Fig 1.
N- or C-terminally GFP labeled CA proteins are efficiently processed in virions and remain associated with HIV-1 replication complexes in the nucleus.
(A) Schematic of C-terminal tagging of CA through a MA-CAeGFP fusion construct. MA drives co-assembly and virion incorporation of MA-CAeGFP with unlabeled Gag/GagPol encoded by the HIV-1 backbone. The protease cleavage site (PC) between MA and CA releases mature fluorescent CAeGFP upon virus maturation. The Vpr-INsfCherry fusion construct supplied in trans is incorporated via interaction with the p6 domain of unlabeled Gag. The resulting viral particles co-labeled with CAeGFP and INsfCherry contain a mixture of native and eGFP-labeled CA proteins. (B) Schematic of CA tagging strategy reported by Burdick et al., PNAS 2020 based on the N-terminal GFP-CA fusion protein expressed in the context of full-length Gag/GagPol. (C) Efficiency of HIV-1 co-labelling by GFP-tagged CA and INmCherry. The % colocalization of CAeGFP with INmCherry (+/- SD from 5 experiments) is shown in gold. Scale bar is 2 μm. (D, E) Western blot of viruses labeled with CAeGFP or HGFP-CA showing efficiently processed Gag/GagPol probed with anti-HIV IgG (D) and release of GFP-tagged CA fusion proteins probed with anti-GFP antibodies (E). (F) Specific infectivity of HIV-1 virions labeled with the different GFP-tagged CA constructs was determined in TZM-bl cells. Viruses were normalized for RT activity and infectivity was determined by luciferase assays in triplicate at 48 hpi. The % infectivity of HGFP-CA and CAeGFP labeled viruses was determined by setting luciferase relative light units (RLUs) of unlabeled HIV-1 to 100% for each experiment. The mean infectivity and SEM from 5 experiments is shown. (G) Fluorescence intensity distribution of GFP-tagged CA proteins associated with INmCherry puncta in intact virions, after virus membrane permeabilization with saponin (cores) and at 4 hpi in the cytoplasm and nucleus. The average object-based colocalization of INsfCherry and GFP-tagged CA from 5 independent experiments is shown above each condition. (H) Single (top panel) and merged (bottom panel) images (1 μm projection of the central plane) showing the distribution of GFP-tagged CA (green) and colocalized INsfCherry (red) labeled HIV-1 VRCs at 12 hpi in the nucleus of TZM-bl cells that stably express SNAP-Lamin (blue). Cells were infected at MOI 0.5. Quantitation of eGFP-intensities in nuclear VRCs between 4 (G) and 12 h (H) are shown in related S1B Fig. Scale bar is 5 μm. Statistics: In (F), data were analyzed using student t-test; in (G), a non-parametric Mann-Whitney Rank Sum test was used. p-values: **<0.01 and ***<0.001.
Fig 2.
GFP-tagged CA is not accessible on the capsid lattice for host-factor interactions.
(A) Strategy to test the localization of viral core-associated GFP-tagged CA. The ability of GFP-tagged CA to render N74D CA virions sensitive to cytosolic CPSF6-358 in TZM-bl cells. Three possible scenarios are illustrated: (i) In the absence of WT CA (tagged or untagged), N74D CA does not bind to CPSF6-358 and therefore resists restriction. (ii) If GFP-tagged WT CA incorporates into the capsid shell consisting of the N74D CA mutant, it is expected to interact with CPSF6-358 and restrict N74D virus infectivity. (iii) Mis-localization of GFP-tagged CA, e.g. with the vRNP inside the viral core, will not sensitize N74D virus to CPSF6-358. (B) Single-round infectivity data shows that GFP-tagged CA is inaccessible to CPSF6-358, while 10% unlabeled WT CA in N74D viruses efficiently interacts with CPSF6-358 resulting in restriction of N74D infectivity. As controls, HIV-1 with 100% unlabeled WT or mutant CA or a mixture of 10% unlabeled WT (psPax2) and 90% N74DCA were used. Infectivity is normalized to that in control cells that do not express CPSF6-358. The average values from 4 experiments and STD are shown. Single Z-slice images (C) and quantification (D) show the lack of nuclear penetration of WT or N74D CA VRCs labeled with N- (HGFP-CA) or C-terminally (CAeGFP) tagged WT CA. Note, N74D CA VRCs labeled with eGFP-tagged CA remain at the nuclear periphery (top left- and middle panels). Control infections with 10% of unlabeled CA (psPAX2) is able to mediate penetration of INsfGFP-labeled N74D VRCs (marked by white dashed circles, top right panel). The distribution of eGFP-tagged CA and INsfGFP-labeled VRCs of HIV-1 WT CA is shown for comparison (bottom panels). Scale bar in (C) is 5 μm. The distribution of fluorescent HIV-1 puncta in (D) is plotted as mean and SEM for >120 nuclei from 3 experiments. Statistics: in (B) student’s t-test was used; data in (D) were analyzed using a non-parametric Mann-Whitney Rank Sum test.
Fig 3.
A subset of GFP-tagged CA localizes inside the conical capsid core.
(A) A strategy to test localization of GFP-tagged CA in single virions. HIV-1 co-labeled with CAeGFP and CypA-DsRed (CDR) are bound to a poly-lysine coated glass (left). Following membrane permeabilization with saponin (SAP; 100 μg/ml) the major fraction of un-polymerized fluid-phase CAeGFP is released, while CDR remains associated with cores through high avidity binding (second from left). Addition of CsA (5 μM) displaces surface-accessible CDR from cores, whereas CDR trapped inside intact cores by virtue of association with CAeGFP in vRNPs is unaffected (second from right) and is displaced only after loss of core integrity or after the initiation of uncoating (right). (B, C) Images of co-labeled WT CA (B) or K203A CA (C) containing HIV-1 before (left) and after treatment with SAP (middle) or SAP + CsA (right). Scale bars in (B) and (C) are 2 μm. (D, E) Analysis of CAeGFP (D) and CDR (E) fluorescence in single virions, after virus membrane permeabilization (+SAP) and after CsA (+SAP+CsA) treatment. The background in eGFP (50 a.u.) and CDR (80 a.u.) channels are marked by dashed blue lines. Data in (D and E) are mean and SEM of the cumulative distribution of fluorescent intensities from 4 experiments. Statistics in (D and E) non-parametric Mann-Whitney Rank Sum test, ns p>0.05; * p<0.05; ** p<0.01 and *** p<0.001.
Fig 4.
A subset of native CA co-precipitates with HIV-1 vRNP proteins.
Virus lysates were subject to IP with antibodies against vRNP components IN, RT, and NC proteins. As controls, anti-CA and anti-gp120 envelope antibodies were used for IP. Mock IP was performed without virus lysate to control for possible secondary antibody bands. Immuno-blots were probed with anti-CA Hyb183 antibody (top) to detect CA/p24 or with HIV-IgG human serum (bottom) to identify HIV-1 proteins based on their respective molecular weight. Viral lysate (2.5 μg p24) was loaded in the last well to detect all viral components. HIV-IgG blots are cropped to show distinct RT, IN, and MA proteins. The fullsize blot is shown in S5 Fig. A representative immunoblot from 4 independent pulldown experiments is shown. In all experiments, a faint CA/p24 band co-immunoprecipitated with vRNP proteins.
Fig 5.
HIV-1 IN becomes accessible at the nuclear pore prior to the nuclear import of VRCs.
Nuclear import and infectivity of VSV-G pseudotyped HIV-1 labeled with INmCherry (red) was tested in MT4 cells stably expressing the dominant-negative NETC-GFP-IBD wild-type (WT) and mutant D366N fusion proteins localized to the nuclear pore complex. (A) Illustration of nuclear pore-associated NETC-GFP-IBD protein binding to INsfCherry and blocking HIV-1 nuclear import. (B) Representative central Z-slice images of MT4 cell nuclei showing the localization of NETC-GFP-IBD constructs (green) and INsfCherry puncta (red). Nuclear INsfCherry VRCs are detected in cells expressing the mutant IBD-D366N (right), but less so in cells expressing the IBD-WT (left) fusion protein. Scale bar is 5 μm. (C, D) Quantification of the fraction of nuclei containing INsfCherry VRCs (C), and the average nuclear import (D) at 6 hpi. Shown are means and SEM from >120 nuclei from 4 independent experiments. (E) Infectivity measured at 72 hpi using a luciferase assay and normalized to control MT4 cells lacking the GFP-IBD proteins. Plotted are means and SEM from 4 independent experiments. Statistical analysis: non-parametric Mann-Whitney rank sum test (D) and student’s t-test (E).
Fig 6.
HIV-1 nuclear import and integration targeting is associated with drastic changes to the structure and composition of the viral core.
(A) The ability of HIV-1 with mixed WT/N74D CA to interact with cytoplasmic CPSF6-358 was determined in TZM-bl cells. The ratio of infection in CPSF6-358 to control permissive TZM-bl cells is plotted. Here, 5% WT/CA incorporated into N74D/CA mutant cores was sufficient for cytoplasmic CPSF6-358 interactions. The average values from 5 independent experiments with SEM are shown. (B) Single Z-plane images showing IN-labeled HIV-1 VRCs (green puncta) colocalized with CPSF6 in the nucleoplasm (yellow dashed lines) and VRCs stuck at the nuclear envelope without CPSF6 recruitment (white arrowheads) in fixed TZM-bl cells at 6 hpi. Endogenous CPSF6 (red) and the nuclear lamin (blue) were detected by immunostaining. Scale bar is 5 μm. (C) The efficiency of nuclear penetration of IN-labeled VRCs (magenta squares/ line) for viruses with mixed CA or a 100% N74D/CA was determined by normalizing to the nuclear penetration of 100% WT/CA at 6 hpi in TZM-bl cells. Nuclear penetration was defined by fraction of IN-VRCs detected inside >0.5 μm of the NE with respect to the total nucleus associated HIV-1 complexes (NE + inside nucleus). The efficiency of SPAD-targeted integration (green circles/line) of the same viral preps was determined in 293T cells after 5 days of infection by normalizing to the SPAD-localized integration sites of a pure WT/CA virus. Nuclear penetration of VRCs and integration targeting into SPADs for viruses with a mixed WT/N74D CA decayed exponentially as a function of fraction of N74D CA. Data are means and standard deviations (too small to be visible) from 2 independent experiments for integration site analysis and 4 independent experiments for VRC penetration analysis. Data without normalization is shown in S7 Fig. (D) IN-associated CPSF6 signal recruited by VRCs in the cytoplasm, NE and >0.5 μm inside the nucleus. The background (BG) CPSF6 fluorescence determined in NE associated N74D-VRCs is shown as dashed grey line. Data is mean and SEM from 4 experiments, n>120 nuclei analyzed for each condition. Statistics in (A, C and D): non-parametric Mann-Whitney rank sum test in black vs. 100% WT/CA control and in red vs. 100% N74D/CA.