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. Author manuscript; available in PMC: 2022 Oct 1.
Published in final edited form as: Virology. 2021 Jul 5;562:19–28. doi: 10.1016/j.virol.2021.07.001

CAPSID-SPECIFIC NANOBODY EFFECTS ON HIV-1 ASSEMBLY AND INFECTIVITY

Ayna Alfadhli 1, CeAnn Romanaggi 1, Robin Lid Barklis 1, Ilaria Merutka 1, Timothy A Bates 1, Fikadu G Tafesse 1,*, Eric Barklis 1,*
PMCID: PMC8419096  NIHMSID: NIHMS1722579  PMID: 34246112

Abstract

The capsid (CA) domain of the HIV-1 precursor Gag (PrGag) protein plays multiple roles in HIV-1 replication, and is central to the assembly of immature virions, and mature virus cores. CA proteins themselves are composed of N-terminal domains (NTDs) and C-terminal domains (CTDs). We have investigated the interactions of CA with anti-CA nanobodies, which derive from the antigen recognition regions of camelid heavy chain-only antibodies. The one CA NTD-specific and two CTD-specific nanobodies we analyzed proved sensitive and specific HIV-1 CA detection reagents in immunoassays. When co-expressed with HIV-1 Gag proteins in cells, the NTD-specific nanobody was efficiently assembled into virions and did not perturb virus assembly. In contrast, the two CTD-specific nanobodies reduced PrGag processing, virus release and HIV-1 infectivity. Our results demonstrate the feasibility of Gag-targeted nanobody inhibition of HIV-1.

Keywords: HIV-1, Gag, capsid, nanobody

INTRODUCTION

From its amino-terminus to carboxy-terminus, the HIV-1 precursor Gag (PrGag) protein is composed of the matrix (MA), capsid (CA), spacer 1 (SP1), nucleocapsid (NC), spacer 2 (SP2) and p6 domains (Swanstrom and Wills, 1997). During viral maturation, the HIV-1 protease (PR) cleaves PrGag into its separate domains, which reorganize to form mature virions (Swanstrom and Wills, 1997). The CA proteins themselves are composed of N-terminal domains (NTDs) and C-terminal domains (CTDs), and play multiple roles in HIV-1 replication (Figure 1a; Pornillos et al., 2009; Barklis, 2013; Carnes et al., 2018; Novikova et al., 2019). They are central to the assembly of immature PrGag hexamers as well as C A hexamers that form the mature cores of HIV-1 particles (Pornillos et al., 2009; Barklis, 2013; Carnes et al., 2018; Novikova et al., 2019). The CA proteins also play important post-entry roles during the viral life cycle. These include cytoplasmic trafficking, nuclear envelope docking and nuclear import (Carnes et al., 2018; Novikova et al., 2019).

Figure 1. HIV-1 capsid hexamer and nanobody binding sites.

Figure 1.

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Panel A shows the HIV-1 CA hexamer structure (PDB 3GV2) with one of the NTDs colored in light green, and CA CTD alpha helices colored as follows: helix 8, blue; helix 9, cyan; helix 10, purple; helix 11 magenta. Note that nanobody CANTDcb1 binds to the CA NTD, but the structure of the complex has not been determined. Panel B shows nanobody VHH9 (in red) bound to the HIV-1 CA CTD, with helices colored as in Panel A (taken from PDB 2XV6). Panel C shows nanobody 59H10 bound to the HIV-1 CA CTD (PDB 5O2U), with coloring as indicated in Panel B. Note that the size bar at the bottom right indicates 20Å, and applies to all panels.

Given its importance in HIV-1 replication, it’s perhaps not surprising that CA proteins are the targets of host restriction factors. Notably, Old World monkey tripartite motif-containing 5 alpha (TRIM5a) proteins inhibit HIV-1 replication by binding CA NTDs of incoming cores in the cytoplasm of newly infected cells (Stremlau et al., 2004; Novikova et al., 2019; Yu et al., 2020). A second restriction factor, the Myxovirus resistance protein 2 (MxB), also binds the CA NTDs of incoming cores, effectively blocking nuclear entry (Novikova et al., 2019). CA also has been the target of many efforts to develop new HIV-1 inhibitors. One set of inhibitors that includes bevirimat (BVM) and PF-46396 prevents CA-SP1 cleavage by stabilizing CA CTD-SP1 conformations in their immature forms (Novikova et al., 2019). The peptide CAI (capsid assembly inhibitor) and its stapled derivative NYAD-1 also target the CTD. In particular, they bind a hydrophobic cavity formed by CA helices 8, 9, and 11 altering the CTD dimer interface, and perturbing viral maturation (Sticht et al., 2005; Zhang et al., 2008). A set of compounds including CAP-1, benzimidazoles (BMs) and benzodiazepines (BDs) also inhibit mature core formation, and also possibly virus release, but bind to CA NTD sites (Tang et al., 2003; Barklis, 2013; Novikova et al., 2019). The compound PF-74 targets yet another site, the intersubunit NTD-CTD interface in mature hexamers, resulting in maturation defects, as well as viral entry defects, due to the inhibition of host factor binding to incoming cores (Carnes et al., 2018). Significantly, the new compounds GS-CA1 and GS-6207 bind close to the PF-74 binding site, and GS-6207 has shown promise in clinical trials (Carnes et al., 2018; Link et al., 2020). Researchers also have explored the possibility of using monoclonal antibodies (mAbs) against CA as therapeutic tools. Notably, conjugation of an anti-CA mAb with a cell penetrating peptide (CPP) has been shown to facilitate mAb entry into T cells and inhibit HIV-1 replication in treated cells (Ali et al., 2016).

As an alternative to the above approaches, we have examined the effects of anti-CA nanobodies on HIV-1. Nanobodies derive from unique 90 kDa heavy chain-only antibodies from camelids and sharks (Muyldermans, 2013; Ingram et al., 2018; Weiss and Verrips, 2019). The antigen recognition regions of the heavy chain-only antibodies are referred to as the variable heavy chain domains (VHHs) or nanobodies. Such nanobodies have a number of unique properties: they are only 15 kDa, are stable over a wide range of conditions, tolerate multiple types of tags, possess long complementarity determining region (CDR) loops and are amenable to bacterial expression, and functioning within animal cells (Muyldermans, 2013; Ingram et al., 2018; Weiss and Verrips, 2019). We have examined three anti-HIV-1 CA nanobodies with respect to their specificities and their effects on HIV-1 assembly and replication. All three of the nanobodies proved convenient, sensitive and specific HIV-1 CA detection reagents. One of the nanobodies, specific for the CA NTD, did not inhibit HIV-1 assembly or replication, but was incorporated into virus particles. The other two nanobodies, specific for different sites on the CA CTD, reduced PrGag processing levels, and virus particle release. They also efficiently inhibited HIV-1 replication when expressed in producer cells, but not when expressed in newly infected cells. Our results offer new avenues for HIV-1 detection and inhibition.

RESULTS

Validation of nanobody specificity

To examine nanobodies as reagents for HIV-1 detection and inhibition, we chose three anti-HIV-1-CA nanobodies for testing: CANTDcb1 (Helma et al., 2012; Pezeshkian et al., 2019), 59H10 (Gray et al., 2017) and VHH9 (Igonet et al.; Tang et al., 2016). Of these, the CANTDcb1 has been shown to bind to an undefined site on the CA NTD (Figure 1A). In contrast, 59H10 and VHH9 bind to different CA CTD regions. In particular, 59H10 binds to the C-terminal helices of (helices 10 and 11) of CA (Figure 1C), while VHH9 binds to the NTD-CTD interdomain linker region and to helix 9, which forms the homodimeric CTD interface that interconnects CA hexamers (Figure 1B).

To validate the binding of the above nanobodies to HIV-1 CA, we generated nanobody fusion constructs (pLVX-puro-CANTDcb1-Fc, pLVX-puro-59H10-Fc, pLVX-puro-VHH9-Fc) composed of N-terminal secretion signal peptides, nanobody sequences, the human IgG Fc region (Capon et al., 1989) and C-terminal histidine tags. When transfected into human embryonic kidney 293T (HEK293T, 293T; DuBridge et al., 1987) cells, all three nanobody-Fc proteins were readily detected within cells using a fluorescently conjugated anti-human IgG antibody, although the 59H10-Fc and VHH9-Fc proteins localized to cell surfaces at higher levels than the CANTDcb1-Fc proteins, which appeared more enriched in intracellular membrane compartments (data not shown). While we do not know the cause for this localization difference, it is noteworthy that, relative to other nanobodies, CANTDcb1-Fc is 12-13 residues longer, and possesses two additional cysteines. By Western immunoblot analysis, we found that 59H10-Fc and VHH9-Fc proteins were secreted into the media of transfected cells at concentrations of approximately 5 pg/ml, while CANTDcb1-Fc protein concentrations were less than 0.2 pg/ml (data not shown). To examine the binding of nanobody-Fc proteins to HIV-1 Gag and CA proteins we performed several experiments (Figure 2). As a first test, the nanobody-Fc proteins were employed in parallel with anti-HIV-1 CA (Hy183) and anti-murine leukemia virus (MuLV; Hy187) mAbs for the immunofluorescent localization of HIV-1 or MuLV Gag proteins in transfected cells. Notably, we found that all three nanobody-Fc proteins bound specifically to HIV-1 Gag-expressing cells and not to cells expressing MuLV Gag (Figure 2, panel A). As a second test, the mAbs and nanobody-Fc proteins were used to probe blots of purified HIV-1 and MuLV CA proteins; and we again observed specific nanobody-Fc binding to HIV-1 CA (Figure 2, panel B). As a third, more stringent test, cell lysate samples from cells expressing HIV-1 or MuLV Gag proteins were probed on immunoblots (Figure 2, panel C). Consistent with predictions, the Hy183 mAb and all three nanobody-Fc proteins did not bind the MuLV PrGag protein, but detected HIV-1 PrGag and CA proteins, along with a 41 kDa polypeptide (p41) that presumably represents the MACA Gag processing intermediate. In contrast, the anti-MuLV Hy187 mAb, as expected, detected only the MuLV PrGag protein. These results verify the HIV-1 CA binding specificities of the CANTDcb1, 59H10 and VHH9 nanobodies; and it is worth noting that the 59H10-Fc and VHH9-Fc concentrations used in immunofluorescence and immunoblotting studies (0.5-1 μg/ml) approximate those used with commercial mAbs.

Figure 2. Nanobody binding specificities.

Figure 2.

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Proteins composed of the indicated nanobodies fused to the human IgG Fc domain were used to analyze nanobody specificities in parallel with mouse monoclonal antibodies to HIV-1 CA (Hy183) and MuLV CA (Hy187). The leftmost two panels (A) show immunofluorescent detection of HIV-1 or MuLV Gag proteins respectively expressed by psPAX2 or pXMGPE expression vectors. Note that the size bar at the bottom left corresponds to 30 microns, and that exposure times for the individual reagents were as follows: Hy183, 25 msec; CANTDcb1, 250 msec; 59H10, 25 msec; VHH9, 20 msec; Hy187, 100 msec. The center two panels (B) show immunoblot detection of 1 microgram purified HIV-1 or MuLV CA. Color reaction development times were as follows: 59H10 and VHH9, 2 min; Hy183, 3 min; Hy187, 5 min; CANTDcb1, 30 min. The rightmost two panels (C) show immunoblot detection of cell lysate samples from cells expressing HIV-1 or MuLV Gag proteins, respectively directed by the psPAX2 or pXMGPE expression vectors. PrGag, p41 and CA bands are as indicated, and color reaction development times were as follows: VHH9, 2 min; Hy183 and 59H10, 3 min; Hy187, 22 min; CANTDcb1, 35 min.

Analysis of nanobody interactions with HIV-1 Gag

Having validated the HIV-1 CA specificity of the CANTDcb1, 59H10 and VHH9 nanobodies, we expressed GFP-tagged control and anti-CA nanobodies in cells in the absence or presence of HIV-1 Gag and GagPol proteins and examined their subcellular localizations (Figure 3). To do so, pInducer20-CANTDcb1-GFP, pInducer20-59H10-GFP, and pInducer20-VHH9-GFP expression constructs (Meerbrey et al., 2011; see Materials and Methods) were transfected into cells in the absence or presence of psPAX2, which expresses wild type (WT) HIV-1 Gag and GagPol proteins (Zuffery et al., 1997; Lopez et al., 2014; Barklis et al., 2018). As a control, we also included the pInducer20-VHH52-GFP plasmid, which produces a GFP fusion protein to a nanobody (VHH52; Ashour et al., 2015; Cavallari, 2017) elicited against the influenza nucleoprotein (NP). Transfected cells were grown on coverslips and processed for fluorescence microscopy at three days post-transfection. When expressed in the absence of HIV-1 proteins, the CANTDcb1 and 59H10 proteins appeared to localize throughout cells, whereas the VHH52 and VHH9 proteins appeared to localize preferentially to cell nuclei (Figure 3A): this is consistent with previous observations of the nuclear localization of GFP-tagged nanobodies (Dong et al., 2019). Interestingly, in the presence of HIV-1 proteins, the control VHH52 protein remained largely in cell nuclei, while the anti-CA nanobody-GFP proteins stained to heterogenous cytoplasmic and cell surface locations that coincided with indirect immunofluorescent staining with the mouse anti-HIV-1 CA mAb (Figure 3B). We calculated correlation coefficients to measure nanobody-CA colocalization, and found that while the localizations of CA and VHH52 were negatively correlated, the three anti-CA nanobodies yielded positive correlation values of 0.5-0.68 (Figure 3B). These results imply that the CANTDcb1, 59H10 and VHH9 nanobody binding to HIV-1 Gag/GagPol proteins causes them to relocalize to sites along the cellular Gag trafficking pathway.

Figure 3. Nanobody colocalization with HIV-1 Gag.

Figure 3.

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A. Cells expressing the indicated nanobody-fusion proteins alone were subjected to GFP (Nano) detection at three days post-transfection. The size bar for all panels is at the bottom and corresponds to 30 microns. B. Cells were transfected with pInducer20 expression vectors for the indicated nanobody-GFP fusion proteins along with an expression vector for HIV-1 Gag and GagPol (psPAX2). Three days post-transfection, cells were subjected to fluorescent detection of GFP (Nano) and indirect immunofluorescent detection of PrGag and CA with the anti-HIV-1-CA primary antibody Hy183 (Capsid), and an Alexafluor594-conjugated secondary antibody. In the rightmost panel of images, GFP and Capsid images were overlaid to show colocalization indicated as yellow. The size bar for all panels at the bottom right corresponds to 30 microns. Pearson’s Correlation Coefficients (correlation), which vary from −1 (inversely correlated) to 0 (uncorrelated) to +1 (completely correlated) were calculated to quantify colocalization values from 21 (VHH52), 29 (CANTDcb1), 30 (59H10), and 25 (VHH9) pairs of cells.

To further examine nanobody-Gag interactions, nanobody-GFP and Gag/GagPol expression constructs were transfected into cells, after which cell and viral samples were collected for Western immunoblot analysis using anti-CA and anti-GFP antibodies (Figure 4). Cells and viruses all showed the expected PrGag, CA and p41 (MACA) processing intermediates (Figure 4, top panels). However, we noted a slight enrichment of the PrGag and p41 bands for the cellular 59H10 and VHH9 samples, and slight reductions in the total viral 59H10 and VHH9 Gag levels. Parallel immunoblots for nanobody-GFP detection (Figure 4, bottom panels) showed approximately 37 kDa nanobody-GFP bands in all cell samples, with a notable reduction in the CANTDcb1-GFP signal. Faint higher molecular weight GFP bands also were evident, possibly representing alternative translation start sites or modifications; and the VHH9-GFP lane also showed evidence of degradation. GFP signals in virus samples also were observed for the anti-CA nanobodies, but not for the control VHH52 nanobody. Interestingly, viral levels of CANTDcb1-GFP appeared significantly higher than those of the other nanobodies, suggesting CANTDcb1-GFP incorporation into virus particles.

Figure 4. Nanobody effects on HIV-1 Gag proteins.

Figure 4.

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HEK293T cells were transfected with the indicated VHH52, CANTDcb1, 59H10 and VHH9 GFP-fusion expression constructs along with the HIV-1 Gag plus GagPol expression construct (psPAX2). Three days post-transfection, cell and viral samples were collected and subjected to immunoblot detection with an anti-CA primary antibody or an anti-GFP primary antibody. The migration positions of the HIV-1 PrGag, p41, and CA proteins, and the nanobody-GFP fusion proteins are indicated. Also shown are the migration positions of 50, 37, and 25 kDa marker proteins that were electrophoresced in parallel lanes.

To determine whether the trends observed in Figure 4 were significant, experiments were repeated multiple times, and cellular CA to PrGag, viral to cellular Gag and viral to cellular nanobody ratios were calculated (Figure 5). As noted for Figure 4, 59H10 and VHH9 PrGag processing levels (Figure 5A) were consistently reduced 2-fold or more relative to cells not expressing nanobodies. Similarly, HIV-1 virus release levels in the presence of these two nanobodies also were reduced (Figure 5B). These results indicate that the two CA CTD-targeted nanobodies both impaired PrGag processing and virus release levels. In contrast, CANTDcb1, which binds the CA NTD, did not appreciably alter processing or release levels, but viral to cellular CANTDcb1 levels were dramatically higher than the other three nanobodies (Figure 5C).

Figure 5. Analysis of Gag processing, particle release, and nanobody incorporation.

Figure 5.

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Tagged nanobodies were co-expressed in cells with HIV-1 Gag plus GagPol expression constructs as illustrated in Figure 4. Panel A: Cellular PrGag processing levels were monitored by measuring CA to PrGag ratios in cells expressing no nanobody (no Nano) or the indicated tagged VHH52, CANTDcb1, 59H10 or VHH9 nanobodies. Averages and standard deviations are as shown, and derive from two (VHH52), three (no Nano, VHH9) or six (CANTDcb1, 59H10) independent experiments. Differences between no Nano and 59H10 and VHH9 samples were highly significant (P < 0.001; ***). Panel B: Viral to cellular Gag levels were monitored by measuring viral versus cellular Gag (PrGag plus p41 plus CA) levels. Averages and standard deviations are as shown, and derive from two (no Nano), three (VHH52, VHH9) or five (CANTDcb1, 59H10) independent experiments. Differences between no Nano and 59H10 and VHH9 samples respectively were highly significant (P < 0.001; ***) and significant (P<0.01;**). Panel C: Viral to cellular tagged nanobody ratios were monitored by measuring viral versus cellular tagged nanobody levels. Averages and standard deviations are as shown, and derive from three (VHH52, VHH9) or four (CANTDcb1, 59H10) independent experiments. The difference between CANTDcb1 and all other samples was highly significant (P < 0.001; ***).

We wished to examine whether the high levels of CANTDcb1 nanobody in our viral fractions (Figure 5C) actually reflected CANTDcb1 incorporation into HIV-1 virions. This was addressed by analysis of virus-like particles (VLPs) produced by cells transfected with psPAX2 plus either pInducer20-CANTDcb1-GFP, the control nanobody (pInducer20-VHH52-GFP), or EGFP-Vpr, which efficiently directs the incorporation of the EGFP-Vpr protein by virtue of Vpr binding to the p6 domain of PrGag (Schaeffer et al., 2001). VLPs so generated were adhered to microscope coverslips and processed for dual fluorescent detection of HIV-1 CA and GFP proteins as described for Figure 3. Consistent with previous studies (Barklis et al., 2018), anti-CA immunofluorescence detection of virus particles yielded images with thousands of bright red fluorescent dots (Figure 6B, D, F). Similarly, EGFP-Vpr tagged viruses appeared as bright green dots (Figure 6A), and our calculation (see Materials and Methods) for the percentage of CA-tagged VLPs that also were GFP-positive was 39%, close to the 36% value we observed previously (Barklis et al., 2018). In contrast, the negative control VHH52-GFP protein showed hardly any GFP-positive VLPs (Figure 6E, 0.12% positive). The CANTDcb1-GFP protein, which is released readily from cells (Figures 4–5), gave an intermediate value. As illustrated in Figure 6C, many GFP-positive VLPs were evident in fluorescent images, and the frequency of GFP-positive VLPs was approximately 13%. This suggests that under some circumstances, CANTDcb1-GFP may be used to tag HIV-1 virions, albeit at one third the efficiency of EGFP-Vpr.

Figure 6. CANTDcb1-GFP nanobody incorporation into HIV-1 particles.

Figure 6.

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EGFP-Vpr (A, B), CANTDcb1-GFP (C, D), or VHH52-GFP (E, F) proteins were co-expressed in HEK293T cells with WT HIV-1 Gag and GagPol proteins. Virus-like particles (VLPs) released by cells were filtered, centrifuged, adhered to polylysine-coated coverslips, and processed for dual fluorescent detection of GFP (A, C, E) and HIV-1 CA (B, D, F) as detailed in the Materials and Methods. As shown, viruses appear as fluorescent green or red dots in images processed in parallel. Note that the 20 micron size bar for all panels is shown at the bottom right of panel F. Note also that original micrograph exposure time for panel A was 375 msec, while exposure times for C and E were 750 msec. Percentages of CA positive particles that were also GFP positive were calculated from five (EGFP-Vpr, VHH52-GFP) or ten (CANTDcb1-GFP) separate images, and yielded the following results: EGFP-Vpr = 39.1 ± 7.9%; CANTDcb1-GFP = 13.1 ± 8.4%; VHH52-GFP = 0.1 ± 0.05%.

Nanobody interactions with MuLV and HIV/Mo Gag proteins

As controls for our studies, we chose to probe potential nanobody interactions with MuLV and HIV/Mo (Hansen and Barklis, 1995; McDermott et al., 2000; Arvidson et al., 2003) Gag proteins. As noted above, anti-HIV-1-CA nanobody-Fc proteins did not recognize murine leukemia virus Gag proteins (Figure 2), so our prediction was that nanobody-GFP proteins would not perturb MuLV Gag protein expression or release from cells. The HIV/Mo Gag construct directs the expression of a chimeric HIV-1/MuLV Gag protein composed of the HIV-1 MA and CA NTD domains fused to the MuLV CA CTD and nucleocapsid (NC) domains (Arvidson et al., 2003). We previously observed that this protein was efficiently released from cells in an unprocessed (PrGag) form, and that the opposite chimera (Mo/HIV) was poorly expressed and not released from cells (Arvidson et al., 2003). As a consequence, we anticipated that CANTDcb1, which binds the HIV-1 NTD, might interact with the HIV/Mo Gag protein, whereas the other nanobodies would not.

We tested our predictions by co-expressing nanobody-GFP proteins with either MuLV or HIV/Mo proteins in cells, and analyzing protein expression and release profiles as we did in Figure 4. Our analysis of MuLV Gag proteins (Figure 7A, top panels) did not show any dramatic alterations of MuLV PrGag cellular processing levels (Figure 7A, top left) or release levels (Figure 7A, top right) by any of the nanobody-GFP proteins. Moreover, while all nanobody-GFP proteins were produced in cells (Figure 7A, bottom left), none of them were efficiently incorporated into virus-like particles (Figure 7A, bottom right). When we examined HIV/Mo proteins in cells (Figure 7B, HIV/MuLV), we saw the predicted ~52 kDa chimeric PrGag (Gag) proteins, along with a higher band of ~60 kDa that we had observed previously (Arvidson et al., 2003). None of the anti-HIV-1-CA nanobody-GFP proteins appeared to affect VLP release, as illustrated by largely equivalent Gag levels in the virus fractions (Figure 7B, upper right). Significantly, all nanobody-GFP proteins were produced in co-transfected cells, and VLP levels for the CANTDcb1-GFP nanobody were high, relative to the other nanobody-GFP proteins (Figure 7B, bottom panels). This observation is consistent with the NTD specificity of CANTDcb1, and with its ability to be incorporated into HIV-1 VLPs (Figures 4–6).

Figure 7. Nanobody effects on MuLV Gag proteins.

Figure 7.

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HEK293T cells were transfected with the indicated VHH52, CANTDcb1, 59H10 and VHH9 GFP-fusion expression constructs along with either the MuLV pXMGPE expression construct (panel A), or the HIV/Mo (HIV/MuLV) construct (panel B) that expresses a PrGag protein composed of the HIV-1 MA and CA NTD regions, combined with the MuLV CA CTD and NC regions. Three days post-transfection, cell and viral samples were collected and subjected to immunoblot detection with an anti-MuLV-CA primary antibody (which recognizes the MuLV CA CTD), or an anti-GFP primary antibody. The migration positions of the MuLV PrGag and CA proteins, the HIV/MuLV Gag protein, and the nanobody-GFP fusion proteins are indicated. Also shown are the migration positions of 50, 37, and 25 kDa marker proteins that were electrophoresced in parallel lanes.

Nanobody effects on viral infectivities

Given that the two CTD-binding nanobodies affected HIV-1 PrGag processing and virus release levels (Figure 5), it was worthwhile to examine whether anti-CA nanobodies also might affect the infection efficiency of HIV-1. For this analysis, we generated two sets of control and anti-CA nanobody-GFP-expessing cell lines via lentivirus vector transduction (see Materials and Methods). One set was generated in the HeLa-based HiJ cell line (Scherer et al., 1953; Kabat et al., 1994), for the purpose of testing nanobody-GFP effects on the infectivities of viruses assembled from virus-producing cells. The second set was generated in TZM-bl cells (Platt et al., 1998; Wei et al., 2002; Alfadhli et al., 2016; Barklis et al., 2021), for the purpose of probing possible nanobody-GFP effects on HIV-1-infected target cells.

To test nanobody effects on HIV-1 viruses assembled in producer cells, nanobody-GFP-expressing HiJ cells were transfected with the WT HIV-1 NL4-3 proviral construct, and the infectivities of viruses so produced were assayed in WT TZM-bl reporter cells. Our results (Figure 8A) showed that the control VHH52 nanobody had little effect on viruses assembled in the producer cells. We also observed a small but significant diminution of HIV-1 infectivity for CANTDcb1-expressing cells. However, greater reductions to 32% and 8% of control levels respectively were seen for the 59H10 and VHH9 nanobodies (Figure 8A). These results appear to reflect PrGag processing and release disturbances noted in Figures 4–5.

Figure 8. Nanobody effects on HIV-1 infection.

Figure 8.

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Panel A: To assess nanobody effects on HIV-1 viruses generated in producer cells, HiJ cells expressing either no nanobody (no Nano), or the indicated nanobody-GFP chimeric proteins were transfected with the expression construct for the WT NL4-3 HIV-1 strain. Three days post-transfection, viruses were collected, normalized for Gag levels by immunoblotting, and used to infect CD4+, CCR5+, CXCR4+ TZM-bl indicator cells, and infectivities were tracked by measuring induced β-galactosidase activities as described in the Materials and Methods section. Infectivities are scored as percentages of the no nanobody control, experiments were performed in tripicate, and averages and standard deviations are as shown. Differences between the no Nano and 59H10 and VHH9 samples were highly significant (P < 0.001; ***), while the difference between the no Nano and CANTDcb1 sample was significant (P<0.01; **). Panel B: To assess nanobody effects on HIV-1 infection of target cells, TZM-bl cells expressing the indicated nanobody-GFP chimeric proteins were infected with WT NL4-3 HIV-1 and infection levels were measured as described in panel A. Infectivities are scored relative to TZM-bl expressing the control VHH52-GFP protein, experiments were performed in triplicate, and averages and standard deviations are as shown. The reduction between the VHH9 and VHH52 samples was significant (P < 0.01; **). Panel C: Nanobody effects on MuLV viruses in HEK293T producer cells were measured by co-transfection of nanobody-GFP expression constructs with expression constructs for MuLV proteins, the VSV G protein, and a β-galactosidase reporter as described in the Materials and Methods section. Viruses so produced were used to infect target HEK293T cells, and infection levels were determined by staining for β-galactosidase positive cells (see Materials and Methods section). Infectivities are scored relative to viruses generated in the presence of the control VHH52-GFP protein, experiments were performed in duplicate, and averages and standard deviations are as shown.

Different results were observed when nanobody-GFP proteins were expressed in TZM-bl target cells that were challenged with WT NL4-3 HIV-1 viruses (Figure 8B). Here, infection rates of TZM-bl cells expressing CANTDcb1 and 59H10 were equal to those in the control VHH52 TZM-bl cells. Target VHH9 cells did show slightly reduced HIV-1 infection levels (Figure 8B), but levels were approximately eight times higher than for viruses assembled in the presence of the VHH9 nanobody. These results may be indicative of a differential access of nanobodies to CA in the assembly versus entry phases of HIV-1 replication (see Discussion). As a final control, we also monitored nanobody effects in producer cells on MuLV infectivities (Figure 8C). Consistent with aforementioned results on nanobody specificities (Figures 2, 7), we did not see any perturbation of MuLV infection levels with any of the anti-HIV-1-CA nanobodies.

DISCUSSION

In our studies, we examined the effects of three previously identified nanobodies (Helma et al., 2012; Igonet et al.; Tang et al., 2016; Gray et al., 2017; Pezeshkian et al., 2019) for their in vitro properties, and their effects on HIV-1 assembly and replication (summarized in Table 1). The HIV-1 CA specificity of the nanobodies was confirmed through the production and analysis of nanobody fusions to the human IgG Fc domain (Figure 2). The nanobody-Fc fusion proteins proved advantageous because they can be recognized by conventional anti-human secondary antibodies, but also because their bivalent structures can increase binding potencies as a consequence of greater binding avidities (Capon et al., 1989; Hanke et al., 2020; Huo et al., 2020; Wrapp et al., 2020). Although the CANTDcb1-Fc protein was poorly released from cells, all three nanobody-Fc proteins were functional in immunoblotting and immunofluorescence assays (Figure 2), and unpurified 59H10-Fc and VHH9-Fc proteins in media supernatants from transfected cells worked as efficiently as commercial mouse mAbs at 0.5-1 μg/ml concentrations.

Table 1. Properties of anti-capsid nanobodies.

Nanobody N-terminal domain (NTD) and C-terminal domain (CTD) specificities were determined previously. In vitro use of nanobody-human IgG Fc fusion proteins for immunoblotting and immunofluorescence was evaluated as shown in Figure 2. Virion incorporation indicates a virus-to-cell signal of greater than three (+++) or less than one (+/−). Processing inhibition corresponds to values equal to controls (−), half of control values (++) or less than half of control values (+++). Assembly inhibition corresponds to equal to controls (−), less than two-thirds of controls (+), or less than half that of controls (++). Infectivity inhibition values correspond to less than controls (+), less than half of control values (++), or one tenth of control values (+++).

CANTDcb1
 59H10
 VHH9
Nanobody specificity NTD CTD CTD
Immunoblotting + +++ +++
Immunofluorescence + +++ +++
Virion Incorporation +++ +/− +/−
Processing inhibition − ++ +++
Assembly inhibition − ++ +
Infectivity inhibition + ++ +++
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Our analysis of the CANTDcb1 nanobody confirmed its colocalization with co-expressed HIV-1 Gag (Helma et al., 2012; Pezeshkian et al., 2019). Previous reports demonstrated that CANTDcb1 can be used to mark HIV-1 assembly sites, but did not indicate whether the nanobody is assembled into virions, or impaired HIV-1 infectivity (Helma et al., 2012; Pezeshkian et al., 2019). Our results clearly demonstrate that CANTDcb1-GFP proteins are readily assembled into HIV-1 VLPs (Figures 4–6); and incorporation into HIV/Mo VLPs, but not MuLV VLPs (Figure 7) attested to the HIV-1 CA NTD specificity of CANTDcb1. Given the affinity of CANTDcb1 for CA, we were somewhat surprised that its expression had little effect on HIV-1 replication. Indeed, when co-expressed with HIV-1 in producer cells, virus infection levels were only slightly reduced (Figure 8A). This may relate to the observation that cyclophilin A binding to HIV-1 CA NTDs in virus particles does not block virus infection (Novikova et al., 2019; Kim et al., 2019). However, when expressed in TZM-bl recipient cells, CANTDcb1 also showed no effect on HIV-1 replication (Figure 8B). Given that both Trim5a and MxB bind to CA NTDs of incoming cores and inhibit HIV-1 replication (Novikova et al., 2019; Yu et al., 2020), it may be that CANTDcb1 failed to localize with post-entry HIV-1 cores, rendering it incapable of HIV-1 inhibition.

As opposed to CANTDcb1, the CA CTD specific nanobodies, 59H10 and VHH9 both perturbed HIV-1 PrGag processing and virus particle release (Figures 4–5). Previously, the structures of 59H10 and VHH9 bound to the CTD were described (Figure 1; Igonet et al.; Gray et al., 2017), and VHH9 has been used as part of a conditionally stable HIV-1 nanobody probe (Tang et al., 2016), but their effects on assembly or replication have not been reported. Consistent with their effects on PrGag processing and viral release, we found that both significantly inhibited the infectivity of HIV-1 virions produced in their presence (Figure 8B), and these are the first observations that intracellular nanobodies (intrabodies; Wu et al., 2017; Wagner and Rothbauer, 2020) can inhibit HIV-1 CA function. Based on their binding sites (Figure 1), we speculate that 59H10 may act to disrupt CA CTD-SP1 interactions in immature virions, and VHH9 may disrupt hexamer-hexamer contacts in immature and/or mature virions. We hypothesize that seemingly minor effects on PrGag processing were amplified when scoring for infection, as has been observed for a number of HIV-1 CA mutants (Wang and Barklis, 1993; Scholz et al., 2005; Noviello et al., 2011; Lopez et al., 2013). In addition to its effects on assembling virions, the VHH9 nanobody showed a small, but significant inhibition of incoming virions in TZM-bl cells (Figure 8B). These results imply that at least some VHH9-GFP proteins localize with HIV-1 cores in infected cells, and can access their CTD targets.

Although the CTD-binding nanobodies do appear to inhibit HIV-1 replication, the pathway to use them in the application of anti-HIV therapies requires further development. Unlike nanobodies that target surface proteins (Koch et al., 2017; Weiss and Verrips, 2019), these CA-binding reagents are intrabodies (Wu et al., 2017; Wagner and Rothbauer, 2020) that need to operate intracellularly. Conceivably, this could be accomplished by viral vector-mediated genetic engineering of specific cell populations in infected individuals (Jin et al., 2021). Alternatively, a variety of methods have been employed to introduce nanobodies into cells to act as intrabodies. These include tagging nanobodies with cell-penetrating peptides or protein transduction domains, or employing nanoparticle, micelle, liposome or oligoaminoamide delivery systems (Ali et al., 2016; Wagner and Rothbauer, 2020). Another approach may be the use of nanobodies and their CTD targets in competition binding screens (Syedbasha et al., 2016) to identify new classes of CA antivirals. At the least, our results demonstrate the feasibility of Gag-targeted nanobody inhibition of HIV-1, and should serve as a springboard for future, related studies on other intracellular protein targets.

MATERIALS AND METHODS

Recombinant DNA constructs.

The following constructs have been described previously: the NL4-3 HIV-1 proviral clone (Adachi et al., 1986), the psPAX2 HIV-1 gag andpol expression construct (Zuffery et al., 1997; Lopez et al., 2014; Barklis et al., 2018), the MuLV pXMGPE gag, pol, and env expession construct (Hansen and Barklis, 1995; McDermott et al., 2000), the HIV/Mo construct that expresses a chimeric Gag protein composed the HIV-1 MA and CA NTD domains fused to the MuLV CA CTD and NC domains (Arvidson et al., 2003), the EGFP-Vpr construct (Schaeffer et al., 2001), and the B2BAG β-galactosidase and LNCX-PH-Btk-GFP murine retrovirus reporter vectors (Berwin and Barklis, 1993; Barklis et al., 2018). Nanobody VHH coding sequences for the anti-HIV-1-CA nanobodies were obtained for CANTDcb1 (Helma et al., 2012), 59H10 (Gray et al., 2017) and VHH9 (Igonet et al.), and DNA fragments corresponding to them were obtained from Integrated DNA Technologies (IDT). The C-terminally HA-tagged nanobody expression constructs pcDNA3.1-CANTDcb1-HA, pcDNA3.1-59H10-HA, and pcDNA3.1-VHH9-HA were prepared by replacing the small NheI-XhoI fragment of pcDNA3.1(+) (Invitrogen) with nanobody-HA coding sequences (see Supplementary Information). Inducible nanobody-GFP constructs derive from a pInducer20 (Meerbrey et al., 2011) variant, in which pInducer nucleotides 2655-3799 were replaced with the GFP cassette sequence provided in the Supplementary Information. The NotI and AscI cloning sites upstream of the GFP coding region in this plasmid were used for the insertion of nanobody sequences (Supplementary Information) to yield the plasmids pInducer20-CANTDcb1-GFP, pInducer20-59H10-GFP and pInducer20-VHH9-GFP, along with a nanobody control (pInducer20-VHH52-GFP) that expresses a VHH that recognizes the influenza virus NP protein (VHH52; Ashour et al., 2015; Cavallari, 2017). Plasmids also were generated for expression of secreted nanobody-human IgG Fc chains from cells. These constructs derive from a pLVX-puro (Clontech) variant, in which the small pLVX-puro XhoI-BamHI fragment was replaced with a DNA fragment (Supplementary Information) encoding a translation initiation codon, a signal peptide, NotI and EcoRI cloning sites, the coding region for the human IgG FC region (Capon et al., 1989), a histidine tag and a termination codon. The plasmids pLVX-puro-CANTDcb1-Fc, pLVX-puro-59H10-Fc and pLVX-puro-VHH9-Fc were generated by insertion of the respective nanobody VHH sequences (Supplementary Information) into the NotI and EcoRI cloning sites.

Cell culture.

Human embryonic kidney 293T cells (HEK293T or 293T; DuBridge et al., 1987) and CD4+, CCR5+, CXCR4+ HIV-1 reporter TZM-bl cells (Platt et al., 1998; Wei et al., 2002; Alfadhli et al., 2016; Barklis et al., 2021) respectively were obtained from the American Type culture Collection and the NIH AIDS Reagent program. HiJ cells are a CD4+ , CXCR4+ variant of HeLa cells (Scherer et al., 1953; Kabat et al., 1994), and were obtained from the laboratory of Dr. David Kabat. Variants of these cells expressing pInducer20-nanobody-GFP proteins were generated by lentivirus vector infection as described below. 293T variants were obtained after high multiplicity of infection (MOI) infections and were screened for GFP expression. TZM-bl and HiJ variants were selected with 0.5mg/ml of geneticin (Genticin, G418; Goldbio, ThermoFisher; corrected for <100% purity). Nanobody expression was induced with 1 microgram/ml doxycycline (Sigma), and cells were screened for GFP expression. All cells were grown in humidified 5% carbon dioxide air at 37°C in Dulbecco’s Modified Eagle’s Media (DMEM) supplemented with 10% fetal bovine sera (FBS) plus 10 mM Hepes, pH 7.3, penicillin and streptomycin.

Transfections.

Transfections of cells were performed using polyethyleneimine (PEI) as described previously (Longo et al., 2013; Barklis et al., 2018). For immunofluorescent detection of nanobody-Fc fusions, confluent 293T cells in six well plates on Fisher 22x22-1.0 coverslips coated with polylysine (0.01%; Sigma P4707) were transfected with 2-4 micrograms of pLVX-puro-nanobody-Fc rapid prep DNAs that had been cleaned by phenol-chloroform extraction, chloroform extraction and ethanol precipitation. For immunofluorescent detection of HIV-1 or MuLV Gag proteins, 24 μg of either psPAX2 or pXMGPE were transfected onto confluent 10 cm plates of 293T cells, and one day post-transfection, transfected cells were seeded onto polylysine coated coverslips and grown an additional two days prior to processing. For colocalization analyses, 10 cm plates were transfected with 12 μg psPAX2 plus 12 μg of pInducer20-nanobody-GFP constructs, and were seeded onto coverslips as described above. In experiments performed for HIV-1 Gag protein gel analyses of viral and cell samples, cells were transfected with 12 pg psPAX2 plus 12 pg of either pInducer20-nanobody-GFP or pcDNA3.1-nanobody-HA constructs, and cell and virus samples were processed at 3 days post-transfection as described below. MuLV Gag analyses and HIV/Mo Gag analyses were performed similarly, but with pXMGPE or HIV/Mo expression constructs used in place of psPAX2.

Transfections also were performed to generate viruses for lentivirus vector transductions, and for infection assays. The pInducer20-nanobody-GFP lentivirus vectors were generated by transfection of 293T cells with 8 μg of the respective pInducer20 plasmids, 8 μg psPAX2 and 8 μg of the VSV G expression plasmid pMD.G (Addgene plasmid #12259, kindly provided by Dr. Didier Trono). Wild type HIV-1 was generated by transfection of cells with 24 μg NL4-3. MuLV-based transducing viruses were generated by transfection of cells with 6 pg each of pXMGPE, pMD.G, pInducer20-nanobody-GFP plasmid and either B2BAG or LNCX-PH-Btkk-GFP. All viruses were collected from medias of cells at three days post-transfection and filtered through 0.45 micron filters prior to storage at −80°C or use.

Infections.

Infection assays with TZM-bl cells or TZM-bl derivatives proceeded as described previously (Alfadhli et al., 2016; Barklis et al., 2021). Briefly, confluent TZM-bl cells in six well plate wells (35 mm diameter) each were incubated with 1 ml virus for 6 h, then supplemented with 1 ml media, and incubated for an additional 42 h. Following infection incubations, media were removed from cells, and cells from each well were scraped into 1.0 ml of phosphate-buffered saline (PBS; 9.5 mM sodium potassium phosphate [pH 7.4], 137 mM NaCl, 2.7 mM KC1) and pelleted. Cell pellets were suspended in 150 μl PBS containing 0.1% sodium dodecyl sulfate (SDS), vortexed, supplemented with 600 μl PM-2 buffer (33 mM NaH2PO4, 66 mM Na2HPO4, 2 mM MgSO4, 0.1 mM MnC12, 40 mM β-mercaptoethanol [BME]), vortexed, supplemented with 150 μl 4 mg/ml 2-nitrophenylβ-d-galactopyranoside (ONPG) in PM-2 buffer and incubated at 37°C. Reactions were stopped by addition of 375 μl 1 M Na2CO3 and flash freezing on dry ice powder. Samples then were thawed, and 420 nm light absorbances were read spectrophotometrically to calculate β-galactosidase (β-gal) activities (1 unit = 1 nMole ONPG hydrolyzed per minute = 420 nm absorbance x 285/minutes of incubation; Jones et al., 1990; Wang et al., 1994) as a measure of infectivity (Alfadhli et al., 2016; Barklis et al., 2021). For the assay of MuLV infections using B2BAG as a reporter, 50% confluent 293T cells were infected as described above, and three days post-infection, cells were fixed 15 min with 0.5% glutaraldehyde (Sigma-Aldrich) in PBS, rinsed with PBS and incubated 4-24 h in PBS containing 5 mM K4Fe(CN)6, 5 mM K3Fe(CN)6, 1 mM MgSO4, 1 mg/ml 5’-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal; Roche 03117073001), and scored for percentages of blue, X-Gal-stained cells. Assays of MuLV infections using the LNCX-PH-Btkk-GFP followed the infection protocol above, but at three days post-infection, cells were simply imaged for fluorescent detection of GFP positive cells. To calculate significance values, means and standard deviations from multiple experiments were converted to Z values, which were used to calculate probabilities.

Proteins and antibodies.

The histidine-tagged HIV-1 and MuLV CA proteins were purified as described previously (Barklis et al., 1997; Barklis et al., 2009). Nanobody-Fc proteins were collected in media of pLVX-puro-nanobody-FC-transfected 293T cells at concentrations ranging from <0.2 (CANTDcb1-Fc) to ~5 μg/ml (59H10, VHH9). Nanobody-Fc proteins were used at 1:10 for HIV-1 and MuLV CA immunoblots. 59H10-Fc and VHH9-Fc were used at 1:5 for immunofluorescence and transfected cell Gag immunoblot detection; and CANTDcb1-Fc was used at 1:2 for immunofluorescence and transfected cell immunoblot detection. Mouse primary antibodies for detection CA proteins were mouse-anti-HIV-CA hybridoma media (Hy183; used at 1:15 for immunoblotting and immunofluorescence) and mouse-anti-MuLV-CA hybridoma media (Hy187; used at 1:15 for immunoblotting and 1:10 for immunofluorescence): both of these cell lines were kindly provided by Dr. Bruce Chesebro. For immunoblot detection of GFP proteins, a rabbit-anti-GFP antibody (Invitrogen A11122) was used at a 1:3,000 dilution. Fluorescent secondary antibodies were Alexa Fluor 594-conjugated goat anti-mouse IgG (Invitrogen A11005) and Alexa Fluor 594-conjugated goat anti-human IgG H&L (Invitrogen A11014), both used at 1:1,000. Secondary antibodies for immunoblot detection were all Promega alkaline phosphatase-conjugated goat antibodies used at 1:15,000 dilutions and directed against IgG H&L chains from mouse (S3728), rabbit (S3738) or human (S3828 or S3821) antibodies.

Protein immunoblot analysis.

To monitor protein levels in virus samples, aliquots of virus were filtered through 0.45 micron filters, concentrated by centrifugation through 2 ml 20% sucrose in PBS cushions (45 min at 197,000 x g; 40,000 rpm, Beckman SW41 rotor), suspended in 0.1 ml PBS, mixed with 0.1 ml of 2 x sample buffer (12.5 mM Tris-HCl [pH 6.8], 2% SDS, 20% glycerol, 0.25% bromphenol blue) plus 0.1 volume of BME and stored frozen prior to analysis. Samples were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) as described previously (Ritchie et al., 2015; Staubus et al., 2019). Typically, 10% viral samples were subjected to electrophoresis in parallel with molecular weight size standards (Bio-Rad). After SDS-PAGE fractionation, proteins were electroblotted and immunoblotted following previously described methods (Ritchie et al., 2015; Staubus et al., 2019). Primary and alkaline phosphatase-conjugated secondary antibodies were employed at the concentrations listed above. Color reactions for visualization of antibody-bound bands employed nitrobluetetrazolium plus 5-bromo-4-chloro-3-indolyl phosphate in AP buffer (100 mM Tris-hydrochloride [pH 9.5], 100 mM NaCl, 5 mM MgCl2).

Protein levels in cell lysate samples also were assayed. To do so, cell lysate samples were prepared by collecting cells in PBS, pelleting 20% of the cell sample, suspension in 50 μl IPB (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM ethylenediamine tetraacetic acid [EDTA], 0.1% SDS, 0.5% sodium deoxycholate, 1.0% TritonX-100, 0.02% sodium azide), vortexing, incubation on ice for 5 min, pelleting 15 min at 13,000 x g to remove insoluble debris, mixing with 50 μl 2 x sample buffer plus 0.1 volume BME and stored frozen prior to analysis as described for viral samples. Typically, 4% of cell samples were subjected to electrophoresis, electroblotting and immunoblotting as described above for viral samples.

For quantitation, immunoblots were air-dried and scanned using an Epson Perfection V600 scanner. Band intensities of scanned TIFF images were determined using NIH Image J software (Schneider et al., 2012), and cellular CA to PrGag, viral to cellular Gag and viral to cellular nanobody ratios were calculated from these intensities. Means and standard deviations from multiple experiments were converted to Z values assuming normal distributions, and statistical significance values were calculated from Z values.

Immunofluorescence.

Cellular immunofluorescence studies were performed as described previously (Barklis et al., 2018). Briefly, cells on polylysine-coated coverslips were fixed, permeabilized, washed and subjected to successive rounds of primary and secondary antibody binding steps, at antibody dilutions described above. Samples were viewed on a Zeiss AxioObserver fluorescence microscope using 63x (Planapochromat; NA=1.4) objective and a Zeiss filter set 10 (excitation bandpass 450-490; beamsplitter Fourier transform 510; emission bandpass 515-565) for green fluorophores, or Zeiss filter set 20 (excitation bandpass 546/12, beamsplitter Fourier transform 560, emission bandpass 575-640) for red fluorophores. Images were collected with Zeiss Axiovision software at 100% gain settings and either the exposure times noted in figure legends (Figure 2), or at exposures taken to maximize brightness levels without overexposure (Figure 3). For the purpose of presenting color images, gray-scale 16 bit TIF images were converted to green or red images using the Image J Image/Lookup Tables function, and then saved as 8 bit RGB images. For overlays of red and green images, RGB TIF images were opened in Adobe Photoshop, layered using the screen option, and flattened. Colocalization analysis of HIV-1 Gag (CA) and nanobody (GFP) signals was performed by determining Pearson’s Correlation Coefficient (PCC) values, which vary from −1 (inversely correlated) to +1 (completely correlated) (Adler and Parmryd, 2010). To do so, matched images in Image J were stacked, single cell areas without background regions were boxed and cropped, destacked and then used as input for the Image J JACoP PCC Plugin (Schneider et al., 2012). Colocalization values were averaged from at least 20 pairs of cell images for each Gag/nanobody combination. Values were evaluated via one-way ANOVA analyses and Tukey comparisons, using GraphPad Prism 5 software.

For virus particle analyses, virus-like particles (VLPs) were adhered to 18 mm circular coverslips in wells of twelve well tissue culture plates that first were incubated rocking 2 h at room temperature in 0.01% poly-L-lysine (Sigma P4707), washed once with water, wicked with filter paper and dried for at least 48 h. VLP samples (5 μl) were applied to the centers of the coverslips, incubated 20 min and then wicked. VLPs were fixed by the gentle addition of 4% PFA in PBS and incubation for 20 min at room temperature. After fixation, coverslips were washed 3 min in PBS, and processed for dual detection of HIV-1 Gag and GFP-tagged nanobodies as described for cell samples. VLP samples were viewed and imaged as for cell images with gain settings at 100%, exposure settings for Gag imaging at 50 msec and GFP exposure settings at 375 msec (Vpr-GFP) or 750 msec (CANTDcb1-GFP and VHH52-GFP).

To determine the percentages of Gag-positive particles that also were GFP-positive, we analyzed VLP images using a combination of Image J and Excel or R software (Barklis et al., 2018). As a first step, images were normalized in Image J to include the full range (0–65535) of brightness values, and then inverted. Following these steps, images were thresholded to include only the top 15% of pixel brightness values (above 55705), and converted to Gag and GFP binary masks. To remove large, GFP+ artifacts from consideration, GFP binary masks were filtered using the Image J Analyze Particle command with a size range of >10 pixels, and then subtracted from the Gag images. VLP positions then were picked from the Gag masks and data for these particles were extracted from the original Gag and background-subtracted GFP images using a combination of the Set Measurements and Analyze Particles (size range 0–4 pixels) commands. Particle data were analyzed using Excel or R, and GFP+ VLPs were defined as VLPs (from Gag images) that had average GFP brightness values of at least 1000. Values represent averages and standard deviations from at least five pairs of images, and at least 40,000 VLPs.

Supplementary Material

1

  • Nanobodies specifically detect HIV-1 capsid proteins via immunotechniques.

  • A nanobody to the capsid N-terminal domain is incorporated into virions.

  • Nanobodies to the capsid C-terminal domain perturb HIV-1 assembly.

  • Capsid nanobodies inhibit HIV-1 infectivity.

ACKNOWLEDGMENTS

E. B. and A. A. gratefully acknowledge support from the Medical Research Foundation of Oregon and from the National Institutes of Health (NIH; R01 AI152579). F. T. was supported by NIH grants R01 AI141549 and T32 AI747225. We thank Drs. David Kabat and Bruce Cheesebro respectively for supplying the HiJ and hybridoma (Hy183, Hy187) cell lines. We also thank all members of Barklis and Tafesse labs, and to the Vollum Institute support staff for their help and assistance.

Footnotes

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Declarations of interest: none

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