Volume 10 Supplement 1
A second generation HIV-IN-EGFP fluorescent viral system to analyze HIV-1 in the nuclear compartment of infected cells
© Francis et al; licensee BioMed Central Ltd. 2013
Published: 19 September 2013
To study the nuclear biology of HIV-1 we recently developed a microscopy based fluorescent HIV-IN-EGFP system . HIV-IN-EGFP exploits the Vpr mediated trans-incorporation to incorporate IN-EGFP in viral particles made with pNL-IN-D64E. The ability to visualize HIV-IN-EGFP within the nucleus makes it an attractive tool to study quantitatively the nuclear import step of HIV-1 [2, 3]. In this work we provide new evidence for efficient visualization of HIV-1 complexes in the nuclei of infected cells through a new optimized system.
In order to optimize HIV-IN-EGFP infectivity various modifications were introduced into the visualization system. The functional tetramerization of IN mutations E11K and K186E  was introduced into Vpr-IN-EGFP constructs and nuclear detection of functional tetramers of HIV-IN-EGFP was evaluated in Hela-P4 cells. In addition the EGFP was replaced in Vpr-IN with super-folder GFR. Infectivity of HIV-IN-EGFP was verified in 293T cells at 3 days post infection.
Since the HIV-IN-EGFP visualization system  is produced by IN-EGFP trans-incorporation through Vpr, we have better analyzed the impact of this manipulation on viral infectivity. We observed that Vpr-IN-EGFP molecules are able to efficiently complement D64E mutant integrase for infectivity, however the infectivity values of trans-incorporated viruses are low with respect to the NL4.3 virus. Through trans-complementation of NL4.3 virus we find that incorporation of IN alone or fused with EGFP has a lethal effect in native wild type virions by severely affecting their infectivity. Further, by modifying the tetramerization properties of IN-EGFP  we attempted to improve viral infectivity. In fact, as previously suggested by Hare et al. , trans-incorporation of integrase molecules mutated in the N-terminus (E11K) and catalytic domain (K186E) induces stabilization of the integrase tetramer leading to a more functional complex. Confocal microscopic analysis revealed that functional tetramerization of IN-EGFP results in a loss of GFP fluorescence that can be recovered through the use of brighter fluorophores such as the super-folder GFP. Finally, the improved infectivity of the trans-incorporated viral particles was complemented with a dual labeling, which allows monitoring the different monomers within the integrase complex.
Until recently the nuclear biology of HIV-1 has been investigated through molecular techniques, with the development of HIV-IN-EGFP it is possible to assess the localization of HIV-1 in the nuclear compartment. This technique allowed the first determination that HIV-1 localizes in the de-condensed regions of the chromatin and near the nuclear rim . In this work we exploited improved tetramerization properties of integrase  to develop a second-generation HIV-IN-EGFP, which preserves the same imaging properties as the first-generation even though the infectivity is significantly improved.
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.