RESUMEN
The Bunyaviridae is the largest family of RNA viruses, with over 350 members worldwide. Several of these viruses cause severe diseases in livestock and humans. With an increasing number and frequency of outbreaks, bunyaviruses represent a growing threat to public health and agricultural productivity globally. Yet, the receptors, cellular factors and endocytic pathways used by these emerging pathogens to infect cells remain largely uncharacterized. The focus of this review is on the early steps of bunyavirus infection, from virus binding to penetration from endosomes. We address current knowledge and advances for members from each genus in the Bunyaviridae family regarding virus receptors, uptake, intracellular trafficking and fusion.
Asunto(s)
Bunyaviridae/fisiología , Interacciones Huésped-Patógeno , Acoplamiento Viral , Internalización del Virus , Animales , HumanosRESUMEN
The hantavirus membrane fusion process is mediated by the Gc envelope glycoprotein from within endosomes. However, little is known about the specific mechanism that triggers Gc fusion activation, and its pre- and post-fusion conformations. We established cell-free in vitro systems to characterize hantavirus fusion activation. Low pH was sufficient to trigger the interaction of virus-like particles with liposomes. This interaction was dependent on a pre-fusion glycoprotein arrangement. Further, low pH induced Gc multimerization changes leading to non-reversible Gc homotrimers. These trimers were resistant to detergent, heat and protease digestion, suggesting characteristics of a stable post-fusion structure. No acid-dependent oligomerization rearrangement was detected for the trypsin-sensitive Gn envelope glycoprotein. Finally, acidification induced fusion of glycoprotein-expressing effector cells with non-susceptible CHO cells. Together, the data provide novel information on the Gc fusion trigger and its non-reversible activation involving lipid interaction, multimerization changes and membrane fusion which ultimately allow hantavirus entry into cells.
Asunto(s)
Infecciones por Hantavirus/virología , Orthohantavirus/fisiología , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/metabolismo , Internalización del Virus , Endosomas/química , Endosomas/virología , Orthohantavirus/química , Orthohantavirus/genética , Humanos , Concentración de Iones de Hidrógeno , Multimerización de Proteína , Proteínas del Envoltorio Viral/genéticaRESUMEN
How hantaviruses assemble and exit infected cells remains largely unknown. Here, we show that the expression of Andes (ANDV) and Puumala (PUUV) hantavirus Gn and Gc envelope glycoproteins lead to their self-assembly into virus-like particles (VLPs) which were released to cell supernatants. The viral nucleoprotein was not required for particle formation. Further, a Gc endodomain deletion mutant did not abrogate VLP formation. The VLPs were pleomorphic, exposed protrusions and reacted with patient sera.
Asunto(s)
Orthohantavirus/metabolismo , Virus Puumala/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Virión/metabolismo , Western Blotting , Reacciones Cruzadas/inmunología , Cartilla de ADN/genética , Ensayo de Inmunoadsorción Enzimática , Orthohantavirus/genética , Humanos , Virus Puumala/genética , Virión/genéticaRESUMEN
The focus assay is currently the most commonly used technique for hantavirus titer determination. This method requires an incubation time of between 5 and 11 days to allow the appearance of foci after several rounds of viral infection. The following work presents a rapid Andes virus (ANDV) titration assay, based on viral nucleocapsid protein (N) detection in infected cells by flow cytometry. To this end, an anti-N monoclonal antibody was used that was developed and characterized previously. ANDV N could be detected as early as 6 h post-infection, while viral release was not observed until 24-48 h post-infection. Given that ANDV detection was performed during its first round of infection, a time reduction for titer determination was possible and provided results in only two days. The viral titer was calculated from the percentage of N positive cells and agreed with focus assay titers. Furthermore, the assay was applied to quantify the inhibition of ANDV cell entry by patient sera and by preventing endosome acidification. This novel hantavirus titration assay is a highly quantitative and sensitive tool that facilitates infectivity titration of virus stocks, rapid screening for antiviral drugs, and may be further used to detect and quantify infectious virus in human samples.
Asunto(s)
Citometría de Flujo/métodos , Viabilidad Microbiana , Orthohantavirus/aislamiento & purificación , Orthohantavirus/fisiología , Carga Viral/métodos , Animales , Anticuerpos Monoclonales , Anticuerpos Antivirales , Chlorocebus aethiops , Proteínas de la Nucleocápside/análisis , Sensibilidad y Especificidad , Factores de Tiempo , Células VeroRESUMEN
Dengue virus envelope protein (E) contains two N-linked glycosylation sites, at Asn-67 and Asn-153. The glycosylation site at position 153 is conserved in most flaviviruses, while the site at position 67 is thought to be unique for dengue viruses. N-linked oligosaccharide side chains on flavivirus E proteins have been associated with viral morphogenesis, infectivity, and tropism. Here, we examined the relevance of each N-linked glycan on dengue virus E protein by removing each site in the context of infectious viral particles. Dengue viruses lacking Asn-67 were able to infect mammalian cells and translate and replicate the viral genome, but production of new infectious particles was abolished. In addition, dengue viruses lacking Asn-153 in the E showed reduced infectivity. In contrast, ablation of one or both glycosylation sites yielded viruses that replicate and propagate in mosquito cells. Furthermore, we found a differential requirement of N-linked glycans for E secretion in mammalian and mosquito cells. While secretion of E lacking Asn-67 was efficient in mosquito cells, secretion of the same protein expressed in mammalian cells was dramatically impaired. Finally, we found that viruses lacking the carbohydrate at position 67 showed reduced infection of immature dendritic cells, suggesting interaction between this glycan and the lectin DC-SIGN. Overall, our data defined different roles for the two glycans present at the E protein during dengue virus infection, highlighting the involvement of distinct host functions from mammalian and mosquito cells during dengue virus propagation.