Generation of bioluminescent HIV-1 reporter vectors.
To generate an HIV-1 reporter virus that enables quantification of the reporter protein directly from virus supernatant, we employed the previously reported strategy (4, 5) of introducing the reporter gene between the MA and CA domains of Gag, a location shown to tolerate genetic insertions with minimal effects on Gag protein expression and processing (4). The NLuc gene was introduced between MA and CA domains of HIV-1 Gag flanked by PR cleavage sites at the N-terminus or C-terminus, or both termini of NLuc or not flanked by any PR cleavage site (Fig. 1A). Upon PR-mediated Gag cleavage, the resulting NLuc products generated from these vectors are the NLuc protein and MA-NLuc, NLuc-CA and MA-NLuc-CA fusion proteins, respectively (Fig. 1A).
To assess the effect of the NLuc insertion into HIV-1 Gag on Gag protein expression and PR-mediated Gag cleavage, we transfected the HIV-1 Gag-NLuc vectors into HEK293T cells and after 48hrs, lysed the cells and pelleted virus from the supernatant. We performed western blot analysis to evaluate the expression of HIV-1 Gag-NLuc fusion proteins in the cells and the pelleted virus. We observed no adverse effect on Gag expression and processing and, importantly, the expected Gag PR cleavage products were generated, including some cleavage intermediates (Fig. 1B). We also measured NLuc activity from the cell lysate and virus supernatant and observed that all the vectors yielded robust NLuc activity; i.e., up to 2.0x107 relative light units (RLUs) (Fig. 1C). Finally, we calculated virus release efficiency (VRE) from the same samples and found release efficiency was similar to that of WT Gag and the Gag-iGFP construct (Fig. 1D).
HIV-1 Gag-iNLuc enables highly sensitive measurement of virus release.
To assess the sensitivity in the detection of virus release using the HIV-1 Gag-iNLuc vector, we transfected HEK293T cells with either an NLuc expression vector, the WT HIV-1 molecular clone pNL4-3, or decreasing amounts of the HIV-1 Gag-iNLuc vector (i.e. 1.0, 0.5, 0.25, 0.125 and 0.0625mg). At 48hrs post-transfection, we lysed the cells and purified virions from the supernatant, analyzed Gag levels by western blot (Fig. 2A), and measured NLuc activity from the cell lysates and supernatants (Fig. 2B). We were able to detect NLuc signal under all conditions tested, including at the lowest DNA input at which virion-associated Gag was undetectable by western blot. pNL4-3 Gag-iNLuc vector-transfected cells produced significantly higher levels of NLuc activity in both the cell lysate (>10-fold) and supernatant (>1000-fold) relative to the NLuc expression vector control. This implies that the NLuc activity in the supernatant is derived from the NLuc protein released with the HIV-1 Gag during virus release. We also measured RT activity (Fig. 2C) and p24 protein levels (Fig. 2D) in the virus supernatant and correlated both with supernatant NLuc activity (Fig. 2E & F). We observed that the supernatant NLuc activity was positively correlated with RT activity and p24 abundance, further reinforcing the specificity of the assay.
The defect in HIV-1 Gag-iNLuc particle infectivity can be rescued by co-expression with WT.
We generated virus using either WT pNL4-3, the HIV-1 Gag-iGFP or the HIV-1 Gag-iNLuc vectors by transfecting them into HEK293T cells and collecting the supernatants containing the progeny virions at 48hrs post-transfection. We quantified the relative amounts of virus in the supernatant by RT activity (Fig. 3A). We observed that RT activity of supernatants from cells transfected with the HIV-1 Gag-iGFP and HIV-1 Gag-iNLuc vectors was about 2-fold less than that of supernatants from cells transfected with WT pNL4-3. To test the infectivity of the virions produced from the HIV-1 Gag-iNLuc vectors, we infected TZM-bl cells with the RT-normalized virus supernatants and measured the infectivity by quantifying the firefly luciferase activity (Fig. 3B). We observed that the HIV-1 Gag-iNLuc viruses were approximately 10-fold less infectious than the WT virus. We also transfected the SupT1 T-cell line with the HIV-1 Gag-iNLuc vectors and monitored virus replication kinetics over several days and observed that replication was significantly impaired compared to the WT HIV-1 (data not shown). We generated viruses using the pNL4-3 Gag-iNLuc vector complemented with different ratios of the WT HIV-1 molecular clone pNL4-3 and tested their infectivity. We observed that infectivity of the viruses generated with the pNL4-3 Gag-iNLuc vector was rescued when complemented with the WT pNL4-3 vector. The infectivity increased with increasing pNL4-3:pNL4-3 Gag-iNLuc ratio; at ratios above 2:1 the infectivity was at WT HIV-1 levels (Fig. 3C).
HIV-1 Gag-iNLuc provides a robust tool for quantifying virus release.
To examine the utility of the HIV-1 Gag-iNLuc vector in functional assays for virus release, we constructed versions of the vector that lacked the PTAP motif in the p6 domain of the HIV-1 Gag protein (HIV-1 Gag-iNLuc-PTAP- ) and the vpu gene (HIV-1 Gag-iNLuc-delVpu) by cloning the iNLuc cassette into previously reported PTAP- and delVpu HIV-1 molecular clones (11, 12). The p6 domain of HIV-1 Gag is required for virus release (12, 13) because of its interaction with the ESCRT machinery (14-17). Vpu is also required for HIV-1 release in the presence of the restriction factor tetherin (also known as BST-2), which blocks release of virions by tethering them to the plasma membrane (18). Vpu counteracts tetherin by targeting it for proteasomal degradation (19). We transfected HEK293T cells with the WT, PTAP- and delVpu versions of the HIV-1 Gag-iNLuc vector with or without varying amounts of tetherin expression vector. At 48hrs post-transfection, we measured NLuc activity in the cell lysates and supernatants. We observed a 2-fold decrease in the NLuc activity in the supernatant of cells transfected with the PTAP- vs. the WT vector, but, as expected, no decrease in NLuc activity in the cell lysates. Likewise, co-transfection with a tetherin expression vector, but not an empty vector control, caused a 4- to 10-fold decrease in NLuc activity in the supernatant of cells transfected with the delVpu vector. The decrease in supernatant NLuc activity was proportional to the amount of tetherin vector transfected (Fig.4A and B). We performed western blot analysis of the cell lysates and the pelleted virions to analyze HIV-1 Gag expression. We observed that virus release measured by virion-associated p24 levels corresponded with the NLuc activity. Finally, we tested the utility of the HIV-1 Gag-iNLuc vector to detect impaired virus release induced by treatment of virus-producer cells with amphotericin B methyl ester (AME), a compound that inhibits HIV-1 particle production (20). We transfected HEK293T cells with the HIV-1 Gag-iNLuc vector and at 24hrs post-transfection treated the cells with either vehicle or increasing amounts (5mM or 10mM) of AME. At 24hrs post-treatment, we collected the supernatant and measured NLuc activity. We observed a decrease in supernatant but not cell-associated NLuc activity in the presence of AME but not vehicle control. Again, the decrease in supernatant NLuc activity corresponded with reduced virion-associated p24 measured by western blot analysis. These results demonstrate that the Gag-iNLuc vector provides a highly sensitive and quantitative tool for measuring the effects of Gag mutations, host cell restriction factors, and small molecule inhibitors on HIV-1 particle assembly and release.