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Title: Host Cofactors and Pharmacologic Ligands Share an Essential Interface in HIV-1 Capsid That Is Lost upon Disassembly}
Article:
Abstract:
The HIV-1 capsid is involved in all infectious steps from reverse transcription to integration site selection, and is the target of multiple host cell and pharmacologic ligands. However, structural studies have been limited to capsid monomers( CA), and the mechanistic basis for how these ligands influence infection is not well understood. Here we show that a multi-subunit interface formed exclusively within CA hexamers mediates binding to linear epitopes within cellular cofactors NUP153 and CPSF6, and is competed for by the antiretroviral compounds PF74 and BI-2. Each ligand is anchored via a shared phenylalanine-glycine( FG) motif to a pocket within the N-terminal domain of one monomer, and all but BI-2 also make essential interactions across the N-terminal domain: C-terminal domain( NTD:CTD) interface to a second monomer. Dissociation of hexamer into CA monomers prevents high affinity interaction with CPSF6 and PF74, and abolishes binding to NUP153. The second interface is conformationally dynamic, but binding of NUP153 or CPSF6 peptides is accommodated by only one conformation. NUP153 and CPSF6 have overlapping binding sites, but each makes unique CA interactions that, when mutated selectively, perturb cofactor dependency. These results reveal that multiple ligands share an overlapping interface in HIV-1 capsid that is lost upon viral disassembly.
Introduction:
The early events in HIV-1 infection proceed through a complex series of steps that include translocation of the viral core through the cytosol, reverse transcription, capsid uncoating, nuclear entry and integration. A number of host cofactors have been identified that regulate these processes, including cyclophilin A( CypA[1]), the karyopherin substrate CPSF6[2],[3], nuclear import/pore proteins TNPO3, NUP153, NUP358[4],[5],[6], and LEDGF[7]. Inhibition of CypA with cyclosporine reduces viral infectivity[8], promotes innate immune sensing[3] and alters the site of viral integration[9]. Depletion of TNPO3[10],[11], NUP358 or NUP153 inhibits nuclear entry and infectivity and, at least in the case of TNPO3 and NUP358, alters integration site targeting[11],[12]. Finally, CPSF6 depletion inhibits infection of primary macrophages by revealing HIV-1 to innate immune sensing[3]. How the virus coordinates recruitment of these cofactors and how they influence each other is not well understood, but there is a growing body of data suggesting that the viral capsid plays a key role. Viral dependence on post-entry cofactors is dictated by CA whilst CA mutations affect all post-entry processes from reverse transcription to integration site selection[9],[10],[13],[14],[15]. CypA and NUP358 bind directly to an exposed loop in the CA NTD[16],[17] and loop mutations such as G89A or P90A that impair CA:CypA interactions phenocopy CypA deletion[18]. HIV-1 utilization of TNPO3 and NUP153 is also CA-dependent, and viruses with CA mutations such as N57A, Q63A/Q67A, K70R and N74D are insensitive to depletion of these proteins[19],[20]. These residues all define a recently identified protein-protein interface on CA that binds CPSF6, suggesting that CPSF6 may act upstream of other nuclear import cofactors and determine their usage[21]. However, CPSF6 binding to CA is critically dependent upon an ‘FG’ motif[21] and NUP153 has recently been shown to interact directly with viral cores using a similar motif[20]. While CPSF6 and NUP153 share a common ‘FG’ binding motif, their interactions with CA are distinct. For example, CA mutant N74D abolishes binding to CPSF6[21] but not NUP153[20]. Moreover, despite this different binding pattern, N74D escapes both CPSF6 and NUP153 dependence suggesting that CPSF6 and NUP153 have distinct roles. A role for NUP153 in HIV-1 nuclear entry is substantiated by depletion experiments in which the production of 2-LTR circles, which form only within the nucleus, and HIV-1 integration, are reduced[22]. CPSF6 may also have a role in nuclear import by recruiting TNPO3 to the virus and providing transport to active nuclear pores[23]. In support of this, it has been shown that TNPO3 is not required for infection when CPSF6 binding is abolished by the CA mutation N74D[2]. Further evidence for a link between CPSF6 and TNPO3 include data showing that mutants of TNPO3 that are selectively impaired for interaction with CPSF6 do not support HIV-1 infection[24], whilst TNPO3-depleted cells are refractory to wild-type HIV-1 infection only in the presence of endogenous CPSF6[25]. Finally, the addition of a nuclear export sequence( NES) onto CPSF6 inhibited HIV-1 in TZM-bl cells[25]. However, the role of CPSF6 and Nup153 is complicated by the fact that perturbing their involvement gives rise to different phenotypes in different cell types. For instance, mutants T54A/N57A and Q63A/Q67A( which are sensitive to the synthetic restriction factor TRIM-CPSF6 but not TRIM-NUP153[20]) have reduced infection in nondividing HeLa cells but only T54A/N57A has reduced infectivity in nondividing macrophages[26]. Meanwhile, neither depletion of CPSF6[2] nor introduction of CPSF6 non-binding CA mutations N74D or T107A[19],[21] reduces infection of HIV-1 in single round replication assays in cell lines, whilst in macrophages N74D or CPSF6 depletion inhibits replication[3]. Importantly, it is not only changes in infection that are associated with an impaired ability of the capsid to engage CPSF6; N74D results in altered integration site targeting in HeLa cells[9]. The antiviral compounds PF-3450074( PF74)[27] and BI-2[28] have been shown to interact with the same pocket in the CA NTD as NUP153 and CPSF6. PF74 inhibits infection, blocks reverse transcription[29] and prevents replication in macrophages[3]. BI-2 has the same reported affinity for CA as PF74 yet does not block reverse transcription nor is as potent an inhibitor. These differences in reverse transcription inhibition are mirrored by NUP153 and CPSF6; NUP153 does not block reverse transcription but expression of a CPSF6 mutant lacking its nuclear localization sequence( hereafter referred to as ‘CPSF6ΔNLS’) inhibits viral DNA synthesis[3],[30]. While complexed crystal structures exist for the four ligands- NUP153, CPSF6, PF74 and BI-1( a BI-2 precursor)- these structures provide no mechanistic basis to explain their differing effects on viral infection[21],[27],[28]. One potential explanation for this is that all three structures were solved using monomeric CA NTD, whereas a fully assembled HIV-1 capsid is comprised exclusively of multimeric CA, predominately hexamers, containing both NTD-NTD and NTD-CTD interfaces[31]. Understanding the effect of ligand binding on intact capsid is of particular interest as there are conflicting reports on the effect of PF74 and BI-2 on capsid stability[28],[29],[32]. Here we report crystal structures of assembled HIV-1 CA hexamers with peptides comprising the linear binding epitopes of NUP153 and CPSF6 and the two antiviral compounds PF74 and BI-2. These structures reveal that the first three ligands make interactions both within and between CA subunits, using a previously unknown binding site at the NTD-CTD interface that significantly increases binding to CPSF6 and PF74 and is required for NUP153 interaction. NUP153, CPSF6 and PF74 utilize a distinct sub-set of residues within this site, providing a molecular basis for their associations with capsid and the resulting effects on HIV-1 infection.
Discussion:
Four ligands for HIV-1 – two antiviral drugs and two host cofactors – have been shown to bind a shared binding site in monomeric CA. Here we show that this binding site is actually part of a larger protein-protein interface that only exists in the context of hexameric CA. Identification of this interface has revealed that, while there are common interactions principally involving CA N57, there are also ligand-specific contacts. An example of such a contact is N74, which our results show is required for binding to CPSF6 and not NUP153. Other CA residues that are important for binding specifically to CPSF6 include A77, S102, T108, Q179 and K182. Of these, we have shown that S102D and K182R escape restriction by CPSF6ΔNLS, are insensitive to TNPO3 depletion and sensitive to TRIM-NUP153. Conversely, NUP153 makes specific CA interactions that are not used by CPSF6, including with P38, S41, Q63 and R143. In agreement with this, we have identified R143A as a capsid mutant that is susceptible to TNPO3 depletion, sensitive to restriction by CPSF6ΔNLS and insensitive to TRIM-NUP153. Our data support a model in which both CPSF6 and NUP153 are important HIV-1 cofactors. Engagement of CPSF6 is not required for HIV-1 infection in transformed cell lines[2] but is tightly correlated with dependence on the nuclear entry cofactors TNPO3, NUP153 and NUP358[38]. There are as yet no examples where perturbing the recruitment of these nuclear entry cofactors( through depletion or CA mutation) reduces infection of viruses that cannot engage CPSF6. Thus, interaction of the capsid with CPSF6 commits HIV-1 to the use of active nuclear pores( as defined by cofactor sensitivity). An important question is therefore why the virus utilizes this pathway if it is not required for infection. However, there are significant consequences for viruses that fail to engage with nuclear entry cofactors. Infection of primary monocyte-derived macrophages depleted of CPSF6 reveals HIV-1 to innate immune sensors, leading to induction of an antiviral state and abrogation of HIV-1 replication[3]. In cells in which innate immune sensing mechanisms are not activated, viruses that are independent of nuclear entry cofactors have altered HIV-1 integration site targeting[9],[11]. Moreover, HIV-1 appears to be under selective pressure in vivo to retain CPSF6 binding[38]. It is noteworthy that CPSF6 and NUP153 not only appear to function in the same nuclear import pathway but also share the same CA binding site. This is reminiscent of CypA and NUP358, which similarly compete for binding[16]. Whether this reflects a sequential pathway, with CypA and CPSF6 operating upstream of NUP358 and NUP153, and what the purpose of this might be, remains unclear. Alternatively, we cannot rule out the possibility that CPSF6 engages HIV-1 during passage through the nuclear pore, after NUP153. The structures presented here allow a protein interaction role to be ascribed to many of the CA mutants that have formed the basis for dissecting HIV-1 post-entry behaviour. However, several CA residues remain for which we cannot currently assign a role in cofactor binding but that nevertheless display specific post-entry defects. Examples of this include E45 and R132, which are located in relative proximity at the top of helices 2 and 7 respectively, between the CPSF6/NUP153 and CypA binding sites. Mutation of R132 results in reduced reverse transcription kinetics[13] and TNPO3 dependence[19] and, in the case of E45A, partial resistance to PF74[37]. These mutations may have a direct effect on intrinsic capsid stability but they may also disrupt the consequences of cofactor binding in an allosteric manner or alter interactions with as yet unidentified cofactors. CPSF6 and NUP153 and the antiviral drug PF74 all bind an interface that includes helices 8 and 9, previously shown to be flexible in both capsid crystal structures and NMR studies[31],[34]. The flexibility in this region may represent a requirement for capsid to be dynamic but it may also make the interface harder to target by restriction factors, analogous to the ‘conformational masking’ employed by gp120 to avoid neutralizing antibodies[39]. Our data suggest that ligand binding is only possible when this region adopts an “open” conformation. The consequences of fixing this conformation in the bound state may involve changes in capsid structure up to and including uncoating. Indeed, this may explain why at saturating doses of PF74 an irreversible block to infection is observed.
The early steps of HIV-1 infection are poorly understood , in part because of the difficulty in obtaining high-resolution information on encapsidated virus and its interaction with host cofactors . This , in turn , has made it difficult to design effective anti-capsid ( CA ) drugs . In our present study , we have used stabilized hexamers of HIV-1 CA to obtain complexed crystal structures with two cellular cofactors that are important for HIV-1 infection . These structures and accompanying virology reveal an essential interface in the capsid of HIV-1 that is lost upon viral uncoating . This interface is used to recruit both the nuclear targeting cofactor CPSF6 and NUP153 , a nuclear pore component that facilitates nuclear entry . The high-resolution information provided by these structures reveals that the interface is degenerate and CA mutations can be made that selectively perturb sensitivity to each cofactor . This interface is also competed by two antiviral drugs , PF74 and BI-2 , whose different mechanisms of action are not fully understood . We show that PF74 , but not BI-2 , binds across monomers within multimerized capsid affecting an inter-hexamer interface that is crucial for maintaining intact virions and that the addition of saturating concentrations of PF74 causes an irreversible block to viral reverse transcription .