please help summary Results Labeling HIV-1 Capsids with a GFP Fluid Phase Marker. To determine whether viral cores in infected cells retained their integrity, we utilized a previously described HIV-1 vector in which GFP was inserted between matrix (MA) and CA (pNL4-3 Gag-iGFP) (35) (Fig. 1A). Proteolytic processing during virion maturation releases GFP from the Gag precursor, some of which remains trapped inside the capsid and is released upon loss of core integrity (Fig. 1B). We also utilized a previously described RNA stem-loop system in which 18 copies of BglG stem loops (BglSLs) were engineered into the HIV-1 genome in place of vif/vpr to facilitate the detection of nascent HIV-1 genomic RNA at the HIV-1 transcription site (Fig. 1A). This system can be used to identify the capsids that lead to productive infection and determine the nuclear location of integration (25). Virions were produced by cotransfection of an HIV-1 vector expressing Gag-iGFP and a vector expressing wild-type gag-pol at a 1:2 ratio (Fig. 1A). Western blot analysis of these virions indicated that nearly all GFP was fully processed from Gag-iGFP and the processing efficiency was similar to that of CA from Gag (Fig. 1 C and D). Treatment of iGFP-labeled virions with saponin detergent in vitro to disrupt the viral membrane reduced the GFP signals to 16.9 ± 11.3% compared to the GFP intensities of intact virions, indicating that nearly ∼20% of the GFP remained inside the capsids (Fig. 1 E and F). The virions retained almost half of their infectivity compared to control virions produced in the absence of iGFP (Fig. 1G). The morphology of iGFP-labeled and unlabeled virions was similar and the ratio of virions with mature and immature morphology was not significantly different (Fig. 1 H and I). To determine whether GFP-labeled capsids were detectable after nuclear import, we generated virions that were colabeled with GFP content marker and core-associated host restriction factor APOBEC3F (A3F) fused to fluorescent protein red-red vine tomato (RRvT) (25). Single virion analysis of HIV-1 particles immunostained with anti-CA antibody showed that most A3F-RRvT+ p24+ virions were labeled with GFP (∼88%, Fig. 1 J and K). In contrast to a previous report indicating the GFP content marker is lost in the cytoplasm ∼25 min after fusion (36), we observed many GFP-labeled capsids at the NE and inside the nucleus at 6 h postinfection (hpi) (Fig. 1 L and M). The percentage of A3F-RRvT-labeled RTCs/PICs in the nucleus at 6 hpi that were GFP+ was ∼47%, indicating many of the capsids that had entered the nucleus remained intact. Interestingly, ∼47% (762/1,608 × 100%) of the A3F-RRvT+ capsids also had detectable GFP content marker levels shortly after in vitro saponin treatment (SI Appendix, Fig. S1A), suggesting that the GFP content marker levels in ∼53% of the nuclear capsids were below the limit of detection. To further analyze the capsid labeling efficiency, we divided the 1,608 GFP+ intact virions into four quartiles based on their GFP intensity and determined the percentage of capsids that had detectable levels of GFP intensity for each quartile (SI Appendix, Fig. S1B). The GFP intensities of the capsids (SI Appendix, Fig. S1B) and the proportion of GFP+ capsids were correlated with the GFP intensities of the intact virions (SI Appendix, Fig. S1C), strongly suggesting that the ∼47% frequency of GFP+ nuclear capsids was expected based on their labeling and detection efficiency. The percentages of A3F-RRvT-labeled RTCs/PICs at the NE and inside the nucleus at 6 hpi were similar for GFP-labeled and unlabeled virions, indicating that GFP labeling of the capsids did not affect their efficiency of docking with the NE or their nuclear import (Fig. 1N).
please help summary
Results Labeling HIV-1 Capsids with a GFP Fluid Phase Marker. To determine whether viral cores in infected cells retained their integrity, we utilized a previously described HIV-1 vector in which GFP was inserted between matrix (MA) and CA (pNL4-3 Gag-iGFP) (35) (Fig. 1A). Proteolytic processing during virion maturation releases GFP from the Gag precursor, some of which remains trapped inside the capsid and is released upon loss of core integrity (Fig. 1B). We also utilized a previously described RNA stem-loop system in which 18 copies of BglG stem loops (BglSLs) were engineered into the HIV-1 genome in place of vif/vpr to facilitate the detection of nascent HIV-1 genomic RNA at the HIV-1 transcription site (Fig. 1A). This system can be used to identify the capsids that lead to productive infection and determine the nuclear location of integration (25). Virions were produced by cotransfection of an HIV-1 vector expressing Gag-iGFP and a vector expressing wild-type gag-pol at a 1:2 ratio (Fig. 1A). Western blot analysis of these virions indicated that nearly all GFP was fully processed from Gag-iGFP and the processing efficiency was similar to that of CA from Gag (Fig. 1 C and D). Treatment of iGFP-labeled virions with saponin detergent in vitro to disrupt the viral membrane reduced the GFP signals to 16.9 ± 11.3% compared to the GFP intensities of intact virions, indicating that nearly ∼20% of the GFP remained inside the capsids (Fig. 1 E and F). The virions retained almost half of their infectivity compared to control virions produced in the absence of iGFP (Fig. 1G). The morphology of iGFP-labeled and unlabeled virions was similar and the ratio of virions with mature and immature morphology was not significantly different (Fig. 1 H and I). To determine whether GFP-labeled capsids were detectable after nuclear import, we generated virions that were colabeled with GFP content marker and core-associated host restriction factor APOBEC3F (A3F) fused to fluorescent protein red-red vine tomato (RRvT) (25). Single virion analysis of HIV-1 particles immunostained with anti-CA antibody showed that most A3F-RRvT+ p24+ virions were labeled with GFP (∼88%, Fig. 1 J and K). In contrast to a previous report indicating the GFP content marker is lost in the cytoplasm ∼25 min after fusion (36), we observed many GFP-labeled capsids at the NE and inside the nucleus at 6 h postinfection (hpi) (Fig. 1 L and M). The percentage of A3F-RRvT-labeled RTCs/PICs in the nucleus at 6 hpi that were GFP+ was ∼47%, indicating many of the capsids that had entered the nucleus remained intact. Interestingly, ∼47% (762/1,608 × 100%) of the A3F-RRvT+ capsids also had detectable GFP content marker levels shortly after in vitro saponin treatment (SI Appendix, Fig. S1A), suggesting that the GFP content marker levels in ∼53% of the nuclear capsids were below the limit of detection. To further analyze the capsid labeling efficiency, we divided the 1,608 GFP+ intact virions into four quartiles based on their GFP intensity and determined the percentage of capsids that had detectable levels of GFP intensity for each quartile (SI Appendix, Fig. S1B). The GFP intensities of the capsids (SI Appendix, Fig. S1B) and the proportion of GFP+ capsids were correlated with the GFP intensities of the intact virions (SI Appendix, Fig. S1C), strongly suggesting that the ∼47% frequency of GFP+ nuclear capsids was expected based on their labeling and detection efficiency. The percentages of A3F-RRvT-labeled RTCs/PICs at the NE and inside the nucleus at 6 hpi were similar for GFP-labeled and unlabeled virions, indicating that GFP labeling of the capsids did not affect their efficiency of docking with the NE or their nuclear import (Fig. 1N).
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