Identification of a second-site substitution that restores replication competence to IP
6
-binding deficient HIV-1 mutants
To identify second site changes that would rescue IP6 binding deficient mutants, we passaged HIV-1 mutants encoding substitutions in IP6 coordinating residues (HIV-1R18A, HIV-1K290A, or HIV-1K359A) in the highly-permissive MT4 T-cell line. Initial attempts, in which MT4 cells were infected with mutant viral stocks, were unsuccessful, likely due to the dramatically impaired fitness of these mutants and consequent inability to establish a sufficiently large population of infected cells to generate revertants. To overcome this problem, we instead co-cultured MT4 cells with virus-producing 293T cells that had been transfected with HIV-1R18A, HIV-1K290A, and HIV-1K359A proviral plasmids that encode GFP in place of nef. After removing the 293T cells, infected MT4 cells were co-cultured with uninfected MT4 cells, until most of the MT4 cells became infected (as monitored by visual inspection of GFP+ cells in the culture). Thereafter, cell-free supernatant was serially passed in MT4 cells (Fig. 1A).
For one mutant, HIV-1K359A, observation of GFP positive cells suggested that an apparently compensatory mutation arose after approximately 2 weeks of passaging. PCR amplification and sequencing of Gag encoding sequences from this culture revealed the presence of a single nucleotide substitution in gag that resulted in a threonine to isoleucine substitution at Gag position 371 (Fig. 1B). No revertant mutants could be obtained for HIV-1R18A or HIV-1K290A. This finding may reflect a greater magnitude of impairment of these particular substitutions, making the generation of revertant mutants more difficult.
To determine whether the T371I mutant rescued the infectivity defect present in HIV-1K359A, we generated a proviral clone, HIV-1K359A/T371I, encoding both mutations and measured the infectious virion yield from proviral plasmid-transfected 293T cells. Addition of the T371I substitution to HIV-1K359A restored infectious virion yield to wild-type levels (Fig. 1C). Although this second-site, apparently compensatory change was identified only in the context of HIV-1K359A, we asked whether the T371I substitution could rescue the HIV-1K290A, given purported similar roles of K290 and K359 in binding IP6. Indeed, we found that HIV-1K290A/T371I, unlike HIV-1K290A, yielded similar levels of infectious HIV-1 virions to wild-type HIV-1.
To determine whether the effects of the T371I mutant were evident outside the context of transfected 293T cells, we performed spreading replication assays of HIV-1WT, HIV-1T371I, HIV-1K359A, and HIV-1K359A/T371I in MT4 cells (Fig. 1D) and CEM cells (Fig. 1E). As expected, HIV-1K359A replicated poorly in both cell types. HIV-1T371I replicated poorly in CEM cells but well in MT4 cells, perhaps reflecting the greater permissiveness of MT4 cells. Importantly however, we found similar phenotypes for HIV-1K359A/T371I in spreading replication assays in both MT4 and CEM cells; namely, addition of the T371I substitution to HIV-1K359A restored replication, with the HIV-1K359A/T371I double mutant exhibiting only a modest delay compared to HIV-1WT in both cell types (Fig. 1D, E).
Infectious HIV-1
K359A/T371I
particle yield is not affected by reduction of IP
6
synthesis in virus producing cells
Because the HIV-1K359A is defective for IP6 binding we next asked whether HIV-1K359A/T371I retained infectiousness when cellular IP6 levels were reduced. Using CRISPR/Cas9 we generated 293T cell lines lacking IPMK, an enzyme in the IP6 synthetic pathway. Previous work has demonstrated IPMK knockout cells have greatly reduced levels of both IP5 and IP6 [13]. To account for potential clonal variation in capacity to generate HIV-1 particles, we used 3 separate IPMK targeting sgRNAs or a corresponding empty vector to generate ten independent single cell clones of IPMK knockout and WT control 293T cells (Fig. 2A). The loss of IPMK was confirmed by DNA sequencing of target loci, which revealed the introduction of frameshift mutations and the absence of intact IPMK alleles. In agreement with previous studies, the yield of infectious HIV-1WT virions from IPMK-deficient 293T cells was significantly decreased, by tenfold (p = 0.0091, Fig. 2A). The yield of HIV-1K359A from 293T cells was greatly reduced compared to wildtype HIV-1 as expected, and was not further reduced by IPMK deficiency (Fig. 2B). Yield of HIV-1T371I was also slightly reduced compared to wildtype but not impacted by IPMK deficiency (Fig. 2C, p = 0.1374). Importantly, the yield of HIV-1K359A/T371I was only marginally reduced compared to wild type HIV-1 and there was no difference in yield of infectious HIV-1K359A/T371I from WT 293T cells versus IPMK deficient 293T cells (Fig. 2D, p = 0.178).
As IP6 deficiency impacts both HIV-1 particle production as well as infectivity, we assayed particle levels in the same supernatants from Fig. 2A–D using detection of reverse transcriptase with the SYBR-PERT assay. Importantly, we see the same phenotype for particle production as we do with infectivity assays: production of HIV-1WT particles is impaired in IPMK KO cells, but there are no production deficits for HIV-1K359A, HIV-1T371I, or HIV-1K359A/T371I (Fig. 2E–H). However, while there was a tenfold reduction in HIV-1WT infectivity from IPMK-deficient 293T cells, we only observed a fivefold reduction in particle production in the exact same supernatants.
Infection of target cells with impaired IP
6
synthesis by HIV-1
WT
or HIV-1
K359A/T371I
It has been proposed that residues K290 and K359 recruit IP6 into HIV-1 virions during assembly, thereby providing the source of the IP6 that binds to and stabilizes the R18 ring in the mature capsid core. The rationale for this idea stems from previous studies which have demonstrated that reduction of cellular IP6 levels in target cells does not impact susceptibility to incoming infection [13, 14]. Because HIV-1K359A/T371I is fully infectious despite encoding a mutation that is predicted to diminish IP6 packaging into virions, we next asked whether HIV-1K359A/T371I requires IP6 in target cells to be maximally infectious. We generated twelve IPMK-deficient MT4 target cell clones and six control clones and performed single cycle infection assays using HIV-1WT and HIV-1K359A/T371I (Fig. 2D, E). In agreement with previous studies [13], there was no difference in the infectiousness of HIV-1WT in WT or IPMK-deficient MT4 cells (Fig. 2D p = 0.3863). Moreover, there was no deficit in the infectiousness of HIV-1K359A/T371I in WT or IPMK-deficient MT4 target cells (Fig. 2E, p = 0.4331), suggesting that HIV-1K359A/T371I either does not require IP6 for replication, or that the T371I mutation rescues both replication and IP6 incorporation.
Bevirimat rescues infectious virion formation by the IP
6
-binding deficient mutant HIV-1
K359A
Notably, The T371I mutation identified herein had been described previously in a different context. Specifically, this substitution was reported to stabilize the immature CA-SP1 lattice, mimicking the effect of maturation inhibitors (MI) [17, 18]. Therefore, we next asked whether maturation inhibitors themselves could rescue the deficit in infectious virion yield exhibited by HIV-1K359A. As a control, we included the previously described assembly-defective, maturation inhibitor-dependent CA mutant HIV-1P289S [18], We found that that BVM indeed rescued the infectivity of HIV-1K359A and HIV-1P289S in both single-cycle and spreading replication. Specifically, in single cycle assays, BVM increased the yield of infectious HIV-1K359A virions, up to 50-fold, and in a dose-dependent manner (Fig. 3A) from transfected 293T cells. In spreading replication assays, BVM restored HIV-1K359A replication to levels similar to that of BVM-treated wildtype virus in MT4 cells (Fig. 3B). BVM also rescued the spreading replication of HIV-1K359A in CEM cells, indeed in this context the effect of BVM on HIV-1K359A spreading was greater than that on the previously described MI-dependent mutant HIV-1P289S (Fig. 3C).
BVM increases release of HIV-1
K359A
virions independently of the viral protease
The interaction between K359 amines and IP6 likely stabilizes the immature Gag lattice, Similarly, maturation inhibitors are known to bind to the immature Gag hexamers at approximal site and stabilize the immature CA-SP1 lattice [19, 20]. Therefore, we hypothesized that BVM rescue particle formation by HIV-1K359A by stabilizing an otherwise destabilized lattice, effectively serving as a functional replacement for IP6. To test this idea, we measured the release of HIV-1K359A virions from BVM-treated 293T cells by western blotting. BVM indeed increased the yield of HIV-1K359A virions, in a dose-dependent manner (Fig. 4A). To confirm that this effect was not due to BVM-mediated inhibition of Gag proteolysis, we performed similar experiments in virions containing an inactivating mutation in protease. We observed a similar dose-dependent increase in immature particle release, even in the context of protease inactivation (Fig. 4B), suggesting that the effect of BVM on HIV-1K359A assembly is due to the direct effect of BVM on the immature lattice, not through inhibition of proteolytic cleavage at the Gag-SP1 junction.
Visualization of BVM-induced HIV-1 assembly observed in real time using live cell fluorescence microscopy
The above data strongly suggested that BVM rescues infectivity and release of HIV-1K359A by facilitating particle assembly. To directly observe effects on virion assembly, we performed fluorescence microscopy using a novel imaging construct based on HIV-1NL4-3, in which Pol has been replaced by an HIV-1 codon-mimicking mNeonGreen, and in which Env and Vpu bear inactivating mutations. The resulting construct, herein referred to as HIV-1 NG, generates Gag-mNeonGreen in place of Gag-Pol during a single cycle of infection, thus allowing visualization of particle assembly as punctae at the plasma membrane.
We generated HIV-1 vectors particle containing this reporter and derivatives (HIV-1 NGWT, HIV-1 NGK359A, and HIV-1 NGP289S) by supplying Gag-Pol and VSV-G in trans. Then, we infected TZM-bl cells in the absence or presence of 5 μM BVM and performed widefield imaging on fixed cells 48 h post infection. In the absence of BVM, cells infected with HIV-1 NGK359A and HIV-1 NGP289S exhibited primarily diffuse cytoplasmic fluorescence, and fewer punctae than for HIV-1 NGWT infected cells, indicating impaired assembly (Fig. 5A). However, when infections were done in the presence of BVM, there were clearly increased numbers of membrane associated punctae in HIV-1 NGK359A and HIV-1 NGP289S infected cells (Fig. 5A) suggesting BVM is able to directly facilitate particle assembly by these mutants.
This ability to induce HIV-1 particle assembly via addition of an exogenous small molecule has potential applications in imaging and other studies, as an inducible particle assembly system. In order to test the possible utility of this approach, we performed live cell widefield imaging studies using HIV-1 NGP289S, as this mutant displayed a greater responsiveness to BVM-induced assembly (see Figs. 3A, B, 4B, and 5A). We infected TZM-bl cells in the absence of BVM and then, at 26 h after infection, added BVM 5 μM and began acquiring images at 30 min intervals. In the absence of BVM, few punctae are apparent in HIV-1 NGP289S infected cells, even after 12 h of imaging (Fig. 5B, Additional files 1, 2). However, in the BVM-treated cells, substantially more punctae were evident, as soon as 30 min after BVM addition (Fig. 5B, Additional files 3, 4).
Given that such striking differences could be observed as early as 30 min post BVM addition, we repeated these experiments with increased time resolution. TZM-bl cells were infected with HIV-1 NGP289S for 26 h and treated with BVM as above, followed by immediate image acquisition at 3 min intervals. Assembly of HIV-1P289S was rapidly induced by BVM (Fig. 5C, Additional files 5, 6) with substantial numbers of punctae forming within 30–120 min. We performed similar experiments using TIR-FM imaging and quantified the presence of Gag-NG punctae over time, with similar results. Specifically, in the absence of BVM there were very few punctae evident at the plasma membrane (Fig. 6A, B, Additional files 7, 8). However, shortly after the addition of BVM, numerous punctae rapidly formed at the plasma membrane (Fig. 6, Additional files 9, 10). These data provide proof of principle that such a system could be used to experimentally manipulate HIV-1 assembly for imaging or functional studies.