 binds to the HPV L2 capsid protein during entry, recruiting HPV to dynein for transport of the virus along the endosome-TGN/Golgi axis to promote infection. In the absence of BICD2 function, HPV accumulates in the endosome and TGN and infection is inhibited. Cell-based and in vitro binding studies identified a short segment near the C-terminus of L2 that can directly interact with BICD2. Our results reveal the molecular basis by which the dynein motor captures HPV to promote infection and identify this virus as a novel cargo of the BICD2 dynein adaptor.
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Proteínas de la Cápside , Papillomavirus Humano 16 , Proteínas Oncogénicas Virales , Infecciones por Papillomavirus , Humanos , Proteínas de la Cápside/metabolismo , Papillomavirus Humano 16/metabolismo , Proteínas Oncogénicas Virales/metabolismo , Infecciones por Papillomavirus/metabolismo , Infecciones por Papillomavirus/virología , Dineínas/metabolismo , Endosomas/metabolismo , Endosomas/virología , Red trans-Golgi/metabolismo , Red trans-Golgi/virología , Internalización del Virus , Unión Proteica , Células HeLa , Proteínas Asociadas a Microtúbulos/metabolismo , Dineínas Citoplasmáticas/metabolismoRESUMEN
The infection caused by porcine epidemic diarrhea virus (PEDV) is associated with high mortality in piglets worldwide. Host factors involved in the efficient replication of PEDV, however, remain largely unknown. Our recent proteomic study in the virus-host interaction network revealed a significant increase in the accumulation of CALML5 (EF-hand protein calmodulin-like 5) following PEDV infection. A further study unveiled a biphasic increase of CALML5 in 2 and 12 âh after viral infection. Similar trends were observed in the intestines of piglets in the early and late stages of the PEDV challenge. Moreover, CALML5 depletion reduced PEDV mRNA and protein levels, leading to a one-order-of-magnitude decrease in virus titer. At the early stage of PEDV infection, CALML5 affected the endosomal trafficking pathway by regulating the expression of endosomal sorting complex related cellular proteins. CALML5 depletion also suppressed IFN-ß and IL-6 production in the PEDV-infected cells, thereby indicating its involvement in negatively regulating the innate immune response. Our study reveals the biological function of CALML5 in the virology field and offers new insights into the PEDV-host cell interaction.
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Calmodulina , Endosomas , Inmunidad Innata , Virus de la Diarrea Epidémica Porcina , Replicación Viral , Animales , Virus de la Diarrea Epidémica Porcina/inmunología , Virus de la Diarrea Epidémica Porcina/fisiología , Porcinos , Calmodulina/metabolismo , Calmodulina/genética , Endosomas/metabolismo , Endosomas/virología , Interacciones Huésped-Patógeno/inmunología , Enfermedades de los Porcinos/virología , Enfermedades de los Porcinos/inmunología , Células Vero , Chlorocebus aethiops , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/virología , Infecciones por Coronavirus/veterinaria , Interleucina-6/genética , Interleucina-6/metabolismo , Interleucina-6/inmunología , Interferón beta/genética , Interferón beta/inmunología , Interferón beta/metabolismoRESUMEN
Feline calicivirus (FCV) is one of the few members of the Caliciviridae family that grows well in cell lines and, therefore, serves as a surrogate to study the biology of other viruses in the family. Conley et al. (14) demonstrated that upon the receptor engagement to the capsid, FCV VP2 forms a portal-like assembly, which might provide a channel for RNA release. However, the process of calicivirus RNA release is not yet fully understood. Our findings suggest that the separation of the FCV capsid from its genome RNA (gRNA) occurs rapidly in the early endosomes of infected cells. Using a liposome model decorated with the FCV cell receptor fJAM-A, we demonstrate that FCV releases its gRNA into the liposomes by penetrating membranes under low pH conditions. Furthermore, we found that VP2, which is rich in hydrophobic residues at its N-terminus, functions as the pore-forming protein. When we substituted the VP2 N-terminal hydrophobic residues, the gRNA release efficacy of the FCV mutants decreased. In conclusion, our results suggest that in the acidic environment of early endosomes, FCV VP2 functions as the pore-forming protein to mediate gRNA release into the cytoplasm of infected cells. This provides insight into the mechanism of calicivirus genome release.IMPORTANCEResearch on the biology and pathogenicity of certain caliciviruses, such as Norovirus and Sapovirus, is hindered by the lack of easy-to-use cell culture system. Feline calicivirus (FCV), which grows effectively in cell lines, is used as a substitute. At present, there is limited understanding of the genome release mechanism in caliciviruses. Our findings suggest that FCV uses VP2 to pierce the endosome membrane for genome release and provide new insights into the calicivirus gRNA release mechanism.
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Calicivirus Felino , Proteínas de la Cápside , Endosomas , ARN Viral , Animales , Gatos , Infecciones por Caliciviridae/virología , Infecciones por Caliciviridae/metabolismo , Calicivirus Felino/genética , Calicivirus Felino/metabolismo , Calicivirus Felino/fisiología , Cápside/metabolismo , Proteínas de la Cápside/metabolismo , Proteínas de la Cápside/genética , Línea Celular , Endosomas/virología , Endosomas/metabolismo , Genoma Viral , Liposomas/metabolismo , ARN Viral/metabolismo , ARN Viral/genética , Liberación del VirusRESUMEN
Foot-and-mouth disease (FMD) is a highly contagious and economically important disease, which is caused by the FMD virus (FMDV). Although the cell receptor for FMDV has been identified, the specific mechanism of FMDV internalization after infection remains unknown. In this study, we found that kinesin family member 5B (KIF5B) plays a vital role during FMDV internalization. Moreover, we confirmed the interaction between KIF5B and FMDV structural protein VP1 by co-immunoprecipitation (Co-IP) and co-localization in FMDV-infected cells. In particular, the stalk [amino acids (aa) 413-678] domain of KIF5B was indispensable for KIF5B-VP1 interaction. Moreover, overexpression of KIF5B dramatically enhanced FMDV replication; consistently, knockdown or knockout of KIF5B suppressed FMDV replication. Furthermore, we also demonstrated that KIF5B promotes the internalization of FMDV via regulating clathrin uncoating. KIF5B also promotes the transmission of viral particles to early and late endosomes during the early stages of infection. In conclusion, our results demonstrate that KIF5B promotes the internalization of FMDV via regulating clathrin uncoating and intracellular transport. This study may provide a new therapeutic target for developing FMDV antiviral drugs.
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Virus de la Fiebre Aftosa , Cinesinas , Internalización del Virus , Replicación Viral , Cinesinas/metabolismo , Cinesinas/genética , Virus de la Fiebre Aftosa/fisiología , Virus de la Fiebre Aftosa/genética , Virus de la Fiebre Aftosa/metabolismo , Animales , Fiebre Aftosa/virología , Fiebre Aftosa/metabolismo , Proteínas de la Cápside/metabolismo , Proteínas de la Cápside/genética , Línea Celular , Humanos , Endosomas/metabolismo , Endosomas/virología , Células HEK293RESUMEN
With a high incidence of acute kidney injury among hospitalized COVID-19 patients, considerable attention has been focussed on whether SARS-CoV-2 specifically targets kidney cells to directly impact renal function, or whether renal damage is primarily an indirect outcome. To date, several studies have utilized kidney organoids to understand the pathogenesis of COVID-19, revealing the ability for SARS-CoV-2 to predominantly infect cells of the proximal tubule (PT), with reduced infectivity following administration of soluble ACE2. However, the immaturity of standard human kidney organoids represents a significant hurdle, leaving the preferred SARS-CoV-2 processing pathway, existence of alternate viral receptors, and the effect of common hypertensive medications on the expression of ACE2 in the context of SARS-CoV-2 exposure incompletely understood. Utilizing a novel kidney organoid model with enhanced PT maturity, genetic- and drug-mediated inhibition of viral entry and processing factors confirmed the requirement for ACE2 for SARS-CoV-2 entry but showed that the virus can utilize dual viral spike protein processing pathways downstream of ACE2 receptor binding. These include TMPRSS- and CTSL/CTSB-mediated non-endosomal and endocytic pathways, with TMPRSS10 likely playing a more significant role in the non-endosomal pathway in renal cells than TMPRSS2. Finally, treatment with the antihypertensive ACE inhibitor, lisinopril, showed negligible impact on receptor expression or susceptibility of renal cells to infection. This study represents the first in-depth characterization of viral entry in stem cell-derived human kidney organoids with enhanced PTs, providing deeper insight into the renal implications of the ongoing COVID-19 pandemic. IMPORTANCE: Utilizing a human iPSC-derived kidney organoid model with improved proximal tubule (PT) maturity, we identified the mechanism of SARS-CoV-2 entry in renal cells, confirming ACE2 as the sole receptor and revealing redundancy in downstream cell surface TMPRSS- and endocytic Cathepsin-mediated pathways. In addition, these data address the implications of SARS-CoV-2 exposure in the setting of the commonly prescribed ACE-inhibitor, lisinopril, confirming its negligible impact on infection of kidney cells. Taken together, these results provide valuable insight into the mechanism of viral infection in the human kidney.
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Enzima Convertidora de Angiotensina 2 , Riñón , Organoides , SARS-CoV-2 , Internalización del Virus , Humanos , Enzima Convertidora de Angiotensina 2/metabolismo , COVID-19/complicaciones , COVID-19/virología , Riñón/citología , Riñón/efectos de los fármacos , Riñón/metabolismo , Riñón/virología , Lisinopril/farmacología , Lisinopril/metabolismo , Organoides/citología , Organoides/efectos de los fármacos , Organoides/metabolismo , Organoides/virología , Pandemias , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidad , Glicoproteína de la Espiga del Coronavirus/metabolismo , Internalización del Virus/efectos de los fármacos , Peptidil-Dipeptidasa A/metabolismo , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Lesión Renal Aguda/etiología , Lesión Renal Aguda/metabolismo , Lesión Renal Aguda/virología , Túbulos Renales Proximales/citología , Túbulos Renales Proximales/efectos de los fármacos , Túbulos Renales Proximales/metabolismo , Túbulos Renales Proximales/virología , Receptores de Coronavirus/metabolismo , Modelos Biológicos , Serina Endopeptidasas/metabolismo , Endosomas/efectos de los fármacos , Endosomas/metabolismo , Endosomas/virología , Regulación de la Expresión Génica/efectos de los fármacos , Células Madre/citologíaRESUMEN
2019 coronavirus disease (COVID-19) is a disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In addition to respiratory illness, COVID-19 patients exhibit neurological symptoms lasting from weeks to months (long COVID). It is unclear whether these neurological manifestations are due to an infection of brain cells. We found that a small fraction of human induced pluripotent stem cell (iPSC)-derived neurons, but not astrocytes, were naturally susceptible to SARS-CoV-2. Based on the inhibitory effect of blocking antibodies, the infection seemed to depend on the receptor angiotensin-converting enzyme 2 (ACE2), despite very low levels of its expression in neurons. The presence of double-stranded RNA in the cytoplasm (the hallmark of viral replication), abundant synthesis of viral late genes localized throughout infected cells, and an increase in the level of viral RNA in the culture medium (viral release) within the first 48 h of infection suggested that the infection was productive. Productive entry of SARS-CoV-2 requires the fusion of the viral and cellular membranes, which results in the delivery of the viral genome into the cytoplasm of the target cell. The fusion is triggered by proteolytic cleavage of the viral surface spike protein, which can occur at the plasma membrane or from endosomes or lysosomes. We found that SARS-CoV-2 infection of human neurons was insensitive to nafamostat and camostat, which inhibit cellular serine proteases, including transmembrane serine protease 2 (TMPRSS2). Inhibition of cathepsin L also did not significantly block infection. In contrast, the neuronal infection was blocked by apilimod, an inhibitor of phosphatidyl-inositol 5 kinase (PIK5K), which regulates early to late endosome maturation. IMPORTANCE COVID-19 is a disease caused by the coronavirus SARS-CoV-2. Millions of patients display neurological symptoms, including headache, impairment of memory, seizures, and encephalopathy, as well as anatomical abnormalities, such as changes in brain morphology. SARS-CoV-2 infection of the human brain has been documented, but it is unclear whether the observed neurological symptoms are linked to direct brain infection. The mechanism of virus entry into neurons has also not been characterized. Here, we investigated SARS-CoV-2 infection by using a human iPSC-derived neural cell model and found that a small fraction of cortical-like neurons was naturally susceptible to infection. The productive infection was ACE2 dependent and TMPRSS2 independent. We also found that the virus used the late endosomal and lysosomal pathway for cell entry and that the infection could be blocked by apilimod, an inhibitor of cellular PIK5K.
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COVID-19 , Células Madre Pluripotentes Inducidas , SARS-CoV-2 , Humanos , Enzima Convertidora de Angiotensina 2 , COVID-19/fisiopatología , Endosomas/metabolismo , Endosomas/virología , Células Madre Pluripotentes Inducidas/metabolismo , Neuronas/metabolismo , Neuronas/virología , Síndrome Post Agudo de COVID-19/fisiopatología , Síndrome Post Agudo de COVID-19/virología , SARS-CoV-2/fisiología , Glicoproteína de la Espiga del Coronavirus/metabolismo , Internalización del Virus/efectos de los fármacos , Fosfotransferasas/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Astrocitos/virología , Células CultivadasRESUMEN
Mechanisms of infection are deciphered at the host-pathogen interface.
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Antivirales , Tratamiento Farmacológico de COVID-19 , Endosomas , Neuropilina-1 , Receptores Virales , SARS-CoV-2 , Antivirales/farmacología , Antivirales/uso terapéutico , Endosomas/virología , Interacciones Huésped-Patógeno/efectos de los fármacos , Neuropilina-1/antagonistas & inhibidores , Receptores Virales/antagonistas & inhibidores , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/fisiología , Red trans-Golgi/virología , Internalización del Virus/efectos de los fármacos , HumanosRESUMEN
Bats are reservoirs for diverse coronaviruses, including swine acute diarrhea syndrome coronavirus (SADS-CoV). SADS-CoV was first identified in diarrheal piglets in 2017. As a novel alphacoronavirus, SADS-CoV shares ~95% identity with bat alphacoronavirus HKU2. SADS-CoV has been reported to have broad cell tropism and inherent potential to cross host species barriers for dissemination. Thus far, no effective antiviral drugs or vaccines are available to treat infections with SADS-CoV. Therefore, knowledge of the protein-coding gene set and a subcellular localization map of SADS-CoV proteins are fundamental first steps in this endeavor. Here, all SADS-CoV genes were cloned separately into Flag-tagged plasmids, and the subcellular localizations of viral proteins, with the exception of nsp11, were detected using confocal microscopy techniques. As a result, nsp1, nsp3-N, nsp4, nsp5, nsp7, nsp8, nsp9, nsp10, nsp14, and nsp15 were localized in the cytoplasm and nuclear spaces, and these viral proteins may perform specific functions in the nucleus. All structural and accessory proteins were mainly localized in the cytoplasm. NS7a and membrane protein M colocalized with the Golgi compartment, and they may regulate the assembly of SADS-CoV virions. Maturation of SADS-CoV may occur in the late endosomes, during which envelope protein E is involved in the assembly and release of the virus. In summary, the present study demonstrates for the first time the location of all the viral proteins of SADS-CoV. These fundamental studies of SADS-CoV will promote studies of basic virology of SADS-CoV and support preventive strategies for animals with infection of SADS-CoV. IMPORTANCE SADS-CoV is the first documented spillover of a bat coronavirus that causes severe diseases in domestic animals. Our study is an in-depth annotation of the newly discovered swine coronavirus SADS-CoV genome and viral protein expression. Systematic subcellular localization of SADS-CoV proteins can have dramatic significance in revealing viral protein biological functions in the subcellular locations. Furthermore, our study promote understanding the fundamental science behind the novel swine coronavirus to pave the way for treatments and cures.
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Alphacoronavirus , Infecciones por Coronavirus , Enfermedades de los Porcinos , Proteínas Virales , Alphacoronavirus/genética , Animales , Núcleo Celular/virología , Quirópteros , Infecciones por Coronavirus/veterinaria , Endosomas/virología , Aparato de Golgi/virología , Porcinos , Enfermedades de los Porcinos/virología , Proteínas Virales/genéticaRESUMEN
Endosomal sorting maintains cellular homeostasis by recycling transmembrane proteins and associated proteins and lipids (termed "cargoes") from the endosomal network to multiple subcellular destinations, including retrograde traffic to the trans-Golgi network (TGN). Viral and bacterial pathogens subvert retrograde trafficking machinery to facilitate infectivity. Here, we develop a proteomic screen to identify retrograde cargo proteins of the endosomal SNX-BAR sorting complex promoting exit 1 (ESCPE-1). Using this methodology, we identify Neuropilin-1 (NRP1), a recently characterized host factor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, as a cargo directly bound and trafficked by ESCPE-1. ESCPE-1 mediates retrograde trafficking of engineered nanoparticles functionalized with the NRP1-interacting peptide of the SARS-CoV-2 spike (S) protein. CRISPR-Cas9 deletion of ESCPE-1 subunits reduces SARS-CoV-2 infection levels in cell culture. ESCPE-1 sorting of NRP1 may therefore play a role in the intracellular membrane trafficking of NRP1-interacting viruses such as SARS-CoV-2.
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COVID-19 , Endosomas , Interacciones Huésped-Patógeno , Neuropilina-1 , SARS-CoV-2 , COVID-19/metabolismo , COVID-19/virología , Sistemas CRISPR-Cas , Endosomas/virología , Eliminación de Gen , Humanos , Nanopartículas , Neuropilina-1/genética , Neuropilina-1/metabolismo , Proteómica , SARS-CoV-2/metabolismo , Nexinas de Clasificación/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismoRESUMEN
Production of infectious HIV-1 particles requires incorporation of the viral envelope glycoprotein (Env) at the plasma membrane (PM) of infected CD4+ T cells. Env trafficking to the PM exposes viral epitopes that can be exploited by the host immune system; however, HIV-1 can evade this response by endocytosis of excess Env from the PM. The fate of Env after internalization remains unclear, with evidence suggesting several different vesicular trafficking steps may be involved, including recycling pathways. To date, there have been very few studies documenting the trafficking pathways of native Env in infected T cells. Furthermore, it remains unclear whether there are T-cell-specific endosomal pathways regulating the fate of endocytic Env. Here, we use a pulse-labeling approach with a monovalent anti-Env Fab probe to characterize the trafficking of internalized Env within infected CD4+ T-cell lines, together with CRISPR/Cas9-mediated endogenous protein tagging, to assess the role of host cell Rab GTPases in Env trafficking. We show that endocytosed Env traffics to Rab14+ compartments that possess hallmarks of late endosomes and lysosomes. We also demonstrate that Env can recycle back to the PM, although we find that recycling does not occur at high rates when compared to the model recycling protein transferrin. These results help to resolve open questions about the fate and relevance of endocytosed Env in HIV-infected cells and suggest a novel role for Rab14 in a cell-type-specific late-endosomal/lysosomal trafficking pathway in T cells. IMPORTANCE HIV-1 envelope glycoprotein (Env) evades immune neutralization through many mechanisms. One immune evasion strategy may result from the internalization of excess surface-exposed Env to prevent antibody-dependent cellular cytotoxicity or neutralization. Characterization of the fate of endocytosed Env is critical to understand which vesicular pathways could be targeted to promote display of Env epitopes to the immune system. In this study, we characterize the endocytic fate of native Env, expressed from infected human T-cell lines. We demonstrate that Env is rapidly trafficked to a late-endosome/lysosome-like compartment and can be recycled to the cell surface for incorporation into virus assembly sites. This study implicates a novel intracellular compartment, marked by host-cell Rab14 GTPases, for the sequestration of Env. Therapeutic approaches aimed at mobilizing this intracellular pool of Env could lead to stronger immune control of HIV-1 infection via antibody-dependent cell-mediated cytotoxicity.
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Endosomas , Infecciones por VIH , VIH-1 , Lisosomas , Linfocitos T , Productos del Gen env del Virus de la Inmunodeficiencia Humana , Línea Celular , Endocitosis , Endosomas/metabolismo , Endosomas/virología , Epítopos , Infecciones por VIH/metabolismo , Humanos , Lisosomas/metabolismo , Lisosomas/virología , Transporte de Proteínas , Linfocitos T/virología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/metabolismo , Proteínas de Unión al GTP rab/metabolismoAsunto(s)
COVID-19/epidemiología , COVID-19/virología , Internacionalidad , SARS-CoV-2/química , SARS-CoV-2/patogenicidad , Glicoproteína de la Espiga del Coronavirus/química , Enzima Convertidora de Angiotensina 2/metabolismo , Microscopía por Crioelectrón , Endosomas/metabolismo , Endosomas/virología , Humanos , Pulmón/metabolismo , Pulmón/virología , Mucosa Nasal/metabolismo , Mucosa Nasal/virología , Faringe/metabolismo , Faringe/virología , SARS-CoV-2/genética , SARS-CoV-2/ultraestructura , Serina Endopeptidasas/metabolismo , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/metabolismoRESUMEN
With more than 80 members worldwide, the Orthobunyavirus genus in the Peribunyaviridae family is a large genus of enveloped RNA viruses, many of which are emerging pathogens in humans and livestock. How orthobunyaviruses (OBVs) penetrate and infect mammalian host cells remains poorly characterized. Here, we investigated the entry mechanisms of the OBV Germiston (GERV). Viral particles were visualized by cryo-electron microscopy and appeared roughly spherical with an average diameter of 98 nm. Labeling of the virus with fluorescent dyes did not adversely affect its infectivity and allowed the monitoring of single particles in fixed and live cells. Using this approach, we found that endocytic internalization of bound viruses was asynchronous and occurred within 30 to 40 min. The virus entered Rab5a-positive (Rab5a+) early endosomes and, subsequently, late endosomal vacuoles containing Rab7a but not LAMP-1. Infectious entry did not require proteolytic cleavage, and endosomal acidification was sufficient and necessary for viral fusion. Acid-activated penetration began 15 to 25 min after initiation of virus internalization and relied on maturation of early endosomes to late endosomes. The optimal pH for viral membrane fusion was slightly below 6.0, and penetration was hampered when the potassium influx was abolished. Overall, our study provides real-time visualization of GERV entry into host cells and demonstrates the importance of late endosomal maturation in facilitating OBV penetration. IMPORTANCE Orthobunyaviruses (OBVs), which include La Crosse, Oropouche, and Schmallenberg viruses, represent a growing threat to humans and domestic animals worldwide. Ideally, preventing OBV spread requires approaches that target early stages of infection, i.e., virus entry. However, little is known about the molecular and cellular mechanisms by which OBVs enter and infect host cells. Here, we developed accurate, sensitive tools and assays to investigate the penetration process of GERV. Our data emphasize the central role of late endosomal maturation in GERV entry, providing a comprehensive overview of the early stages of an OBV infection. Our study also brings a complete toolbox of innovative methods to study each step of the OBV entry program in fixed and living cells, from virus binding and endocytosis to fusion and penetration. The information gained herein lays the foundation for the development of antiviral strategies aiming to block OBV entry.
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Endosomas , Orthobunyavirus , Internalización del Virus , Animales , Microscopía por Crioelectrón , Endosomas/virología , Mamíferos , Orthobunyavirus/fisiologíaRESUMEN
African swine fever virus (ASFV) infectious cycle starts with the viral adsorption and entry into the host cell. Then, the virus is internalized via clathrin/dynamin mediated endocytosis and macropinocytosis. Similar to other viruses, ASF virion is then internalized and incorporated into the endocytic pathway. While the endosomal maturation entails luminal acidification, the decrease in pH acts on the multilayer structure of the virion dissolving the outer capsid. Upon decapsidation, the inner viral membrane is exposed to interact with the limiting membrane of the late endosome for fusion. Viral fusion is then necessary for the egress of incoming virions from endosomes into the cytoplasm, however this remains an intriguing and yet essential process for infection, specifically for the egress of viral nucleic acid into the cytoplasm for replication. ASFV proteins E248R and E199L, located at the exposed inner viral membrane, might be implicated in the fusion step. An interaction between these viral proteins and cellular endosomal proteins such as the Niemann-Pick C type 1 (NPC1) and lysosomal membrane proteins (Lamp-1 and -2) was shown. Furthermore, the silencing of these proteins impaired ASFV infection. It was also observed that NPC1 knock-out cells using CRISPR jeopardized ASFV infection and that the progression and endosomal exit of viral cores was arrested within endosomes at viral entry. These results suggest that the interactions of ASFV proteins with some endosomal proteins might be important for the membrane fusion step. In addition to this, reductions on ASFV infectivity and replication in NPC1 KO cells were accompanied by fewer and smaller viral factories. Our findings pave the way to understanding the role of proteins of the endosomal membrane in ASFV infection.