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• W2176753208 abstract "•ViewHIV visualizes the HIV-1 capsid and viral DNA in the cytosol and nucleus•ViewHIV can be done with any HIV-1 strain and primary cells•ViewHIV shows that CA enters the nucleus and associates with vDNA•CA’s interaction with CPSF6 promotes HIV-1’s nuclear entry and integration Direct visualization of HIV-1 replication would improve our understanding of the viral life cycle. We adapted established technology and reagents to develop an imaging approach, ViewHIV, which allows evaluation of early HIV-1 replication intermediates, from reverse transcription to integration. These methods permit the simultaneous evaluation of both the capsid protein (CA) and viral DNA genome (vDNA) components of HIV-1 in both the cytosol and nuclei of single cells. ViewHIV is relatively rapid, uses readily available reagents in combination with standard confocal microscopy, and can be done with virtually any HIV-1 strain and permissive cell lines or primary cells. Using ViewHIV, we find that CA enters the nucleus and associates with vDNA in both transformed and primary cells. We also find that CA’s interaction with the host polyadenylation factor, CPSF6, enhances nuclear entry and potentiates HIV-1’s depth of nuclear invasion, potentially aiding the virus’s integration into gene-dense regions. Direct visualization of HIV-1 replication would improve our understanding of the viral life cycle. We adapted established technology and reagents to develop an imaging approach, ViewHIV, which allows evaluation of early HIV-1 replication intermediates, from reverse transcription to integration. These methods permit the simultaneous evaluation of both the capsid protein (CA) and viral DNA genome (vDNA) components of HIV-1 in both the cytosol and nuclei of single cells. ViewHIV is relatively rapid, uses readily available reagents in combination with standard confocal microscopy, and can be done with virtually any HIV-1 strain and permissive cell lines or primary cells. Using ViewHIV, we find that CA enters the nucleus and associates with vDNA in both transformed and primary cells. We also find that CA’s interaction with the host polyadenylation factor, CPSF6, enhances nuclear entry and potentiates HIV-1’s depth of nuclear invasion, potentially aiding the virus’s integration into gene-dense regions. How HIV-1 overcomes our defenses and infects our cells has been studied intensively for over 30 years. While much has been learned, there remain events in the viral life cycle that resist interrogation. One such area is the initial intra-nuclear portion of infection, from the virus’s nuclear entry to its integration into chromatin. We reasoned that an image-based method would be useful for investigating this phase, and so we set about to develop methods that would permit the direct visualization of these early events. Infection commences with HIV-1’s binding to the host receptors, progressing to fusion of the host and viral membranes and entry of the viral core into the cytosol. A conical-shaped assembly comprised of ∼250 hexamers and 12 pentamers of capsid protein (CA; Ganser-Pornillos et al., 2007Ganser-Pornillos B.K. Cheng A. Yeager M. Structure of full-length HIV-1 CA: a model for the mature capsid lattice.Cell. 2007; 131: 70-79Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar), the core contains two copies of the virus’s RNA genome, reverse transcriptase (RT) and integrase (IN). After entry, the core partially uncoats to produce the reverse transcription complex (RTC), wherein RT synthesizes the viral DNA genome (vDNA). HIV-1s with CA mutations have shown that the efficiency of reverse transcription depends on the kinetics of core uncoating (Hulme et al., 2015bHulme A.E. Kelley Z. Okocha E.A. Hope T.J. Identification of capsid mutations that alter the rate of HIV-1 uncoating in infected cells.J. Virol. 2015; 89: 643-651Crossref PubMed Scopus (45) Google Scholar, Xu et al., 2013Xu H. Franks T. Gibson G. Huber K. Rahm N. Strambio De Castillia C. Luban J. Aiken C. Watkins S. Sluis-Cremer N. Ambrose Z. Evidence for biphasic uncoating during HIV-1 infection from a novel imaging assay.Retrovirology. 2013; 10: 70Crossref PubMed Scopus (59) Google Scholar, Yang et al., 2013Yang Y. Fricke T. Diaz-Griffero F. Inhibition of reverse transcriptase activity increases stability of the HIV-1 core.J. Virol. 2013; 87: 683-687Crossref PubMed Scopus (73) Google Scholar). The vDNA and its accompanying proteins are referred to as the preintegration complex (PIC). Associating with microtubules, the PIC travels toward the nucleus, gaining access via the nuclear pore complex (NPC). Once within the nucleus, IN interacts with LEDGF, a chromatin-associated factor, resulting in viral integration into actively transcribed genes (Ciuffi et al., 2005Ciuffi A. Llano M. Poeschla E. Hoffmann C. Leipzig J. Shinn P. Ecker J.R. Bushman F. A role for LEDGF/p75 in targeting HIV DNA integration.Nat. Med. 2005; 11: 1287-1289Crossref PubMed Scopus (490) Google Scholar). Recent studies have suggested that after 4 days of infection of CD4+ T cells, HIV-1 is found to be predominantly integrated into chromatin located at the nuclear periphery (Marini et al., 2015Marini B. Kertesz-Farkas A. Ali H. Lucic B. Lisek K. Manganaro L. Pongor S. Luzzati R. Recchia A. Mavilio F. et al.Nuclear architecture dictates HIV-1 integration site selection.Nature. 2015; 521: 227-231Crossref PubMed Scopus (214) Google Scholar). Current models estimate that the final stages of core uncoating occur at the NPC, with the PIC-associated proteins, i.e., CA, being shed prior to nuclear entry (Ambrose and Aiken, 2014Ambrose Z. Aiken C. HIV-1 uncoating: connection to nuclear entry and regulation by host proteins.Virology. 2014; 454-455: 371-379Crossref PubMed Scopus (122) Google Scholar, Hilditch and Towers, 2014Hilditch L. Towers G.J. A model for cofactor use during HIV-1 reverse transcription and nuclear entry.Curr. Opin. Virol. 2014; 4: 32-36Crossref PubMed Scopus (70) Google Scholar). While the existence of nuclear PIC-associated CA is under active study, one group’s data suggest that CA complexes with nuclear vDNA in primary macrophages, but not in HeLa cells (Peng et al., 2014Peng K. Muranyi W. Glass B. Laketa V. Yant S.R. Tsai L. Cihlar T. Müller B. Kräusslich H.G. Quantitative microscopy of functional HIV post-entry complexes reveals association of replication with the viral capsid.eLife. 2014; 3: e04114Crossref Scopus (126) Google Scholar), while a second work reports CA in HeLa cell nuclei (Hulme et al., 2015aHulme A.E. Kelley Z. Foley D. Hope T.J. Complementary Assays Reveal a Low Level of CA Associated with Viral Complexes in the Nuclei of HIV-1-Infected Cells.J. Virol. 2015; 89: 5350-5361Crossref PubMed Scopus (76) Google Scholar). Several findings argue that CA plays a role in the intra-nuclear viral life cycle (Ambrose et al., 2012Ambrose Z. Lee K. Ndjomou J. Xu H. Oztop I. Matous J. Takemura T. Unutmaz D. Engelman A. Hughes S.H. KewalRamani V.N. Human immunodeficiency virus type 1 capsid mutation N74D alters cyclophilin A dependence and impairs macrophage infection.J. Virol. 2012; 86: 4708-4714Crossref PubMed Scopus (69) Google Scholar, Lee et al., 2010Lee K. Ambrose Z. Martin T.D. Oztop I. Mulky A. Julias J.G. Vandegraaff N. Baumann J.G. Wang R. Yuen W. et al.Flexible use of nuclear import pathways by HIV-1.Cell Host Microbe. 2010; 7: 221-233Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar). The polyadenylation factor CPSF6 and the NPC proteins NUP153 and NUP358/RANBP2 interact with CA, thereby influencing nuclear entry and integration sites, although the mechanism is unclear for the latter (Bhattacharya et al., 2014Bhattacharya A. Alam S.L. Fricke T. Zadrozny K. Sedzicki J. Taylor A.B. Demeler B. Pornillos O. Ganser-Pornillos B.K. Diaz-Griffero F. et al.Structural basis of HIV-1 capsid recognition by PF74 and CPSF6.Proc. Natl. Acad. Sci. USA. 2014; 111: 18625-18630Crossref PubMed Scopus (166) Google Scholar, Matreyek et al., 2013Matreyek K.A. Yücel S.S. Li X. Engelman A. Nucleoporin NUP153 phenylalanine-glycine motifs engage a common binding pocket within the HIV-1 capsid protein to mediate lentiviral infectivity.PLoS Pathog. 2013; 9: e1003693Crossref PubMed Scopus (177) Google Scholar, Price et al., 2012Price A.J. Fletcher A.J. Schaller T. Elliott T. Lee K. KewalRamani V.N. Chin J.W. Towers G.J. James L.C. CPSF6 defines a conserved capsid interface that modulates HIV-1 replication.PLoS Pathog. 2012; 8: e1002896Crossref PubMed Scopus (187) Google Scholar, Schaller et al., 2011Schaller T. Ocwieja K.E. Rasaiyaah J. Price A.J. Brady T.L. Roth S.L. Hué S. Fletcher A.J. Lee K. KewalRamani V.N. et al.HIV-1 capsid-cyclophilin interactions determine nuclear import pathway, integration targeting and replication efficiency.PLoS Pathog. 2011; 7: e1002439Crossref PubMed Scopus (333) Google Scholar). CA mutant viruses that fail to interact with NUP358 or CPSF6 (for example, N74D/A) undergo aberrant integration and, in some instances, exhibit poor fitness (Ambrose et al., 2012Ambrose Z. Lee K. Ndjomou J. Xu H. Oztop I. Matous J. Takemura T. Unutmaz D. Engelman A. Hughes S.H. KewalRamani V.N. Human immunodeficiency virus type 1 capsid mutation N74D alters cyclophilin A dependence and impairs macrophage infection.J. Virol. 2012; 86: 4708-4714Crossref PubMed Scopus (69) Google Scholar, Krishnan et al., 2010Krishnan L. Matreyek K.A. Oztop I. Lee K. Tipper C.H. Li X. Dar M.J. Kewalramani V.N. Engelman A. The requirement for cellular transportin 3 (TNPO3 or TRN-SR2) during infection maps to human immunodeficiency virus type 1 capsid and not integrase.J. Virol. 2010; 84: 397-406Crossref PubMed Scopus (147) Google Scholar, Lee et al., 2010Lee K. Ambrose Z. Martin T.D. Oztop I. Mulky A. Julias J.G. Vandegraaff N. Baumann J.G. Wang R. Yuen W. et al.Flexible use of nuclear import pathways by HIV-1.Cell Host Microbe. 2010; 7: 221-233Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar, Schaller et al., 2011Schaller T. Ocwieja K.E. Rasaiyaah J. Price A.J. Brady T.L. Roth S.L. Hué S. Fletcher A.J. Lee K. KewalRamani V.N. et al.HIV-1 capsid-cyclophilin interactions determine nuclear import pathway, integration targeting and replication efficiency.PLoS Pathog. 2011; 7: e1002439Crossref PubMed Scopus (333) Google Scholar). The majority of these insights were obtained using established molecular virology and biochemistry methods evaluating cell populations. Importantly, antibody-based imaging of HIV-1 and fluorescence in situ hybridization (FISH) strategies to visualize the viral RNA genome (vRNA) and vDNA (Pezzella et al., 1987Pezzella M. Pezzella F. Galli C. Macchi B. Verani P. Sorice F. Baroni C.D. In situ hybridization of human immunodeficiency virus (HTLV-III) in cryostat sections of lymph nodes of lymphadenopathy syndrome patients.J. Med. Virol. 1987; 22: 135-142Crossref PubMed Scopus (21) Google Scholar, Singer et al., 1989Singer R.H. Byron K.S. Lawrence J.B. Sullivan J.L. Detection of HIV-1-infected cells from patients using nonisotopic in situ hybridization.Blood. 1989; 74: 2295-2301PubMed Google Scholar), as well as the use of chimeric viral proteins (Campbell and Hope, 2008Campbell E.M. Hope T.J. Live cell imaging of the HIV-1 life cycle.Trends Microbiol. 2008; 16: 580-587Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, Francis et al., 2014Francis A.C. Di Primio C. Quercioli V. Valentini P. Boll A. Girelli G. Demichelis F. Arosio D. Cereseto A. Second generation imaging of nuclear/cytoplasmic HIV-1 complexes.AIDS Res. Hum. Retroviruses. 2014; 30: 717-726Crossref PubMed Scopus (23) Google Scholar), also have improved our knowledge of HIV-1 infection at a single-cell level. Some of the acknowledged limitations of these approaches are a lack of sensitivity and disruptive preparative conditions (i.e., conventional FISH) and/or an inability to readily distinguish replication-competent viruses from replication-defective viruses (i.e., fluorescent viral fusion proteins). Several methods address such issues, including one employing modified dinucleotide triphosphates (dNTPs) that label reverse-transcribed vDNA (Peng et al., 2014Peng K. Muranyi W. Glass B. Laketa V. Yant S.R. Tsai L. Cihlar T. Müller B. Kräusslich H.G. Quantitative microscopy of functional HIV post-entry complexes reveals association of replication with the viral capsid.eLife. 2014; 3: e04114Crossref Scopus (126) Google Scholar) and another using single-cell imaging of HIV provirus (SCIP, Di Primio et al., 2013Di Primio C. Quercioli V. Allouch A. Gijsbers R. Christ F. Debyser Z. Arosio D. Cereseto A. Single-cell imaging of HIV-1 provirus (SCIP).Proc. Natl. Acad. Sci. USA. 2013; 110: 5636-5641Crossref PubMed Scopus (50) Google Scholar), which introduces a restriction enzyme cut site into the vDNA, permitting proviruses to be detected with an exogenous endonuclease. In a complementary approach, we adapted existing technologies and reagents. Specifically, we use a sensitive branch-chain DNA (bDNA) variant of FISH, ViewRNA (Yang et al., 2006Yang W. Maqsodi B. Ma Y. Bui S. Crawford K.L. McMaster G.K. Witney F. Luo Y. Direct quantification of gene expression in homogenates of formalin-fixed, paraffin-embedded tissues.Biotechniques. 2006; 40: 481-486Crossref PubMed Scopus (27) Google Scholar), in combination with immunolabeling using an established anti-CA monoclonal antibody (Simm et al., 1995Simm M. Shahabuddin M. Chao W. Allan J.S. Volsky D.J. Aberrant Gag protein composition of a human immunodeficiency virus type 1 vif mutant produced in primary lymphocytes.J. Virol. 1995; 69: 4582-4586Crossref PubMed Google Scholar), to visualize events in early HIV-1 infection in fixed cells. By combining bDNA technology and sandwich hybridization, this approach enhances the detection of nucleic acids (Yang et al., 2006Yang W. Maqsodi B. Ma Y. Bui S. Crawford K.L. McMaster G.K. Witney F. Luo Y. Direct quantification of gene expression in homogenates of formalin-fixed, paraffin-embedded tissues.Biotechniques. 2006; 40: 481-486Crossref PubMed Scopus (27) Google Scholar). The ViewRNA probes can be generated to specifically recognize much shorter targets than traditional FISH. This approach also uses a conventional confocal microscope. Among the HIV-1 life cycle events made more appreciable with this approach, we observed that the majority of reverse transcription occurs in the cytosolic periphery of primary macrophages and that loss of the nuclear importer TNP03 prevents PIC nuclear entry via mislocalization of TNP03’s cargo, the polyadenylation factor CPSF6. We find that HIV-1 CA enters the nuclei of HeLa cells, U20S cells, and monocyte-derived macrophages (MDMs), and it associates in part with the vDNA, suggesting that CA plays a functional role in HIV-1’s intra-nuclear life cycle in both primary and transformed cells. Consistent with this notion, our methods show that either the loss of CPSF6 or point mutations in CA that prevent interaction with CPSF6 decreases nuclear entry as well as the distance that the PIC penetrates into the nucleus, revealing a viral dependency on the host for intra-nuclear trafficking and integration into more centrally located actively transcribed genes. In studying influenza A virus genome trafficking, we used an established bDNA-based sandwich hybridization assay, ViewRNA (Yang et al., 2006Yang W. Maqsodi B. Ma Y. Bui S. Crawford K.L. McMaster G.K. Witney F. Luo Y. Direct quantification of gene expression in homogenates of formalin-fixed, paraffin-embedded tissues.Biotechniques. 2006; 40: 481-486Crossref PubMed Scopus (27) Google Scholar, Feeley et al., 2011Feeley E.M. Sims J.S. John S.P. Chin C.R. Pertel T. Chen L.M. Gaiha G.D. Ryan B.J. Donis R.O. Elledge S.J. Brass A.L. IFITM3 inhibits influenza A virus infection by preventing cytosolic entry.PLoS Pathog. 2011; 7: e1002337Crossref PubMed Scopus (284) Google Scholar). We estimated that this approach might be useful for studying HIV-1 because (1) it uses a pool of small probes that must anneal in a template-directed manner for signal amplification to occur, (2) the small probes can assemble on less accessible targets, and (3) the probes anneal strand specifically so that they could be designed to preferentially bind to the de novo synthesized vDNA of functional virions rather than the vRNA of inactive particles (Figure 1A). We created four probe sets against the indicated nucleotides of the cDNA of two cloned HIV-1 genomes, NL4-3 and HX2B (Figure 1B); the targeted regions are highly homologous across viral strains. The probe sets are denoted by the viral genes they were designed against as follows: gag, pol (two sets, A and B), and envelope (env). The env probe set was used in an infection time course (Figure 1C). HeLa-T4 cells were incubated on ice with HIV-IIIB, a CXCR-4-tropic viral population, and then warmed to 37°C to synchronize infection. At the indicated times, the cells were fixed and permeabilized, treated with protease, heated to denature vDNA, hybridized with the env probes, and subsequently confocally imaged. The vDNA was first seen at 4–6 hr post-infection (p.i.), increasing until 12 hr with a decrease at 24 hr. The vDNA signals were seen in the nuclei at 6 hr, with the peak nuclear signal at 12 hr. The mean number of vDNA signals present per nucleus for each condition is provided based on the analysis of ≥10 cells/condition over three experiments. While the exact number of vDNA molecules per signal cannot be determined using this approach, it stands to reason that the magnitude of the signal is proportional to the number of vDNA molecules each one contains; thus, this approach permits a relative, rather than absolute, comparison to be made across samples and experiments. Control samples used heat-killed (HK) virus, an IN inhibitor (entegravir, EVG), or an RT inhibitor (azidothymidine, AZT). The AZT-treated samples had ∼20-fold less vDNA signal at 12 hr p.i. than the untreated samples, indicating that the probe preferentially recognized de novo RT products. By 24 hr p.i., when the majority of the HIV-1 has integrated (Butler et al., 2001Butler S.L. Hansen M.S. Bushman F.D. A quantitative assay for HIV DNA integration in vivo.Nat. Med. 2001; 7: 631-634Crossref PubMed Scopus (577) Google Scholar), the vDNA signal of the HIV-1 alone samples had a ∼9-fold decrease in vDNA signals compared to the EVG samples, suggesting that the integration of the vDNA may prevent the probe set’s hybridization. In instances of many vDNA signals, there may be overlap so the level detected may be an underestimate. The low level of vDNA in the 24 hr p.i. AZT samples was likely from incomplete RT inhibition. These data demonstrate that the ViewHIV assay preferentially detects the vDNA products of functional viruses carrying out reverse transcription. Because the ViewHIV probe only detects the newly synthesized vDNA, and not the host DNA, this method allows the tracking over time of the vDNA component of the PIC, from its synthesis by RT in the cytosol to its subsequent nuclear entry. As noted, several lines of evidence point toward a nuclear role for CA. Both the level and timing of CA dissociation from the viral genome is a topic of active inquiry, with published data now suggesting that CA enters the nuclei of MDMs and HeLa cells (Hulme et al., 2015aHulme A.E. Kelley Z. Foley D. Hope T.J. Complementary Assays Reveal a Low Level of CA Associated with Viral Complexes in the Nuclei of HIV-1-Infected Cells.J. Virol. 2015; 89: 5350-5361Crossref PubMed Scopus (76) Google Scholar, Peng et al., 2014Peng K. Muranyi W. Glass B. Laketa V. Yant S.R. Tsai L. Cihlar T. Müller B. Kräusslich H.G. Quantitative microscopy of functional HIV post-entry complexes reveals association of replication with the viral capsid.eLife. 2014; 3: e04114Crossref Scopus (126) Google Scholar). In a complementary effort, we visualized CA along with the vDNA using the ViewHIV approach in conjunction with an anti-CA antibody. We tested several antibodies under the ViewHIV conditions with or without the protease treatment step (Figure S1A). Only the AG3.0 monoclonal antibody, which detects p24 from HIV-1, HIV-2, or SIV (Simm et al., 1995Simm M. Shahabuddin M. Chao W. Allan J.S. Volsky D.J. Aberrant Gag protein composition of a human immunodeficiency virus type 1 vif mutant produced in primary lymphocytes.J. Virol. 1995; 69: 4582-4586Crossref PubMed Google Scholar), produced a signal with or without protease treatment, with the former condition providing a markedly stronger result (Figure S1B). The baseline signal (no protease treatment) is consistent with data from publications using the AG3.0 antibody under similar conditions to those here (Fricke et al., 2014Fricke T. White T.E. Schulte B. de Souza Aranha Vieira D.A. Dharan A. Campbell E.M. Brandariz-Nuñez A. Diaz-Griffero F. MxB binds to the HIV-1 core and prevents the uncoating process of HIV-1.Retrovirology. 2014; 11: 68Crossref PubMed Scopus (123) Google Scholar, Lukic et al., 2014Lukic Z. Dharan A. Fricke T. Diaz-Griffero F. Campbell E.M. HIV-1 uncoating is facilitated by dynein and kinesin 1.J. Virol. 2014; 88: 13613-13625Crossref PubMed Scopus (96) Google Scholar). In addition, only protease treatment together with AG3.0 showed CA in the nucleus. We conclude that protease treatment enhances a weaker baseline AG3.0 signal (Supplemental Experimental Procedures). The ViewHIV assay was done in combination with anti-CA AG3.0 immunolabeling in HeLa-T4 cells synchronously infected with HIV-IIIB (Figure 2A). The vDNA and CA were detectable in the cytosol, with 67% ± 8% of vDNA colocalizing with CA in the cytosol at 12 hr p.i. (Figures 2B and 2C). CA signal decreased when an RT inhibitor, nevaripine (NVP), was used, suggesting that the CA epitope recognized by AG3.0 is more accessible upon reverse transcription, potentially via core uncoating. Thus, reverse transcription and proteolysis both enhance CA detection by AG3.0. The approach showed that CA entered the nuclei of HeLa cells by 12 hr p.i., with the majority of intra-nuclear CA colocalizing with vDNA (61% ± 9%, Figures 2B and 2C). These data are inconsistent with that of Peng et al., 2014Peng K. Muranyi W. Glass B. Laketa V. Yant S.R. Tsai L. Cihlar T. Müller B. Kräusslich H.G. Quantitative microscopy of functional HIV post-entry complexes reveals association of replication with the viral capsid.eLife. 2014; 3: e04114Crossref Scopus (126) Google Scholar, but similar to data from another group (Hulme et al., 2015aHulme A.E. Kelley Z. Foley D. Hope T.J. Complementary Assays Reveal a Low Level of CA Associated with Viral Complexes in the Nuclei of HIV-1-Infected Cells.J. Virol. 2015; 89: 5350-5361Crossref PubMed Scopus (76) Google Scholar); such differences may arise from the use of distinct protocols and antibodies. Staining for the nuclear envelope protein, Lamin B1, revealed no difference in the number of viral signals (HIV-IIIB) assigned to the nucleus using boundaries defined with either Lamin B1 or DAPI (Figure S1C). Therefore, we used the DAPI signal to define nuclear boundaries throughout. We examined the possibility that the CA detected in these assays is not brought in by the incoming virus, but instead represents de novo protein synthesis occurring post-infection. Assays performed with or without the protein translation inhibitor Lactimidomycin (LACT) (Schneider-Poetsch et al., 2010Schneider-Poetsch T. Ju J. Eyler D.E. Dang Y. Bhat S. Merrick W.C. Green R. Shen B. Liu J.O. Inhibition of eukaryotic translation elongation by cycloheximide and lactimidomycin.Nat. Chem. Biol. 2010; 6: 209-217Crossref PubMed Scopus (571) Google Scholar) detected no difference in CA signals (Figure S1D), demonstrating that the CA detected is brought by the incoming virus. These data show that CA enters the nucleus and colocalizes with vDNA in HeLa cells. The Leica SP-5 confocal microscope used for this work captures image sections in the transverse plane (xy plane, parallel to the adherent surface) that are 754–978 nm deep in the z plane, depending on the wavelength of light used (z sections, Figure 2D; Experimental Procedures). For HeLa and U2OS cells, images comprising one complete nucleus consist of 12–17 sequential z sections, with the most centrally located z section in this stack being defined as the central z section. To determine the best method for the detection of viral signals, we compared the number of vDNA or CA signals detected in the central z section with the total number of signals detected in a 3D reconstruction of the corresponding nucleus using all of the DAPI-containing z sections (vDNA, Figures 3A–3F) or in the 3D reconstruction of the entire cell (CA, Figures S2A–S2C), over a wide range of MOIs; this comparison showed a near linear relationship between the respective central z section and 3D reconstruction values. These experiments also showed that serially diluting the viral supernatant led to proportional changes in the viral signals detected, even down to the detection of a single viral signal per nucleus or cell. Therefore, the viral signals detected in central z sections permit comparisons over a wide range of MOIs to be made between cells and samples, similar to the results obtained by evaluating complete nuclei or cells. Using the 3D nuclear reconstructions as a start, we also generated image sections that are orthogonal to the central z section transverse plane and of comparable volumes, referred to as central orthogonal sections (Figures 3B–3E); these would be analogous to coronal plane images in an anatomic setting. This was done to evaluate if CA and vDNA were clearly within the nucleus in both planar surface orientations, as well as to determine in what regions of the nucleus these viral signals were detected. The latter analysis was structured by dividing the nucleus into three regions of equivalent volumes as follows: peripheral nuclear (PN), middle nuclear (MN), and central nuclear (CN, Figure 2D; Experimental Procedures); these studies found that CA and vDNA were detected within the nucleus using both the central z and central orthogonal sections, and with the same relative regional distributions (Figures 3F and S2). To further test the validity of using the central z section for these analyses, both the number and distribution of viral signals (CA and vDNA) in the central orthogonal section of each nucleus (shown as zy or zx planes, Figure S3A) were compared with these same values obtained by analyzing the central z section (xy plane) of the corresponding nucleus (Figures S3A–S3E); as seen with the 3D reconstruction versus central z section comparisons, both the number and position of the CA and vDNA signals were very similar between the paired central orthogonal and central z sections. Therefore, when viral signals per nucleus are provided in the text or figures, those values represent the signals detected in the central z section. To determine if this approach can detect CA entering the nucleus in cells that are relevant to HIV-1 infection in vivo, we tested the ViewHIV assay on MDMs from patient donors. Using HIV-1 BaL virus, a CCR5-tropic viral population, we initiated a synchronized infection and then processed the cells at the indicated times (Figures 3G and 4A ). The vDNA was detected in the untreated and EVG samples, but not in the HK virus or NVP samples. Similar to the data in HeLa cells, vDNA first appeared in the peripheral cytosol of the MDMs at 4–6 hr, with the greatest level of vDNA signals being detected at 12 hr in the untreated samples and at 24 hr in the EVG samples (Figure 4A). The ViewHIV probe set was used in combination with the anti-CA antibody in assays with MDMs (Figures 3G and 4B). Similar to HeLa cells, CA entered the nuclei of MDMs and colocalized in part with vDNA (Figures 3G, 4B, and 4C). These data are consistent with the results of Peng et al., 2014Peng K. Muranyi W. Glass B. Laketa V. Yant S.R. Tsai L. Cihlar T. Müller B. Kräusslich H.G. Quantitative microscopy of functional HIV post-entry complexes reveals association of replication with the viral capsid.eLife. 2014; 3: e04114Crossref Scopus (126) Google Scholar; 59% ± 10% of the nuclear CA colocalized with vDNA (Figures 4D and S3F). Similar to the HeLa cells, most of the vDNA and CA signals were lost by 24 hr p.i. The inhibition of integration also increased the level of nuclear vDNA and CA. An RT inhibitor (AZT) resulted in not only less vDNA signal but also less CA signal. We note that although the levels of both CA and vDNA signals were considerably lower in MDMs than in HeLa cells, their presence supports the relevance of results using transformed cells. We next assessed the usefulness of the ViewHIV approach to investigate the role of host factors (MxB, WNK1, and COG2 and 3) in viral replication (Supplemental Experimental Procedures; Figures S4A–S4H and S5A–S5G). The host factors NUP153, TNPO3, and CPSF6 all modulate HIV-1 nuclear entry and integration (Brass et al., 2008Brass A.L. Dykxhoorn D.M. Benita Y. Yan N. Engelman A. Xavier R.J. Lieberman J. Elledge S.J. Identification of host proteins required for HIV infection through a functional genomic screen.Science. 2008; 319: 921-926Crossref PubMed Scopus (1177) Google Scholar, Christ et al., 2008Christ F. Thys W. De Rijck J. Gijsbers R. Albanese A. Arosio D. Emiliani S. Rain J.C. Benarous R. Cereseto A. Debyser Z. Transportin-SR2 imports HIV into the nucleus.Curr. Biol. 2008; 18: 1192-1202Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, Lee et al., 2010Lee K. Ambrose Z. Martin T.D. Oztop I. Mulky A. Julias J.G. Vandegraaff N. Baumann J.G. Wang R. Yuen W. et al.Flexible use of nuclear import pathways by HIV-1.Cell Host Microbe. 2010; 7: 221-233Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar). Hypotheses regarding the role of TNPO3 in HIV-1 replication include it bei" @default.
• W2176753208 created "2016-06-24" @default.
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• W2176753208 date "2015-11-01" @default.
• W2176753208 modified "2023-10-14" @default.
• W2176753208 title "Direct Visualization of HIV-1 Replication Intermediates Shows that Capsid and CPSF6 Modulate HIV-1 Intra-nuclear Invasion and Integration" @default.
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