Identification of a Human Immunodeficiency Virus Type 2 (HIV-2) Encapsidation Determinant and Transduction of Nondividing Human Cells by HIV-2-Based Lentivirus Vectors

Replication-defective retrovirus vectors are advantageous for gene transfer because they permit permanent chromosomal integration and stable gene expression. Following entry into target cells, however, murine retrovirus and retrovirus vectors require mitosis-dependent dissolution of the nuclear envelope to achieve integration (36, 43). Therefore, these vectors can stably transduce dividing cells but possess limited utility for gene delivery to quiescent or postmitotic cells that are important targets for gene therapy. In contrast, lentiviruses infect nondividing cells (36). For human immunodeficiency virus type 1 (HIV-1), this property has been mapped to establishment of a stable preintegration complex and to virion proteins that mediate transport of the preintegration complex across an intact nuclear envelope (6, 17, 18, 22, 55). Accordingly, retrovirus vectors derived from HIV-1 (35) can transduce growth-arrested and terminally differentiated, postmitotic cells. Naldini et al. (45, 46) established that these lentivirus-specific biological properties hold for HIV-1-derived vectors and showed their capacity for in vitro and in vivo gene delivery. HIV-1 vectors have now been engineered to reduce both the risks for recombination and the complement of genes needed for transduction of neuronal cells (65). However, some safety concerns remain incompletely explored, since the determinants of the severe pathogenicity of HIV-1 in humans remain uncertain and no animal model amenable to testing of disease causation by the parental lentivirus exists (16). In addition, some of the accessory genes of HIV-1 appear to be required for targeting of some tissues in vivo—vif and vpr for hepatocytes, for example (25). In this regard, simian immunodeficiency virus (SIV) with multiple nonstructural gene deletions has been shown to cause disease in infant primates (2) and more recently even in adult animals (51). Safety of HIV-2 vectors can be tested in primates susceptible to HIV-2/SIV pathogenicity (50). In addition, HIV-2 accounts for less than 1% of human HIV infections worldwide and has now been documented to be both less transmissible sexually and less pathogenic in longitudinally studied West African human populations (26, 39). In the most comprehensive, prospective natural history study, all indices of virulence, including HIV-related morbidity and CD4+ lymphocyte depletion, were much lower for HIV-2-infected than HIV-1-infected subjects in the same West African population; 5-year AIDS-free survival was 100% in the HIV-2 cohort (39). These considerations prompted us to study the feasibility of a replication-defective lentivirus vector system derived from HIV-2. To further enhance safety potential, we have used HIV-2KR (57), a molecular clone that was apathogenic following infection established by high-dose intravenous challenge in pig-tailed macaques and either delayed or prevented disease induction by subsequent challenge with highly virulent HIV-2EHO (38). Genomic regions that determine mRNA encapsidation are crucial to retrovirus vector system design but have received very limited study for HIV-2. In the present work, we initially focused on studying the effects of deleting the region between the major splice donor (SD) and the gag start codon (following convention, in this paper this segment of retroviral genomes is designated “ψ”). In murine oncoretroviruses such as Moloney murine leukemia virus (Mo-MuLV), ψ is relatively long (351 nucleotides [nt]), and deletions in ψ markedly attenuate genomic mRNA encapsidation. In addition, attachment of ψ (and more optimally, the ψ′ segment, which includes ψ plus a portion of gag) to heterologous test RNAs confers nearly wild-type levels of encapsidation (3, 37). In HIV-1, ψ is considerably shorter (44 nt), and a 21-nt deletion in the region also suffices to greatly reduce or prevent HIV-1 encapsidation (34). However, requirements for HIV-1 encapsidation appear more complex than for murine retroviruses: an important distinction is that neither ψ nor ψ′ from HIV-1 can confer efficient encapsidation when attached to heterologous test RNAs; involvement of regions outside of ψ and ψ′ in HIV-1 RNA packaging have been suggested (4, 5, 10, 20, 27, 35, 40, 62). A complete description of packaging determinants has not been achieved for any lentivirus: interaction of multiple regions distributed widely within the HIV-1 genome has been proposed (5). HIV-2 packaging determinants are potentially even more complex. For example, deletions in HIV-2 ψ were reported to variably increase or decrease HIV-2 genome encapsidation without inhibiting infectivity and to produce an increase in HIV-2 LTR expression in a transient proviral transfection assay (19). In one study of the closely related lentivirus SIVmac, the leader sequence upstream of the major 5′ SD was reported to be the principal packaging determinant (52). Recently, deletions within the ψ region of HIV-2 were reported to have minimal effects on encapsidation or replication, while regions in the 5′ leader (that are also present in all spliced RNAs) severely reduced gag/pol mRNA packaging (41); this finding implies the existence of other genomic encapsidation signals, as some means of discriminating the full-length mRNA from spliced viral messages must be available to the packaging machinery. Another level of complexity stems from the functional intron within the HIV-2 long terminal repeat (LTR) (9, 12, 61, 63), a situation which is unique in retroviruses and which provides another potential means of distinguishing genomic from subgenomic mRNAs. In this study, regions of the HIV-2 genome that, when deleted, prevent genomic encapsidation and replication but not viral protein expression were identified. These data were then used to construct an HIV-2-based retrovirus vector system, and vectors were tested for the ability to transduce dividing, growth-arrested, and terminally differentiated human cells. Aphidicolin-arrested cells, monocyte-derived macrophages, and a terminally differentiated postmitotic human neuronal cell culture model (NTN2 neurons) were transduced efficiently with these vectors. NTN2 neurons are a polarized human neuronal cell system derived from NT2 teratocarcinoma cells by a 6-week process using retinoic acid and several mitotic inhibitors (13, 14). Third-replate cells, used in this study, are irreversibly postmitotic, morphologically resemble primary neurons, express a number of neuron-specific markers, and can be maintained on a basement membrane matrix as clumps of neurons that elaborate functional axons and dendrites (13, 14, 29, 48, 49). The ability to lentivirus vectors to transduce quiescent hematopoietic cells or their pluripotent precursors remains uncertain. Indeed, a large body of literature suggests that resting T cells (cells in G0, which represent the majority of peripheral blood T lymphocytes) cannot be productively infected with HIV; arrest of reverse transcription at various stages and variable rescuability of such intermediates by subsequent cycling have been reported (55, 56, 58, 64). We compared the abilities of HIV-2 vectors and Mo-MuLV vectors to target primitive (CD34+ CD38−) hematopoietic progenitor cells that are not actively cycling. These cells have proven elusive in transduction experiments using murine retrovirus vectors (1, 47). Hematopoietic stem cells possess dual properties of self-renewal and multilineage differentiation (44). These rare cells are largely quiescent and divide stochastically in vivo. Since no specific assay exists for true stem cells, they have been operationally defined by the CD34 antigen (expressed by 1 to 3% of bone marrow cells) and lack of expression of the CD38 antigen in combination with other lineage markers. A preponderance of studies have indicated that human pluripotent marrow-repopulating ability resides within the small fraction of total CD34+ cells that are CD38− (23, 44, 47). A consensus stem cell phenotype has been suggested by numerous studies: CD34+ CD38− CD33− HLA-DR− Thy-1lo Lin− CD45RO+ rhodamine-123dull (44, 47, 60). Since ex vivo manipulation and retrovirus vector transduction may trigger cell cycling and differentiation of CD34+ cells, we performed multiparameter flow cytometry after transduction to sort these cells according to surface expression of CD34 and CD38 as well as cell division history and then assayed sorted subsets for the marker gene. The results showed a preference of HIV-2 vectors over murine vectors to establish proviral DNA in this cell population. However, the integration status of the vector DNA remains to be determined.