Método de las Necesidades Básicas Insatisfechas

Assembly of HIV particles is dependent on the viral Gag protein. It is well established that HIV-1 Gag budding and assembly occurs predominantly within cholesterol- enriched microdomains (detergent resistant membranes or lipid rafts) at the plasma membrane of T-cells (Ono and Freed, 2001). However, it was recently discovered that in macrophages and dendritic cells, a multivesicular late-endosome derived compartment is the site of particle assembly, prior to release from the PM (Pelchen- Matthews, 2003; Ono and Freed, 2004). These MVB-like sites of assembly in macrophages are also described as VCCs (virion containing compartmentd), which are non-acidic and linked to the PM by very narrow channels (Gaudin et al., 2013).

Live imaging of Gag by confocal microscopy and electron microscopy studies in HeLa and HEK 293 cells found the multivesicular body (MVB) was used for particle

44 assembly within these cell lines as well (Sherer et al., 2003). To support this, the use of a biarsenic-based imaging technique demonstrated that nascent Gag can travel through the MVB and perinuclear compartments as an intermediate, prior to arriving at the PM in HeLa cells (Perlman and Resh, 2006). This was suggestive of an involvement with endosomal pathways as trafficking routes for Gag in non-macrophage cells as well as macrophages. It has been demonstrated that Gag may form particles inside the endosomal lumen, which are then later released via fusion of the endosome-like structure with the PM by exploiting the pre-existing exosome release pathway, which was named the “Trojan exosome hypothesis” (Nguyen et al., 2003).

Consistent with this, a number of proteins involved in endosomal trafficking and recycling were found to interact with Gag. Specifically, the MA of Gag was found to interact with the clathrin adaptor proteins AP-1, AP-2 and AP-3 (see chapters 1.5.2i-iii, 3.1 and 5.1 for more detail). AP-1 and AP-3 were found to facilitate particle release, whereas AP-2 inhibits particle release (Batonick et al., 2005; Dong et al., 2005; Camus et al., 2007; Liu et al., 2012). The MA region of Gag has been found to be critical for Gag trafficking and localisation. More detail on the interaction of Gag with the adaptor protein pathways will be discussed in chapters 3 and 5.

The involvement of endosomes in the trafficking of Gag in T cells is, however, complicated by the fact that Gag present in these compartments may be derived from assembling particles which have been internalised from the PM. For example, TIRF (total internal reflection fluorescence) microscopy demonstrated that the Gag population which associated with late endosomal markers was indeed as a result of PM internalised Gag (Ivanchenko et al., 2009). Studies using a Gag-GFP construct also discovered that Gag reaches the PM prior to being found within intracellular compartments in HEK 293T cells (Jouvenet et al., 2006). Therefore, there still remains

45 some discrepancy over the route newly synthesised Gag adopts prior to assembly in T cells.

After trafficking to sites of assembly, Gag and Gag-Pol polyproteins sort into lipid rafts (Ono and Freed, 2001), which are rich in cholesterol and sphingolipids, with which the HIV-1 particle membrane is highly enriched (Ono, 2009). HIV-1 Gag does not extensively polymerise prior to reaching assembly sites; instead molecules are thought to arrive as soluble, monomeric proteins, folded into compact arrangements which subsequently undergo conformational changes which allow MA-membrane, NC-RNA and Gag-Gag interactions. Gag molecules arriving at assembly sites are most likely monomers/dimers, which extensively polymerise onto nucleation sites (Sundquist and Kräusslich, 2012).

Targeting of Gag to membranes requires a combination of factors; myristoylation (Gag is cotranslationally modified with the 14 carbon fatty acid myristate) (Resh, 2005), and

the interaction of MA and PI(4,5)P2, which is found on lipid rafts on the inner leaflet of

the PM, where Gag is concentrated (Ono et al., 2004; Gousset et al., 2008; Ghanam et al., 2012). Upon MA binding to membranes, the myristoyl group becomes exposed during an event called the “myristoyl switch”; this anchors Gag to the inner leaflet of the membrane (see Figure 17). The inositol head group and unsaturated 2’-fatty acid of

PI(4,5)P2 bind to MA, allosterically inducing extrusion of the myristoyl group

(Sundquist and Kräusslich, 2012). Once Gag has reached assembly sites, it dimerises. This process is principally driven by the C-terminus of the CA region of Gag. This, together with the N-terminus of spacer peptide p2, forms a continuous α-helix, which is critical for particle assembly (Gӧttlinger, 2001).

46 As described earlier, Env trimers travel independently of Gag to the PM. The long intracellular tail of gp41 (TM) is used to sort the Env spikes into raft-like domains and also assists specific interactions with the MA of Gag (Sundquist and Kräusslich, 2012).

Figure 17: Myristoyl switch mechanism. MA is shown in yellow with the myristoyl (brown), sequestered within the soluble protein (left) and with the myristoyl group embedded into the membrane when bound to PI(4,5)P2 (red) (right) (adapted

from Sundquist and Kräusslich, 2012).

HIV-1, like all retroviruses, packages two copies of genomic (gRNA) RNA into each virion as a dimer, joined by non-covalent linkages at the 5’ UTR. The gRNA is coated by NC at a density of roughly 1 NC molecule per 5-8 RNA bases (Johnson and Telesnitsky, 2010). Dimerisation initiates through formation of a “kissing loop” structure mediated by Watson-Crick base pairing of the self-complementary sequences found within the dimer initiation site (DIS). Efficient packaging is dependent on a packaging site (Ψ) located at the 5’ end of the genome, which spans the splice donor (SD) site and the Gag initiation codon. These are part of 4 stable stem loop structures within the highly structured 5’ UTR (see Figure 18 overleaf) (Sundquist and Kräusslich, 2012). Myristoyl group Soluble MA Membrane- bound MA Myristoyl group Lipid raft

47

Figure 18: The 5’ UTR of HIV-1 and its interactions with NC. The 4 stem loops critical to packaging are highlighted. 3 different NC/RNA complexes are shown; NC protein is shown in red, whilst viral RNA is shown in blue. TAR (Trans- activation response), PBS (primer binding site), DIS (dimer initiation site), SD (splice donor) are indicated (adapted from Sundquist and Kräusslich, 2012).