Recent advances in fluorescence microscopy provided tools for the investigation and the analysis of the viral replication steps in the cellular context. In the HIV field, the current visualization systems successfully achieve the fluorescent labeling of the viral envelope and proteins, but not the genome. Here, we developed a system able to visualize the proviral DNA of HIV-1 through immunofluorescence detection of repair foci for DNA double-strand breaks specifically induced in the viral genome by the heterologous expression of the I-SceI endonuclease. The system for Single-Cell Imaging of HIV-1 Provirus, named SCIP, provides the possibility to individually track integrated-viral DNA within the nuclei of infected cells. In particular, SCIP allowed us to perform a topological analysis of integrated viral DNA revealing that HIV-1 preferentially integrates in the chromatin localized at the periphery of the nuclei.T echnical developments in imaging-based techniques have greatly improved our understanding of HIV-host cell interactions. HIV-1 virions labeled with fluorophores were pivotal in shedding light onto multiple aspects of the virus-host interplay during all steps of HIV-1 replication cycle (1-13). Nevertheless, few optical approaches have been so far developed to visualize viral particles within the nuclear compartment (14, 15), which limits our comprehension of the interaction between HIV-1 and the nuclear architecture. Moreover, the existing detection tools are based on the visualization of the viral protein complexes or envelope but not of the viral DNA with the only exception of the fluorescence in situ hybridization (FISH) technique. Even though FISH is a powerful technique, it is not very sensitive for HIV-1 detection and moreover disrupts the native architecture of the nuclear compartment as it requires harsh denaturation conditions. In addition, this technique does not allow the discrimination between integrated and nonintegrated viral DNA (16, 17). Here we describe a fluorescent approach to visualize HIV-1 DNA in the nuclear compartment of infected cells. We exploited a site-specific genome engineering technique that represents one of the most promising approaches to detect specific genome regions in modified organism (18) allowing for their spatial localization in the cell (19,20). This technique couples endogenous repair pathways, induced by rare cutting endonuclease, with immunofluorescence analysis. Rare cutting endonucleases, such as the yeast-homing endonuclease I-SceI, specifically cuts target sequences that cannot be found in the mammalian genome. By engineering DNA to contain the I-SceI cleavage site, it is thus possible to induce endogenous repair mechanism for double-strand breaks (DSBs) at specific genomic positions. DSB repair leads to the formation of distinct subnuclear structures that are generally referred to as "foci" (21). The first sensor of the DSB is the histone H2AX, which becomes massively phosphorylated at serine 139 (γ-H2AX). Foci of DNA repair are thus visible through immu...

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GFP mutants are known to display fluorescence flickering, a process that occurs in a wide time range. Because serine 65, threonine 203, glutamate 222, and histidine 148 have been indicated as key residues in determining the GFP fluorescence photodynamics, we have focused here on the role of histidine 148 and glutamate 222 by studying the fluorescence dynamics of GFPmut2 (S65A, V68L, and S72A GFP) and its H148G (Mut2G) and E222Q (Mut2Q) mutants. Two relaxation components are found in the fluorescence autocorrelation functions of GFPmut2: a 10-100 micros pH-dependent component and a 100-500 micros laser-power-dependent component. The comparison of these three mutants shows that the mutation of histidine 148 to glycine induces a 3-fold increase in the protonation rate, thereby indicating that the protonation-deprotonation of the chromophore occurs via a proton exchange with the solution mediated by the histidine 148 residue. The power-dependent but pH-independent relaxation mode, which is not affected by the E222Q and H148G mutations, is due to an excited-state process that is probably related to conformational rearrangements of the chromophore after the photoexcitation, more than to the chromophore excited-state proton transfer.

New probes for kinetic intracellular measurements in the millisecond range are desirable to monitor protein biochemical dynamics essential for catalysis, allosteric regulation, and signaling. Good candidates to this aim are the photoswitchable mutants of the green fluorescent protein, whose anionic fluorescence, primed by blue light, is markedly enhanced under an additional excitation at a shorter wavelength and relaxes within a few milliseconds. The aim of this report is to study how the brightness enhancement kinetics depends on the physical-chemical and spectroscopic parameters and to provide proof-of-concept experiments for the use of the fluorescence enhancement in conditions in which the protein diffusion is hindered and thereby photobleaching can be a limiting critical issue. Future, direct applications of photochromic mutants for modulated excitation imaging would in fact require such a detailed knowledge. We present here an extensive study of the photoswitching mechanism of the E222Q mutant of GFPMut2 (Mut2Q), pumped by visible 488 nm light and probed at 400-420 nm, as a function of pH, viscosity, temperature, and light intensity. In solution, two characteristic photoswitching times are found by means of modulated double beam fluorescence correlation spectroscopy in the 1-30 ms range, depending on the solution pH. The photoswitching kinetics is solved in terms of the eigenvalues and the eigenvectors of a specific energy diagram and used directly to fit the data, suggesting that the observed photoswitching amplitudes and kinetics are related to a single three-level transition loop. Finally, we give in vitro examples of the use of modulated excitation microscopy, based on fluorescence enhancement amplitude and kinetics detection, on Mut2Q protein samples immobilized in acrylamide gels.

The ability to visualize fluorescent HIV-1 particles within the nuclei of infected cells represents an attractive tool to study the nuclear biology of the virus. To this aim we recently developed a microscopy-based fluorescent system (HIV-IN-EGFP) that has proven valid to efficiently visualize HIV-1 complexes in the nuclear compartment and to examine the nuclear import efficiency of the virus. Detailed confocal microscopy analysis revealed that the newly generated viral particles resulted in HIV-1 complexes significantly smaller in size, thus requiring the use of brighter fluorophores for nuclear visualization [HIV-IN(K)sfGFP_IN(E)]. The second-generation visualization system HIV-IN(K)sfGFP_IN(E), in addition to allowing direct visualization of HIV-1 nuclear entry and other viral events related to nuclear import, preserves intact viral properties in terms of nuclear entry and improved infectivity.