Occasional spikes in fluorescence (for example, at the 38?min time point) are due to a transient overlap of the particle of interest with either another particle or with cells autofluorescent features YFP-Vpr released from a post-fusion core accumulates in the nucleus Since Vpr has two nuclear localization signals , the YFP-Vpr marker released from post-fusion cores is expected to enter the nucleus. nucleus. More than 10,000 Vpr molecules can be delivered into the cell nucleus within 45?min of infection by HIV-1 particles pseudotyped with the avian sarcoma and leukosis virus envelope glycoprotein. The fraction of Vpr from cell-bound viruses that accumulated in the nucleus was proportional to the extent of virus-cell fusion and was fully blocked by viral fusion inhibitors. Entry of virus-derived Vpr into the nucleus occurred independently of envelope glycoproteins or target cells. Fluorescence correlation spectroscopy revealed two forms of nuclear Vprmonomers and very large complexes, likely involving host factors. The kinetics of viral Vpr entering the nucleus after fusion was not affected by point mutations in the capsid protein that alter the stability of the viral core. Conclusions The independence of Vpr shedding of capsid stability and its relatively rapid dissociation from post-fusion cores suggest that this process may precede capsid uncoating, which appears to occur on a slower time scale. Our results thus demonstrate that a bulk of fluorescently labeled Vpr incorporated into HIV-1 particles is released shortly after fusion. Future studies will address the question whether the quick and efficient nuclear delivery of Vpr derived from incoming viruses can regulate subsequent steps of HIV-1 infection. Electronic supplementary material The online version of this article (doi:10.1186/s12977-015-0215-z) contains supplementary material, which is available to authorized users. in a and d). show Veliparib dihydrochloride the boundaries of cell nuclei. b, c Fluorescence intensity profiles (total fluorescence of YFP-Vpr and Gag-imCherry) obtained by single ASLVpp tracking in CV-1-derived cells. e, f Fluorescence intensity profiles for YFP-Vpr and Gag-imCherry obtained by single ASLVpp tracking in an A549-derived cell. g An example of YFP-Vpr and Gag-imCherry signals from a non-fusing particle selected from an experiment carried out in the presence of the ASLV fusion inhibitor R99 (50?g/ml). outline different YFP decay profiles occurring without (c, HYAL2 e) and with a lag (b, f) after the release of mCherry. Here and in Fig.?2, the abrupt ending of fluorescence traces occurs due to the inability to track faint YFP/GFP-Vpr puncta using particle tracking Veliparib dihydrochloride software, as the signal approaches the background level Interestingly, the initial increase in the YFP-Vpr signal at the time of fusion with CV-1- or A549-derived cell lines was followed by fluorescence decay over the course of several minutes (Fig.?1aCf). All single ASLVpp that we were able to track in these two cell lines, using tracking software or by visual observation (370 particles total), lost YFP-Vpr within about 15C20?min after fusion (Fig.?1aCf). This characteristic gradual decrease in the YFP signal after fusion has also been observed in our previous study . The loss of YFP-Vpr was not caused by photobleaching, since the mCherry and YFP signals from non-fusing particles did not change considerably throughout the imaging experiments (Fig.?1g). Also, because post-fusion viral cores are expected to Veliparib dihydrochloride reside in the cytosol, acidification of the viral interior as the reason for the vanishing YFP signal can also be ruled out. The YFP-Vpr decay started either immediately (Fig.?1c, e) or several minutes after the release of mCherry (compare Fig.?1b, f). A delayed decay of YFP-Vpr fluorescence suggests the existence of an additional post-fusion step that triggers dissociation of YFP-Vpr from the viral core. Single virus tracking demonstrated that a gradual loss of YFP-Vpr signal after viral fusion was universally observed for particles pseudotyped with HXB2 Env glycoprotein (Fig.?2). As observed previously, the pH-independent fusion mediated by HXB2 Env occurred at delayed time-points after initiation of entry, compared to low pH-triggered fusion mediated by VSV-G or ASLV Env ([10, 29C31] and see below). However, in all cases, the formation of the fusion pore was manifested in an abrupt loss of mCherry and transient increase in the YFP-Vpr signal followed by a slow decay (Figs.?1, ?,22). Open in a separate window Fig.?2 Loss of YFP-Vpr after viral fusion mediated by HXB2 envelope glycoprotein. a Snapshots of entry and fusion of an HXB2 Env-pseudotyped particle co-labeled with YFP-Vpr (traces show sum fluorescence of mCherry and GFP channels, respectively, obtain by tracking the virus shown in a. For comparison, fluorescence intensities of mCherry and GFP for a non-fusing particle are shown (traces, respectively). c Single virus tracking results of another fusing VSVpp. Occasional spikes in fluorescence (for example, at the 38?min time point) are due to a transient overlap of the particle of interest with either another particle or with cells autofluorescent features YFP-Vpr released from a Veliparib dihydrochloride post-fusion core accumulates in the nucleus Since Vpr has two nuclear localization signals , the YFP-Vpr marker released from post-fusion cores is expected to enter the nucleus. Indeed, progressive YFP-Vpr accumulation in the nuclei was observed within 45?min incubation of ASLVpp and cell at 37?C (Fig.?4a; see also Additional file 1: Movie 1). Spatial redistribution of Gag-imCherry and YFP-Vpr as time passes is definitely obvious through the linear.