9 Separación para polímeros tipo III plastómeros.  109

The virus particles released from the cells are in a very stable form made of complex intermolecular interactions. They must be able to resist the harsh conditions in the extracellular environment during the process of transmission to a new cell or host. Despite their stable conformation the viruses must also be able to disassemble at specific sites and at specific time points during the entry process to release their genome into the cells. This is made possible by the metastable conformations of the viral coat proteins so that they can undergo major

conformational changes when triggered by specific signals during entry (Steven et al., 2005).

Virus entry starts with interactions of viral factors with the cell surface components and end with decondensation of the genome at the site of replication. This process involves penetration, capsid destabilization and uncoating of the genome resulting from conformational changes in

metastable virus structure that are triggered by receptor binding, exposure to low pH, reducing environment, proteolytic cleavage or other cellular factors ensuring that each step occurs at the right point in the sequence, at the right time, and at the right place (Earp et al., 2005; Harrison, 2005; Hogle, 2002; Smith and Helenius, 2004).

Viruses generally exploit the existing cellular mechanisms to enter their host cells. Some of them enter through the ongoing endocytic activities while majority of them induce their uptake through various mechanisms. Virus entry through clathrin-mediated endocytosis,

macropinocytosis and caveolar-dependent endocytosis are well studied, although there are other clathrin- and caveolin-independent mechanisms that are less well understood. Once internalized the incoming viruses are trafficked through the endosomal network to their sites of replication.

The endosomal network consists of early endosomes, late endosomes, recycling endosomes and maturing endosomes (Bonifacino and Glick, 2004). The endosomal network is also connected to

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the secretory pathway via the vesicles shuttling between endosomes, trans-Golgi network and plasma membrane (Bonifacino and Glick, 2004). The different classes of endosomes are

heterogeneous in composition. The early endosomes have a complex structure with long, narrow, often-branched tubes and they are defined by different Rabs and their effectors (Rab5 positive early endosomes, Rab5 and Rab7 positive maturing endosomes, Rab7 positive late endosomes) (Novick and Zerial, 1997).

Viruses exploit cellular signaling pathways to induce changes in the cell to promote virus entry, early cytoplasmic events and also some later events in the replication cycle. These

signaling events promotes the cell to initiate endocytosis process (Marsh and Helenius, 2006).

The interaction of adenovirus pentons with integrins activates phosphtidyl-inositol 3-kinase which in turn activates Rac and Cdc42 resulting in the polymerization of actin and clathrin-mediated endocytosis (Nemerow and Stewart, 1999). SV40 internalization by caveolar/raft endocytosis is regulated by five different kinases (Pelkmans et al., 2005) and inhibition of tyrosine kinases blocks the internalization reducing infection (Chen and Norkin, 1999; Pelkmans et al., 2002).

Although various pathways are utilized by different viruses to enter the cell, the endocytic pathway is the most widely used by majority of viruses as it offers the benefit of carrying the viruses in endocytic vesicles into the cytoplasm bypassing cellular barriers (Marsh and Bron, 1997). Clathrin-mediated endocytosis is the most common endocytic route used. The viruses along with their receptors are rapidly transported along the early and/or late endosomes and during this transit the incoming viruses use the decreasing pH of endocytic organelles to activate uncoating mechanisms and escape from the endosomes before they reach the lysosomes (Helenius et al., 1980). The site of endosomal escape can be either early endosomes (pH 6.5-6.0)

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or late endosomes (pH 6.0-5.5) depending on the virus. Some viruses like ebola virus, SARS coronavirus and nonenveloped mammalian reoviruses enter through clathrin-mediated

endocytosis and require acidic pH and proteolytic cleavage of viral proteins by cathepsin L and B for their process of uncoating and endosomal escape (Chandran et al., 2005; Ebert et al., 2002b; Simmons et al., 2005). Several studies have reported the use of clathrin-independent pathways by several viruses. Caveolae-dependent endocytosis is a well characterized mechanism for biological processes such as virus entry, internalization of

glycophosphatidylinositol-anchored proteins and certain signaling cascades including p38 kinase, JNK signaling as well as caspases activation. Caveolae are clathrin-independent raft-dependent endocytosis characterized by cholesterol, sphingolipid and caveolin-rich plasma membrane invaginations (Simons and Toomre, 2000). The caveolar pathway bring viruses including SV40, to caveosomes that are pH-neutral, from which they continue and by a second vesicle transport step reach the ER where penetration occurs in the redox conditions (Anderson et al., 1996; Pelkmans et al., 2001; Stang et al., 1997). Echovirus 1 entry by caveolar/raft endocytosis involves protein kinase C and

penetration occurs at caveosomes (Pietiainen et al., 2005; Upla et al., 2004). Macropinocytosis requires actin remodeling mediated by Rac-GTPase and its effector p21-activated kinase (Pak-1) producing membrane ruffles and blebs from cell surface that fold/drop back enclosing the target material (Swanson, 2008) and mediate the uptake of extracellular fluid and bulky materials (Mercer and Helenius, 2008; Mercer et al., 2010).

Macropinocytosis occurs constitutively in dendritic cells and is inducible in other cells by tyrosine kinases like EGFR (Swanson, 2008). Attachment of vaccinia virus activates EGFR, Rho-GTPases and actin remodeling initiating macropinocytosis (Mercer and Helenius, 2008;

Mercer et al., 2010). Other viruses that are internalized by this process include Kaposi’s

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sarcoma-associated herpesvirus, adenovirus, echovirus 1, Ebola virus and Vaccinia virus (Amstutz et al., 2008; Liberali et al., 2008; Mercer and Helenius, 2008; Mercer et al., 2010;

Nanbo et al., 2010; Raghu et al., 2009; Saeed et al., 2010; Schmidt et al., 2011). In spite of the heterogeneity in the formation of primary vesicles the later steps in intracellular trafficking involve either endosomes or caveosomes and may require cholesterol (Imelli et al., 2004) and depending on the virus and cell type, penetration reactions occur in five locations: the plasma membrane, early and late endosomes, caveosomes, and the endoplasmic reticulum. Various mechanism of virus entry is illustrated in figure 1-3.