Literature review
1.10 Gag structure, function and its role in PI resistance
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= matrix, CA = capsid and NC = nucleocapsid. PDB codes: 2H3I (Matrix), 1L6N (N-terminal Capsid), 1A43 (C-terminal Capsid), 1U57 (p2), 1A1T (Nucleocapsid) and 2C55 (p6).
1.10.1.1 Matrix
The HIV-1 matrix (MA; p17) protein is a thin layer lining the inner surface of the virion lipid bilayer after maturation (Doherty et al., 2005). Structurally, the 132 amino acid protein (Hill et al., 1996) consists of an amino-terminal (N-terminal) globular head and a flexible carboxyl- terminal (C-terminal) tail. Globular structures are typically spherical or “globe-shaped” water soluble proteins. In matrix, the head is composed of a 310 helix (Freed, 2015) and four alpha helices that is capped by a 3-stranded beta sheet. A 5th α-helix connects the capsid and MA domains (Freed, 2015), projecting away from the β-sheet to expose the C-terminal residues and forms the tail (Fiorentini et al., 2006). Based on the ionic strength and concentration of the protein buffer, MA may exist as either monomers, dimers, timers or oligomers of monomers, dimers and trimers (Forster et al., 2000).
Functionally, MA facilitates the targeting of Gag-Pol and Gag to the plasma membrane via myristoylation and PIP2 signals8. Additionally, MA is also involved in the nuclear import of the pre-integration complex (PIC) (Freed, 1998). The PIC is a large nucleoprotein composed of several viral and cellular proteins that allows integration during replication (Thierry et al., 2015).
Interestingly, MA also has cytokine-like functions that act on pre-activated human T cells encouraging proliferation, viral replication after p17R host cellular receptor binding and pro- inflammatory cytokine release. Therefore, MA may have an important role in the virus- and host- derived factors that contribute to favourable HIV-1 infection and replication (Fiorentini et al., 2006).
1.10.1.2 Capsid
In a mature virion, the HIV capsid (CA, p24) protein oligomerizes to form a shell around the virus’s RNA and core-associated proteins (Freed, 1998). All morphological variances of the CA domain are derived from a collection of hexameric (rings) CA monomers (Maillard et al., 2011) that has the same tertiary structure (Ganser-Pornillos et al., 2007). CA comprises two separate domains: the N-terminal domain (NTD 1–145) and the C-terminal domain (CTD 146–231) (Gitti et al., 1996), connected by a flexible linker (Lingappa et al., 2014). Structural studies revealed
8 Plasma membrane-specific phosphatidylinositol-4,5-bisphosphate (PIP2) and myristoylation facilitates Gag membrane targeting to the plasma membrane.
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that CA is predominantly composed of α-helices, with seven and four α-helices in the NTD and CTD, respectively (Bharat et al., 2012). Moreover, the NTD of CA contains an amino-terminal β-hairpin and resembles an arrowhead whereas the globular CTD comprises one 310 helix (Berthet-Colominas et al., 1999). Extensive intermolecular NTD-NTD interactions and to a lesser extent NTD-CTD interactions stabilize the hexameric rings. This stabilization occurs predominantly between helices 8–11 on the C-terminal side and 4–7 on the N-terminal side.
Flexible CTD-CTD interactions link the adjacent hexameric rings, allowing for the formation of the curved lattice (Fassati, 2012) while inter-hexamer angle variation between the longitudinal or lateral positions or at the broad and narrow ends of CA has been suggested. Furthermore, the asymmetrical structure infers that no two CAs are identical (Zhao et al., 2013). Therefore, the flexibility and complex molecular interactions supports a variety of conformations that contribute to structural variation in CA (Gres et al., 2015).
Additionally, each CA domain has a different function in HIV-1 morphogenesis. Although the NTD of CA is not necessary for immature virion assembly, it is indispensable for mature core formation (Borsetti et al., 1998). Contastingly, the CTD of CA is important for both the formation of the core and in virion assembly (McDermott et al., 1996). Additionally, all retroviruses comprise a conserved region of 20 AAs known as the major homology region (MHR) which is located in the CTD of CA (Wills and Craven, 1991). While this region is not clearly understood (Bell and Lever, 2013), its presence has been suggested to have a role at post-assembly and during assembly stages (Cairns and Craven, 2001). The entire MHR region forms a compacted strand turned helix stabilized by hydrogen bonds (Gamble et al., 1997). The MHR has been suggested to have a role CTD dimerization contributing to the stability of the viral shell (Ivanov et al., 2005).
As such, mutations in this region may affect viral assembly.
1.10.1.3 Nucleocapsid
The 55 amino acid nucleocapsid (NC; p7) protein (Bell and Lever, 2013) packages two copies of the viral RNA genome into the assembling virions (Berkowitz et al., 1996). A prominent feature of NC is the presence of two highly conserved Cys-X2-Cys-X4-His-X4-Cys (CCHC) signatures that resemble zinc-finger motifs. The zinc-fingers, each containing an aromatic residue (F16 in the N-terminal zinc finger and W37 in the C-terminal zinc finger) (Darlix et al., 2011) coordinate a zinc ion (Lingappa et al., 2014) and are divided by a RAPRKKG basic domain linker (Godet et al., 2012). These separately folded zinc-fingers resemble beads attached to a string (Summers et al., 1992).
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Functionally, in addition to genome packaging, NC can renature nucleic acids with catalytic rates of about four orders of magnitude. In reverse transcription, NC can also stimulate tRNALys binding to the primer binding site found at the N-terminal region of the genome, initiate reverse transcription from the bound tRNALys and partake in strand transfer (Rodriguez-Rodriguez et al., 1995; Guo et al., 1997; Freed, 1998). Furthermore, annealing by NC enhances the formation of the converted loop-loop interaction into a stable duplex and the formation of RNA dimers (Lu et al., 2011).
1.10.1.4 p6
The C-terminal region of Gag comprises a proline-rich (Freed, 1998), 52 amino acid p6 domain (Votteler et al., 2011) where two amino acid sequences are translated, the -1 frameshift Gag-Pol p6 and the in-frame Gag p6 domain (Bell and Lever, 2013). Although the structure of the p6 domain of Gag remains unclear (Freed, 2015), it is known that the domain displays little, if any secondary structure (Stys et al., 1993), instead serving as a flexible docking site for cellular host factors (Gottlinger et al., 1991). The p6 domain is also necessary for the incorporation of viral accessory protein, Vpr into the virions (Solbak et al., 2013). Vpr is associated with trans- activation of the LTR, import of the pre-integration complex (PIC) in non-dividing cells, the induction of apoptosis9 and cell halt at the G2/M transition pathway10.
1.10.1.5 p2 and p1 spacer peptides
The 14 amino acid p2 peptide is found wedged between the N-terminal MA and C-terminal NC proteins (Bell and Lever, 2013). The C- and N-termini of the CA and p2 domains respectively, were shown to form two parts of an α-helix. Electron cryotomography analyses show that p2 forms a six α-helical bundle that stabilizes Gag hexamers in immature virus particles (Wright et al., 2007). As a result, cleavage of p2 from NC is necessary for the formation of ribonucleoprotein within RNA and condensation of the CA domain. The late cleavage of CA|p2 allows morphogenesis through the cadence of CA-CA interactions. The importance of the integrity of the CA|p2 segment in formation of immature CA was shown in an electron microscopic study (Gross et al., 2000).
The 16 amino acid p1 peptide lies between the CTD of NC and the NTD of p6. This peptide contains two highly conserved proline residues, namely P445 and P439. Hill et al. (2007)
9 Programmed cell death
10 The G2/M transition is a point in the cell’s lifecycle where after the second growth phase (G2) and DNA replication (S phase), mitosis occurs (M phase) to separate the cell into identical daughter cells.
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implicated the importance of p1 for Pol and Gag incorporation into the virus particles. The
“slippery site” involved in the Gag-Pol ribosomal frameshift overlaps with the end of p1 leading to potentially complex effects of mutations at this site (Bell and Lever, 2013). The substitution of either proline (P445 and P439) by leucine results in lower stability of the NC-RNA complex and rescinds infectivity (Hill et al., 2002). Moreover, mutations at the cleavage sites at either end of p1 have discrepant effects. For example, mutations in NC|p1 have no effect on proviral integration, whereas mutations at the C-terminal that produces p15 (NC|p1|p6) or p8 (p1|p6) peptides decreases the amount of integrated provirus on subsequent infection. This differential effect may suggest the role of NC|p1 in proviral integration as opposed to p15 and p8 (Coren et al., 2007).
1.10.2 Resistance
The collection of resolved structures on individual Gag domains contributes to the intrinsic and functional behaviour of Gag at the molecular level (Novikova et al., 2018). As a result, it is now evident that Gag and its individual domains have significant roles in the lifecycle and perseverance of HIV (Freed, 1998; Mailler et al., 2016; Tomasini et al., 2018). Even so, an extensive review by Fun et al. (2012) highlights several Gag mutations within and outside the CSs that are associated with drug resistance or exposure. Mutations in Gag, particularly at NC|p1 and p1|p6 CSs have been extensively reported (Borman et al., 1996; Nijhuis et al., 1999; Maquire et al., 2002; Prado et al., 2002; Dam et al., 2009). Additionally, while mutations in Gag can restore substrate processing (Myint et al., 2004; Tamiya et al., 2004; Ho et al., 2008; Parry et al., 2009), it has also been shown that Gag can confer resistance in the absence of mutations in the viral PR (Nijhuis et al., 2007; Gupta et al., 2010).