Members of Venezuelan Equine Encephalitis complex entry into host cells by clathrin-mediated endocytosis in a pH-dependent manner.

Pixuna virus (PIXV) and Río Negro virus (RNV) are mosquito-borne alphaviruses belonging to the Venezuelan Equine Encephalitis (VEE) complex, which includes pathogenic epizootic and enzootic subtypes responsible for life-threatening diseases in equines. Considering that the first steps in viral infection are crucial for the efficient production of new progeny, the aim of this study was to elucidate the early events of the replication cycle of these two viruses. To this end, we used chemical inhibitors and the expression of dominant-negative constructs to study the dependence of clathrin and endosomal pH on PIXV and RNV internalization mechanisms. We demonstrated that both viruses are internalized primarily via clathrin-mediated endocytosis, where the low pH in endosomes is crucial for viral replication. Contributing knowledge regarding the entry route of VEE complex members is important to understand the pathogenesis of these viruses and also to develop new antiviral strategies.

Pixuna virus modifies host cell cytoskeleton to secure infection.

Pixuna virus (PIXV) is an enzootic member of the Venezuelan Equine Encephalitis Virus complex and belongs to the New World cluster of alphaviruses. Herein we explore the role of the cellular cytoskeleton during PIXV replication. We first identified that PIXV undergoes an eclipse phase consisting of 4 h followed by 20 h of an exponential phase in Vero cells. The infected cells showed morphological changes due to structural modifications in actin microfilaments (MFs) and microtubules (MTs). Cytoskeleton-binding agents, that alter the architecture and dynamics of MFs and MTs, were used to study the role of cytoskeleton on PIXV replication. The virus production was significantly affected (p < 0.05) after treatment with paclitaxel or nocodazole due to changes in the MTs network. Interestingly, disassembly of MFs with cytochalasin D, at early stage of PIXV replication cycle, significantly increased the virus yields in the extracellular medium (p < 0.005). Furthermore, the stabilization of actin network with jasplakinolide had no effect on virus yields. Our results demonstrate that PIXV relies not only on intact MTs for the efficient production of virus, but also on a dynamic actin network during the early steps of viral replication.

Participación de las mitocondrias, los endosomas y el citoesqueleto en la replicación del virus pixuna

El género Alphavirus es de gran interés epidemiológico ya que sus miembros, incluyendo los virus del "Complejo de Encefalitis Equina Venezolana", pueden provocar importantes enfermedades, tanto en animales domésticos con en el hombre. Durante las últimas décadas se ha registrado un aumento en la incidencia mundial de virus transmitidos por artrópodos (arbovirus), particularmente aquellos transmitidos por mosquitos, como los virus de Encefalitis Equina Venezolana, el Virus Rio Negro, Moyaro, PIXV y Chikunguya, entre otros.

Abstract: The present work is based on the study of the replication mechanisms of Pixuna virus (PIXV), including the participation of the endosomal pathway during viral stripping as well as the mitochondria and cytoskeleton, both microtubules (MTs) and microbilaments (MFLS), in the replication process for the correct location on your replication sites.