Mary Peretz

LEDGF/p75 Determines Cellular Trafficking of Diverse Lentiviral but Not Murine Oncoretroviral Integrase Proteins and Is a Component of Functional Lentiviral Preintegration Complexes

ABSTRACT Human immunodeficiency virus type 1 (HIV-1), feline immunodeficiency virus (FIV), and Moloney murine leukemia virus (MoMLV) integrases were stably expressed to determine their intracellular trafficking. Each lentiviral integrase localized to cell nuclei in close association with chromatin while the murine oncoretroviral integrase was cytoplasmic. Fusions of pyruvate kinase to the lentiviral integrases did not reveal transferable nuclear localization signals. The intracellular trafficking of each was determined instead by the transcriptional coactivator LEDGF/p75, which was required for nuclear localization. Stable small interfering RNA expression eliminated detectable LEDGF/p75 expression and caused dramatic, stable redistribution of each lentiviral integrase from nucleus to cytoplasm while the distribution of MoMLV integrase was unaffected. In addition, endogenous LEDGF/p75 coimmunoprecipitated specifically with each lentiviral integrase. In vitro integration assays with preintegration complexes (PICs) showed that endogenous LEDGF/p75 is a component of functional HIV-1 and FIV PICs. However, HIV-1 and FIV infection and replication in LEDGF/p75-deficient cells was equivalent to that in control cells, whether cells were dividing or growth arrested. Two-long terminal repeat circle accumulation in nondividing cell nuclei was also equivalent to that of LEDGF/p75 wild-type cells. Virions produced in LEDGF/p75-deficient cells had normal infectivity. We conclude that LEDGF/p75 fully accounts for cellular trafficking of diverse lentiviral, but not oncoretroviral, integrases and is the main lentiviral integrase-to-chromatin tethering factor. While lentiviral PIC nuclear import is unaffected by LEDGF/p75 knockdown, this protein is a component of functional lentiviral PICs. A role in HIV-1 integration site distribution merits investigation.

Identification of the LEDGF/p75 HIV-1 integrase-interaction domain and NLS reveals NLS-independent chromatin tethering

To investigate the basis for the LEDGF/p75 dependence of HIV-1 integrase (IN) nuclear localization and chromatin association, we used cell lines made stably deficient in endogenous LEDGF/p75 by RNAi to analyze determinants of its location in cells and its ability to interact with IN. Deletion of C-terminal LEDGF/p75 residues 340-417 preserved nuclear and chromatin localization but abolished the interaction with IN and the tethering of IN to chromatin. Transfer of this IN-binding domain (IBD) was sufficient to confer HIV-1 IN interaction to GFP. HRP-2, the only other human protein with an identifiable IBD domain, was found to translocate IN to the nucleus of LEDGF/p75(–) cells. However, in contrast to LEDGF/p75, HRP-2 is not chromatin bound and does not tether IN to chromatin. A single classical nuclear localization signal (NLS) in the LEDGF/p75 N-terminal region (146RRGRKRKAEKQ156) was found by deletion mapping and was shown to be transferable to pyruvate kinase. Four central basic residues in the NLS are critical for its activity. Strikingly, however, stable expression studies with NLS(+/–) and IBD(+/–) mutants revealed that the NLS, although responsible for LEDGF/p75 nuclear import, is dispensable for stable, constitutive nuclear association of LEDGF/p75 and IN. Both wild-type LEDGF/p75 and NLS-mutant LEDGF/p75 remain entirely chromatin associated throughout the cell cycle, and each tethers IN to chromatin. Thus, these experiments reveal stable nuclear sequestration of a transcriptional regulator by chromatin during the nuclear-cytosolic mixing of cell division, which additionally enables stable tethering of IN to chromatin. LEDGF/p75 is a multidomain adaptor protein that interacts with the nuclear import apparatus, lentiviral IN proteins and chromatin by means of an NLS, an IBD and additional chromatin-interacting domains.

Unintegrated lentivirus DNA persistence and accessibility to expression in nondividing cells: analysis with class I integrase mutants.

ABSTRACT The circumstances under which unintegrated lentivirus DNA can persist and be a functional template for transcription and protein expression are not clear. We constructed and validated the first class I (nonpleiotropic) integrase (IN) mutants for a non-human lentivirus (feline immunodeficiency virus [FIV]) and analyzed both these and known class I human immunodeficiency virus type 1 IN mutants. The FIV IN mutants (D66V and D66V/D118A) had class I properties: Gag/Pol precursor expression, proteolytic processing, particle formation, and reverse transcriptase (RT) production were normal, while the transduction of dividing fibroblasts was prevented and integration was blocked. When injected into rat retinas, the wild-type (WT) vector produced extensive, persistent transgene expression, compared with only rare positive neuronal cells for the IN mutant vector. In contrast, both WT and mutant vectors produced entirely equivalent, effective transduction levels of primary rat neurons (retinal ganglion cells). By testing the hypothesis that the unexpected retinal neuron transduction was related to cell cycle status, we found that when fibroblasts were growth arrested, transduction and internally promoted transgene expression were not inhibited at all by the class I FIV or HIV-1 IN mutations. Cells were then transduced under aphidicolin arrest and were released from the block 48 h later. Vector expression was stable and durable during repeated passaging in WT vector-transduced cells, while the release of cells transduced with equivalent RT units of class I IN mutant FIV or HIV vector resulted in a steady decline of expression, from 97 to 0% of cells by day 10. Southern blot and PCR analyses showed a lack of integration, irrespective of cell cycle, for the class I mutants and an increase in one- and two-long terminal repeat circular and linear unintegrated DNAs in growth-arrested cells. We conclude that if cell division is prevented, unintegrated FIV and HIV-1 vector DNAs can produce high-level internally promoted transgene expression equivalent to WT vectors. The expression correlates with the unintegrated DNA levels. These observations may facilitate the study of the roles of IN and other preintegration complex components in preintegration phases of infection by (i) providing an alternative way to monitor unintegrated nuclear cDNA forms, (ii) restricting ascertainment to the transcriptionally functional subset of unintegrated DNA, (iii) enabling analysis in individual, nondividing cells, and (iv) uncoupling other potential functions of IN from integration.

Genetic Modification of Human Trabecular Meshwork with Lentiviral Vectors

Glaucoma, a group of optic neuropathies, is the leading cause of irreversible blindness. Neuronal apoptosis in glaucoma is primarily associated with high intraocular pressure caused by chronically impaired outflow of aqueous humor through the trabecular meshwork, a reticulum of mitotically inactive endothelial-like cells located in the angle of the anterior chamber. Anatomic, genetic, and expression profiling data suggest the possibility of using gene transfer to treat glaucomatous intraocular pressure dysregulation, but this approach will require stable genetic modification of the differentiated aqueous outflow tract. We injected transducing unit-normalized preparations of either of two lentiviral vectors or an oncoretroviral vector as a single bolus into the aqueous circulation of cultured human donor eyes, under perfusion conditions that mimicked natural anterior chamber flow and maintained viability ex vivo. Reporter gene expression was assessed in trabecular meshwork from 3 to 16 days after infusion of 1.0 x 10(8) transducing units of each vector. The oncoretroviral vector failed to transduce the trabecular meshwork. In contrast, feline immunodeficiency virus and human immunodeficiency virus vectors produced efficient, localized transduction of the trabecular meshwork in situ. The results demonstrate that lentiviral vectors permit efficient genetic modification of the human trabecular meshwork when delivered via the afferent aqueous circulation, a clinically accessible route. In addition, controlled comparisons in this study establish that feline and human immunodeficiency virus vectors are equivalently efficacious in delivering genes to this terminally differentiated human tissue.

LEDGF/p75 Proteins with Alternative Chromatin Tethers Are Functional HIV-1 Cofactors

LEDGF/p75 can tether over-expressed lentiviral integrase proteins to chromatin but how this underlies its integration cofactor role for these retroviruses is unclear. While a single integrase binding domain (IBD) binds integrase, a complex N-terminal domain ensemble (NDE) interacts with unknown chromatin ligands. Whether integration requires chromatin tethering per se, specific NDE-chromatin ligand interactions or other emergent properties of LEDGF/p75 has been elusive. Here we replaced the NDE with strongly divergent chromatin-binding modules. The chimeras rescued integrase tethering and HIV-1 integration in LEDGF/p75-deficient cells. Furthermore, chromatin ligands could reside inside or outside the nucleosome core, and could be protein or DNA. Remarkably, a short Kaposis sarcoma virus peptide that binds the histone 2A/B dimer converted GFP-IBD from an integration blocker to an integration cofactor that rescues over two logs of infectivity. NDE mutants were corroborative. Chromatin tethering per se is a basic HIV-1 requirement and this rather than engagement of particular chromatin ligands is important for the LEDGF/p75 cofactor mechanism.

Identification of a Central DNA Flap in Feline Immunodeficiency Virus

ABSTRACT A duplication of the polypurine tract (PPT) at the center of the human immunodeficiency virus type 1 (HIV-1) genome (the cPPT) has been shown to prime a separate plus-strand initiation and to result in a plus-strand displacement (DNA flap) that plays a role in nuclear import of the viral preintegration complex. Feline immunodeficiency virus (FIV) is a lentivirus that infects nondividing cells, causes progressive CD4+ T-cell depletion, and has been used as a substrate for lentiviral vectors. However, the PPT sequence is not duplicated elsewhere in the FIV genome and a central plus-strand initiation or strand displacement has not been identified. Using Southern blotting of S1 nuclease-digested FIV preintegration complexes isolated from infected cells, we detected a single-strand discontinuity at the approximate center of the reverse-transcribed genome. Primer extension analyses assigned the gap to the plus strand, and mapped the 5′ terminus of the downstream (D+) segment to a guanine residue in a purine-rich tract in pol(AAAAGAAGAGGTAGGA). RACE experiments then mapped the 3′ terminus of the upstream plus (U+)-strand segment to a T nucleotide located 88 nucleotides downstream of the D+ strand 5′ terminus, thereby identifying the extent of D+ strand displacement and the central termination sequence of this virus. Unlike HIV, the FIV cPPT is significantly divergent in sequence from its 3′ counterpart (AAAAAAGAAAAAAGGGTGG) and contains one and in some cases two pyrimidines. An invariant thymidine located −2 to the D+ strand origin is neither required nor optimal for codon usage at this position. Although the mapped cPPTs of FIV and HIV-1 act in cis, they encode homologous amino acids in integrase.

Comparison of wild-type and class I integrase mutant-FIV vectors in retina demonstrates sustained expression of integrated transgenes in retinal pigment epithelium.

In neonatal and adult rodent retina, substantial lentiviral vector expression has been detected primarily in retinal pigment epithelium (RPE), except in very young animals (2–5 days post‐natal). In non‐retinal tissues, studies of lentiviral vectors have utilized various controls. Among the most stringent are class I integrase mutants, which selectively block the integration reaction while leaving all other gag/pol‐encoded functions intact. For HIV‐1 vectors injected into brain, these have been used to simultaneously control for pseudotransduction and verify that long‐term expression requires integration. Such experiments compare particles that differ only in a single amino acid within a single enzyme that forms a very small molar fraction of the virion. Class I integrase mutants have not been described for feline immunodeficiency virus (FIV) integrase, or tested in the eye for any lentiviral vector.

Productive Replication of vif-Chimeric HIV-1 in Feline Cells

ABSTRACT Nonprimate animal models of HIV-1 infection are prevented by missing cellular cofactors and by antiviral actions of species-specific host defense factors. These blocks are profound in rodents but may be less abundant in certain Carnivora. Here, we enabled productive, spreading replication and passage of HIV-1 in feline cells. Feline fibroblasts, T-cell lines, and primary peripheral blood mononuclear cells supported early and late HIV-1 life cycle phases in a manner equivalent to that of human cells, except that produced virions had low infectivity. Stable expression of feline immunodeficiency virus (FIV) Vif-green fluorescent protein (GFP) in HIV-1 entry receptor-complemented feline (CrFK) cells enabled robust spreading HIV-1 replication. FIV Vif colocalized with feline APOBEC3 (fA3) proteins, targeted them for degradation, and prevented G→A hypermutation of the HIV-1 cDNA by fA3CH and fA3H. HIV-1 Vif was inactive against fA3s as expected and even paradoxically augmented restriction in some assays. In an interesting contrast, simian immunodeficiency virus SIVmac Vif had substantial anti-fA3 activities, which were complete against fA3CH and partial against fA3H. Moreover, both primate lentiviral Vifs colocalized with fA3s and could be pulled down from cell lysates by fA3CH. HIV-1 molecular clones that encode FIV Vif or SIVmac Vif (HIV-1VF and HIV-1VS) were then constructed. These viruses replicated productively in HIV-1 receptor-expressing CrFK cells and could be passaged serially to uninfected cells. Thus, with the exception of entry receptors, the cat genome can supply the dependency factors needed by HIV-1, and a main restriction can be countered by vif chimerism. The results raise the possibility that the domestic cat could yield an animal model of HIV-1 infection.

The HIV-1 Central Polypurine Tract Functions as a Second Line of Defense against APOBEC3G/F

ABSTRACT HIV-1 and certain other retroviruses initiate plus-strand synthesis in the center of the genome as well as at the standard retroviral 3′ polypurine tract. This peculiarity of reverse transcription results in a central DNA “flap” structure that has been of controversial functional significance. We mutated both HIV-1 flap-generating elements, the central polypurine tract (cPPT) and the central termination sequence (CTS). To avoid an ambiguity of previous studies, we did so without affecting integrase coding. DNA flap formation was disrupted but single-cycle infection was unaffected in all target cells tested, regardless of cell cycle status. Spreading HIV-1 infection was also normal in most T cell lines, and flap mutant viruses replicated equivalently to the wild type in nondividing cells, including macrophages. However, spreading infection of flap mutant HIV-1 was impaired in non-vif-permissive cells (HuT78, H9, and primary human peripheral blood mononuclear cells [PBMCs]), suggesting APOBEC3G (A3G) restriction. Single-cycle infections confirmed that vif-intact flap mutant HIV-1 is restricted by producer cell A3G/F. Combining the Δvif and cPPT-CTS mutations increased A3G restriction synergistically. Moreover, RNA interference knockdown of A3G in HuT78 cells released the block to flap mutant HIV-1 replication. Flap mutant HIV-1 also accrued markedly increased A3G-mediated G→A hypermutation compared to that of wild-type HIV-1 (a full log10 in the 0.36 kb downstream of the mutant cPPT). We suggest that the triple-stranded DNA structure, the flap, is not the consequential outcome. The salient functional feature is central plus-strand initiation, which functions as a second line of defense against single-stranded DNA editing by A3 proteins that survive producer cell degradation by Vif.

Blockade of Human Immunodeficiency Virus Type 1 Expression by Caveolin-1

ABSTRACT Caveolin-1 (Cav-1) is a major protein constituent of caveolae, a type of plasma membrane raft. We observed that coexpression of human Cav-1 with human immunodeficiency virus type 1 (HIV-1) blocked virion production from cells that are ordinarily highly permissive. Further investigation showed that this effect is specific, occurs at low ratios of Cav-1 to HIV-1 DNA, depends on expression of Cav-1 protein, and involves severely impaired expression of HIV-1 proteins. Cav-1 also blocked HIV-2 expression. In contrast, Cav-1 did not inhibit protein expression by a paramyxovirus and did not induce apoptosis or affect cellular morphology, cell viability, or cell cycle progression. Although only small amounts of HIV-1 virions were released from Cav-1-transfected cells, these were fully infectious. Deletion mutagenesis showed that the C-terminal 78 residues were as active as the full-length (178-amino-acid) protein in producing the block. In contrast, the 100 most N-terminal amino acids of Cav-1, which include the previously identified oligomerization and scaffolding domains, were shown to be dispensable. Study of single-amino-acid-exchange mutants of Cav-1 established that palmitoylation was not required. Additional deletion mutants then identified the hydrophobic, membrane-associated domain (residues 101 to 135) as the main determinant. Cellular distribution of wild-type and mutant proteins correlated with ability to block HIV-1 expression. Finally, Cav-2 also blocked HIV-1 expression. These data show that coexpression of caveolins can markedly inhibit expression of HIV proviral DNA and establish that the inhibition is mediated by the hydrophobic, membrane-associated domain.