Infections with dengue virus (DENV) are a significant public health concern in tropical and subtropical regions. However, little detail is known about how DENV interacts with the host-cell machinery to facilitate its translation and replication. In DENV-infected HepG2 cells, an increase in the level of LC3-II (microtubule-associated protein 1 light chain 3 form II), the autophagosomal membrane-bound form of LC3, was observed, and LC3 was found to co-localize with dsRNA and DENV NS1 protein, as well as ribosomal protein L28, indicating the presence of at least some of the DENV translation/replication machinery on autophagic vacuoles. Inhibition of fusion of autophagic vacuoles with lysosomes resulted in an increase in both intracellular and extracellular virus, and co-localization observed between mannose-6-phosphate receptor (MPR) and dsRNA and between MPR and LC3 identified the autophagic vacuoles as amphisomes. Amphisomes are formed as a result of fusion between endosomal and autophagic vacuoles, and as such provide a direct link between virus entry and subsequent replication and translation. INTRODUCTIONWith an estimated 100 million infections per year worldwide, dengue virus (DENV), which is spread to humans by the bite of female Aedes mosquitoes, represents a significant public health threat in tropical and subtropical countries (Guzman & Kouri, 2002). The DENV complex comprises four antigenically distinct viruses termed DENV serotypes 1, 2, 3 and 4 (DENV-1 to -4), all of which can cause a wide spectrum of disease presentation, from a relatively mild febrile disease to a life-threatening haemorrhagic syndrome (Malavige et al., 2004). DENV is an enveloped, positivesense, single-stranded RNA virus of approximately 11 kb that encodes three structural proteins (core, pre-membrane and envelope) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) in one open reading frame (Chang, 1997). Entry of DENV into a susceptible cell occurs primarily through receptormediated endocytosis via clathrin-coated pits (Krishnan et al., 2007) and the virus is then trafficked to endosomes (Krishnan et al., 2007;van der Schaar et al., 2007) and fuses with the endosomal membranes (Heinz et al., 2004) through a pH-dependent conformational change in the envelope protein (Modis et al., 2004;Mukhopadhyay et al., 2005). The fate of the released nucleocapsid is largely obscure, although it is known that, after uncoating of the virus genome, the host-cell translational machinery is utilized to synthesize a polyprotein precursor that is processed co-and post-translationally by a virus-encoded protease (NS3) and host signallases (Cahour et al., 1992). Translation and subsequent replication of the DENV genome occur in tight association with intracellular membranous structures that are believed to be endoplasmic reticulum (ER)-derived (Clyde et al., 2006;Miller & Krijnse-Locker, 2008;Salonen et al., 2005), although the exact origin of these membranes remains unclear.Autophagy is a lysosomal degradation pathway involve...
Chikungunya virus (CHIKV) has recently re-emerged causing millions of infections in countries around the Indian Ocean. While CHIKV has a broad host cell range and productively infects a number of different cell types, macrophages have been identified as a potential viral reservoir serving to increase the duration of symptoms. To date no CHIKV interacting protein has been characterized and this study sought to identify CHIKV binding proteins expressed on target cell membranes. Two-dimensional virus overlay identified prohibitin (PHB) as a microglial cell expressed CHIKV binding protein. Co-localization, co-immunoprecipitation as well as antibody and siRNA mediated infection inhibition studies all confirmed a role for PHB in mediating internalization of CHIKV into microglial cells. PHB is the first identified CHIKV receptor protein, and this study is evidence that PHB may play a role in the internalization of multiple viruses.
We have recently proposed that amphisomes act as a site for translation and replication of dengue virus (DENV)-2 and that DENV-2 entry and replication are linked through an ongoing association with membranes of an endosomal-autophagosomal lineage. In this report, we present the results of an investigation into the interaction between DENV-3 and the autophagy machinery. Critically, treatment with the lysosomal fusion inhibitor L-asparagine differentiated the interaction of DENV-3 from that of DENV-2. Inhibition of fusion of autophagosomes and amphisomes with lysosomes resulted in decreased DENV-3 production, implying a role for the autophagolysosome in the DENV-3 life cycle. Evidence based upon the co-localization of LC3 and cathepsin D with double stranded RNA and NS1 protein, as assessed by confocal microscopy, support a model in which DENV-3 interacts with both amphisomes and autophagolysosomes. These results demonstrate that the interactions between DENV and the host cell autophagy machinery are complex and may be determined in part by virus-encoded factors.
While several studies have shown a role for autophagy in the replication of dengue virus (DENV), these studies have been performed in directly infected cells. However, in severe cases of DENV infection the critical cell in the disease is believed to be monocytes which are poorly infected directly, but are highly susceptible to antibody enhanced infection. This study sought to determine the involvement of autophagy in the DENV infection of monocytic cells, using U937 cells as a model system. While the induction of autophagy was seen in response to DENV-2 infection, biochemical induction of autophagy resulted in a significant decrease in virus output. Down regulation of autophagy resulted in only a very slight increase in intracellular virus levels. In monocytic cells autophagy is not a significant part of the DENV replication mechanism, and there are distinct cell type specific differences in the DENV-autophagy interaction.
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