Derivation and Characterization of an inositol phosphate-independent HIV-1

A critical step in the HIV-1 replication cycle is the assembly of Gag proteins to form virions at the plasma membrane. Virion assembly and maturation is facilitated by the cellular polyanion inositol hexaphosphate (IP6), which is proposed to stabilize both the immature Gag lattice and the mature capsid lattice by binding to rings of primary amines at the center of Gag or capsid protein (CA) hexamers. The amino acids comprising these rings are critical for proper virion formation and their substitution results in assembly deficits or impaired infectiousness. To better understand the nature of the deficits that accompany IP6-deficiency, we passaged HIV-1 mutants that had substitutions in IP6-coordinating residues to select for compensatory mutations. We found a mutation, a threonine to isoleucine substitution at position 371 (T371I) in Gag, that restored replication competence to an IP6-binding-deficient HIV-1 mutant. Notably, unlike wild-type HIV-1, the assembly and infectiousness of resulting virus was not impaired when IP6 biosynthetic enzymes were genetically ablated. Surprisingly, we also found that the maturation inhibitor Bevirimat (BVM) could restore the assembly and replication of an IP6-binding deficient mutant. Moreover, using BVM-dependent mutants we were able to image the BVM-inducible assembly of individual HIV-1 particles assembly in living cells. Overall these results suggest that IP6-Gag and Gag-Gag contacts are finely tuned to generate a Gag lattice of optimal stability, and that under certain conditions BVM can functionally replace IP6. Author Summary A key step in HIV-1 replication is the assembly of virions that are released from the infected cell. Previous work has suggested that a small molecule called IP6 is critical role in this process, promoting both HIV-1 assembly and the stability of mature fully infectious virions. Since IP6 is required for multiple steps in HIV-1 assembly and maturation, it is a candidate for the development of anti-retroviral therapies. Here, we identify an HIV-1 mutant that replicates independently of IP6, and show that a different small molecule can functionally substitute for IP6 under certain conditions. These findings suggest that IP6 regulates the stability of protein interactions during virion assembly and that the precise degree stability of these interactions is finely tuned and important for generating infectious virions. Finally, our work identifies an inducible virion assembly system that can be utilized to visualize HIV-1 assembly events using live cell microscopy.


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To determine whether the T371I mutant rescued the infectivity defect present in HIV-135 1 K359A , we generated a proviral clone, HIV-1 K359A/T371I , encoding both mutations and measured 136 the infectious virion yield from proviral plasmid-transfected 293T cells. Addition of the T371I 7 138 this second-site, apparently compensatory change was identified only in the context of HIV-139 1 K359A , we asked whether the T371I substitution could rescue the HIV-1 K290A , given purported 140 similar roles of K290 and K359 in binding IP 6 . Indeed, found that HIV-1 K290A/T371I , unlike HIV-141 1 K290A , yielded similar levels of infectious HIV-1 virions to wild-type HIV-1.

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143 Infectious HIV-1 K359A/T371I particle yield is not affected ablation of IP 6 synthesis in virus 144 producing cells 145 Because the HIV-1 K359A is defective for IP 6 binding we next asked whether HIV-1 K359A/T371I 146 retained infectiousness when cellular IP 6 levels were reduced. Using CRISPR/Cas9 we 147 generated 293T cell lines lacking IPMK, an enzyme in the IP 6 synthetic pathway. Previous work 148 has demonstrated IPMK knockout cells have greatly reduced levels of both IP 5 and IP 6 (13). To 149 account for potential clonal variation in capacity to generate HIV-1 particles, we used 3 separate 150 IPMK targeting sgRNAs or a corresponding empty vector to generate ten independent single 151 cell clones of IPMK knockout and WT control 293T cells (Figure 2A). The loss of IPMK was 152 confirmed by DNA sequencing of target loci, which revealed the introduction of frameshift 153 mutations into both alleles of the IPMK coding sequences, and the absence of intact IPMK 154 alleles. In agreement with previous studies, the yield of infectious HIV-1 WT virions from IPMK-155 deficient 293T cells was significantly decreased, by 10-fold (p=0.0091, Figure 2A). The yield of 156 HIV-1 K359A from 293T cells was greatly reduced compared to wildtype HIV-1 as expected, and 157 was not further reduced by IPMK deficiency ( Figure 2B). Importantly, the yield of HIV-1 K359A/T371I 158 was only marginally reduced compared to wild type HIV-1 and there was no difference in yield  164 It has been proposed that residues K290 and K359 selectively recruit IP 6 into HIV-1 virions 165 during assembly, thereby providing the source of the IP 6 that binds to and stabilizes the R18 166 ring in the mature capsid core. The rationale for this idea stems from previous studies which 167 have demonstrated that reduction of cellular IP 6 levels in target cells does not impact 168 susceptibility to incoming infection (13). Because HIV-1 K359A/T371I is fully infectious despite 169 encoding a mutation that diminishes IP 6 packaging into virions, we next asked whether HIV-170 1 K359A/T371I requires IP 6 in target cells to be maximally infectious. We generated multiple twelve  179 Notably, The T371I mutation identified herein had been described previously in a different 180 context. Specifically, this substitution was reported to stabilize the immature CA-SP1 lattice, 181 mimicking the effect of maturation inhibitors (MI) (16,17). Therefore, we next asked whether 182 maturation inhibitors themselves could rescue the deficit in infectious virion yield exhibited by 183 HIV-1 K359A . As a control, we included the previously described assembly-defective, maturation 184 inhibitor-dependent CA mutant HIV-1 P289S . We found that that BVM indeed rescued the 185 infectivity of HIV-1 K359A and HIV-1 P289S in both single-cycle and spreading replication assays. 222 infected cells suggesting BVM is able to directly facilitate particle assembly by these mutants.

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224 This ability to induce HIV-1 particle assembly via addition of an exogenous small molecule has 225 potential applications in imaging and other studies, as an inducible particle assembly system. In

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260 HIV-1 K359A/T371I is fully infectious despite containing a mutation (K359A) that renders VLPs 261 unresponsive to IP 6 in vitro and substantially impairs IP 6 incorporation into virions (9,13). Indeed, 262 we found no significant reduction in yield of HIV-1 K359A/T371I from IPMK KO 293T cells, in contrast 263 to WT HIV-1. This finding suggests that HIV-1 K359A/T371I is no longer dependent on IP 6 in virus 264 producing cells, and that the lattice stabilizing mutation T371I can functionally substitute for IP 6 . 265 267 stabilizing the mature lattice and promoting viral DNA synthesis by binding to a positively 268 charged pore formed by R18 residues in the mature CA lattice (10,20). Previously it was 269 proposed that the source of the IP 6 required to stabilize the mature lattice in virions is selective 270 recruitment by K290 and K359 residues, with IP 6 being liberated to bind R18 residues following 271 disruption of the immature lattice after proteolysis (9,10). The fact that HIV-1 K359A/T371I has no 272 infectivity deficit, despite bearing a substitution known to decrease IP 6 incorporation into virions 273 argues against a requirement for IP 6 post assembly. While we cannot formally exclude the 274 possibility that HIV-1 K359A/T371I has regained the ability to efficiently package IP 6 , this is highly 275 unlikely as 1) our data shows that HIV-1 K359A/T371I is not dependent on cellular IP 6 levels in  293 Because the T371I substitution, apparently mimics the effect of MIs, we hypothesized that MIs 294 might also rescue the infectivity of HIV-1 K359A . Strikingly, we found that this was the case, and 295 found that BVM can stimulate the assembly and release of HIV-1 K359A . That the stabilizing 296 effects of both the T371I substitution and BVM can compensate for the lack of IP 6 coordination 297 in HIV-1 K359A provides in vivo mechanistic support for the model proposed by Dick et al: i.e. that 298 binding of IP 6 to K290 and K359 residues stabilizes the immature lattice to drive particle 299 assembly.

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308 Together, these data support a model whereby stability of the immature CA lattice is finely 309 tuned, with IP 6 coordinating and stabilizing the otherwise repulsive positive charges of K290 and 310 K359 to drive assembly. Manipulations that cause IP 6 binding deficiency, either mutagenesis of 311 K359 or decreasing IP 6 levels in producer cells, destabilize the immature lattice and decrease 312 production of progeny virions. Conversely, manipulations such as the T371I substitution or 313 treatment of HIV-1 WT with BVM, hyper-stabilize the immature lattice in the wild type context and 314 decrease HIV-1 infectivity. However, either the T371I substitution or BVM treatment are able to 315 functionally substitute for IP 6 in the context of HIV-1 K359A and rescue virion assembly and 316 infectiousness. Understanding how small molecules such as BVM or IP 6 can enhancing Gag