Nuclear localization of human immunodeficiency virus type 1 preintegration complexes (PICs): V165A and R166A are pleiotropic integrase mutants primarily defective for integration,not PIC nuclear import

Retroviral replication requires the integration of reverse-transcribed viral cDNA into a cell chromosome. A key barrier to forming the integrated provirus is the nuclear envelope, and numerous regions in human immunodeficiency virus type 1 (HIV-1) have been shown to aid the nuclear localization of viral preintegration complexes (PICs) in infected cells. One region in integrase (IN), composed of Val-165 and Arg-166, was reportedly essential for HIV-1 replication and nuclear localization in all cell types. In this study we confirmed that HIV-1(V165A) and HIV-1(R166A) were replication defective and that less mutant viral cDNA localized to infected cell nuclei. However, we present three lines of evidence that argue against a specific role for Val-165 and Arg-166 in PIC nuclear import. First, results of transient transfections revealed that V165A FLAG-tagged IN and green fluorescent protein-IN fusions carrying either V165A or R166A predominantly localized to cell nuclei. Second, two different strains of previously described class II IN mutant viruses displayed similar nuclear entry profiles to those observed for HIV-1(V165A) and HIV-1(R166A), suggesting that defective nuclear import may be a common phenotype of replication-defective IN mutant viruses. Third, V165A and R166A mutants were defective for in vitro integration activity, when assayed both as PICs isolated from infected T-cells and as recombinant IN proteins purified from Escherichia coli. Based on these results, we conclude that HIV-1(V165A) and HIV-1(R166A) are pleiotropic mutants primarily defective for IN catalysis and that Val-165 and Arg-166 do not play a specific role in the nuclear localization of HIV-1 PICs in infected cells.     

The HIV-1 passage from cytoplasm to nucleus: the process involving a complex exchange between the components of HIV-1 and cellular machinery to access nucleus and successful integration

The human immunodeficiency virus 1 (HIV-1) synthesizes its genomic DNA in cytoplasm as soon as it enters the cell. The newly synthesized DNA remains associated with viral/cellular proteins as a high molecular weight pre-integration complex (PIC), which precludes passive diffusion across intact nuclear membrane. However, HIV-1 successfully overcomes nuclear membrane barrier by actively delivering its DNA into nucleus with the help of host nuclear import machinery. Such ability allows HIV-1 to productively infect non-dividing cells as well as dividing cells at interphase. Further, HIV-1 nuclear import is also found important for the proper integration of viral DNA. Thus, nuclear import plays a crucial role in establishment of infection and disease progression. While several viral components, including matrix, viral protein R, integrase, capsid, and central DNA flap are implicated in HIV-1 nuclear import, their molecular mechanism remains poorly understood. In this review, we will elaborate the role of individual viral factors and some of current insights on their molecular mechanism(s) associated with HIV-1 nuclear import. In addition, we will discuss the importance of nuclear import for subsequent step of viral DNA integration. Hereby we aim to further our understanding on molecular mechanism of HIV-1 nuclear import and its potential usefulness for anti-HIV-1 strategies.     

Identification of a central DNA flap in feline immunodeficiency virus

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.     

Nuclear export of Vpr is required for efficient replication of human immunodeficiency virus type 1 in tissue macrophages

Retroviruses must gain access to the host cell nucleus for subsequent replication and viral propagation. Human immunodeficiency virus type 1 (HIV-1) and other primate lentiviruses are distinguished from the gammaretroviruses by their ability to infect nondividing cells such as macrophages, an important viral reservoir in vivo. Rather than requiring nuclear membrane breakdown during cell division, the HIV-1 preintegration complex (PIC) enters the nucleus by traversing the central aqueous channel of the limiting nuclear pore complex. The HIV-1 PIC contains three nucleophilic proteins, matrix, integrase, and Vpr, all of which have been implicated in nuclear targeting. The mechanism by which Vpr can display such nucleophilic properties and yet also be available for incorporation into virions assembling at the plasma membrane is unresolved. We recently characterized Vpr as a nucleocytoplasmic shuttling protein that contains two novel nuclear import signals and an exportin-1-dependent nuclear export signal (NES). We now demonstrate that mutation of this NES impairs the incorporation of Vpr into newly formed virions. Furthermore, we find that the Vpr NES is required for efficient HIV replication in tissue macrophages present in human spleens and tonsils. These findings underscore how the nucleocytoplasmic shuttling of Vpr not only contributes to nuclear import of the HIV-1 PIC but also enables Vpr to be present in the cytoplasm for incorporation into virions, leading to enhancement of viral spread within nondividing tissue macrophages.     

Transportin-SR2 imports HIV into the nucleus

BACKGROUND: The human immunodeficiency virus type 1 (HIV-1) and other lentiviruses have the capacity to infect nondividing cells like macrophages. This requires import of the preintegration complex (PIC) through the nuclear pore. Although many cellular and viral determinants have been proposed, the mechanism leading to nuclear import is not yet understood. RESULTS: Using yeast two-hybrid and pull-down, we identified and validated transportin-SR2 (TRN-SR2) as a bona fide binding partner of HIV-1 integrase. We confirmed the biological relevance of this interaction by RNAi. Depletion of TRN-SR2 interfered with the replication of HIV-1 and HIV-2 but not MoMLV in HeLaP4 cells. Knockdown of TRN-SR2 in primary macrophages likewise interfered with HIV-1 replication. Using Q-PCR, we pinpoint this block in replication to the early steps of the viral lifecycle. A reduction in 2-LTR formation suggests a block in PIC nuclear import upon siRNA-mediated knockdown. Different lines of evidence clearly proved that the late steps of viral replication are not affected. In an in vivo nuclear-import assay using labeled HIV-1 particles, the defect in nuclear import after depletion of TRN-SR2 was directly visualized. In comparison with control cell lines, the great majority of siRNA-treated cells did not contain any PIC in the nucleus. CONCLUSION: Our data clearly demonstrate that TRN-SR2 is the nuclear-import factor of HIV.     

Nuclear import defect of human immunodeficiency virus type 1 DNA flap mutants is not dependent on the viral strain or target cell type

We have previously established, using human immunodeficiency virus type 1 (HIV-1) strain LAI, that the HIV-1 central DNA Flap acts as a cis determinant of viral genome nuclear import. Although the impact of the DNA Flap on nuclear import has already found numerous independent confirmations in the context of lentivirus vectors, it has been claimed that it may be nonessential for infectious virus strains LAI, YU-2 (J. D. Dvorin et al., J. Virol. 76:12087-12096, 2002), HXB2, and NL4-3 (A. Limon et al., J. Virol. 76:12078-12086, 2002). We conducted a detailed analysis of virus infectivity using the provirus clones provided by the authors and analogous target cells. In contrast to published data, our results show that all cPPT mutant viruses exhibit reduced infectivity corresponding to a nuclear import defect irrespective of the viral genetic background or target cell.     

Identification of Critical Motifs within HIV-1 Integrase Required for Importin α3 Interaction and Viral cDNA Nuclear Import

The viral cDNA nuclear import is an important requirement for human immunodeficiency virus type 1 (HIV-1) replication in dividing and nondividing cells. Our recent study identified a specific interaction of importin α3 (Impα3) with HIV-1 integrase (IN) and its involvement in viral cDNA nuclear import. In this study, we have performed a more detailed investigation on the molecular mechanism of how HIV-1 IN interacts with Impα3. Our results revealed a reduced interaction between the two IN mutants INKK215,9AA (IN215,9) and INRK263,4AA (IN263,4) with Impα3, while an IN double mutant, IN215,9/263,4, was severely impaired for its Impα3-binding ability, even though it was still found interacting with other cofactors, IN interactor I and Transportin3. Immunostaining and fractionation analysis have shown that YFP-IN215,9/263,4 failed to localize in the nucleus of transfected cells. Also, we found that both major and minor nuclear localization signal binding grooves of Impα3 are involved in interaction with IN. All of these results suggest a cargo protein-import receptor type of interaction. Finally, the effect of IN215,9/263,4 mutations on HIV-1 replication was evaluated, and real-time quantitative PCR analysis showed that, while mutant virus (v215,9/263,4) had a slightly lowered total viral DNA, the 2-long-terminal-repeat DNA, a marker for nuclear import, was greatly reduced during v215,9/263,4 infection in both dividing and nondividing cells. Also, by cell fractionation assay, we found that a significant proportion of viral cDNA was still retained in cytoplasmic fraction of v215,9/263,4-infected cells. Overall, our study provides strong evidence that ²¹¹KELQKQITK and ²⁶²RRKAK regions of IN C-terminal domain are required for Impα3 interaction and HIV-1 cDNA nuclear import.     

Subcellular localization of feline immunodeficiency virus integrase and mapping of its karyophilic determinant

Feline immunodeficiency virus (FIV), like other members of the lentivirus subfamily, such as human immunodeficiency virus type 1 (HIV-1), can infect nondividing and terminally differentiated cells. The transport of the preintegration complex into the nucleus is cell cycle-independent, but the mechanism is not well understood. Integrase is a key component of the complex and has been suggested to play a role in nuclear import during HIV-1 replication. To determine its karyophilic property, FIV integrase fused with glutathione S-transferase and enhanced green fluorescent protein was expressed in various feline and human cells and the subcellular localization was visualized by fluorescence microscopy. Wild-type FIV integrase was karyophilic in all cell lines tested and capable of targeting the fusion protein to the nuclei of transfected cells. Analysis of deletion and point mutation variants of FIV integrase failed to reveal any canonical nuclear localization signal, and the karyophilic determinant was mapped to the highly conserved N-terminal zinc-binding HHCC motif. A region near the C-terminal domain enriched with basic amino acid residues also affected the nuclear import of integrase. However, the role of this region is only modulatory in comparison to that of the zinc-binding domain. The N-terminal zinc-binding domain does not bind DNA and instead is essential in integrase multimerization. We therefore postulate that the karyophilic property of FIV integrase requires subunit multimerization promoted by the HHCC motif. Alternatively, the HHCC motif may directly promote interaction between FIV integrase and cellular proteins involved in nuclear import.     

Nucleocytoplasmic shuttling by human immunodeficiency virus type 1 Vpr

Human immunodeficiency virus type 1 (HIV-1) is capable of infecting nondividing cells such as macrophages because the viral preintegration complex is able to actively traverse the limiting nuclear pore due to the redundant and possibly overlapping nuclear import signals present in Vpr, matrix, and integrase. We have previously recognized the presence of at least two distinct and novel nuclear import signals residing within Vpr that, unlike matrix and integrase, bypass the classical importin alpha/beta-dependent signals and do not require energy or a RanGTP gradient. We now report that the carboxy-terminal region of Vpr (amino acids 73 to 96) contains a bipartite nuclear localization signal (NLS) composed of multiple arginine residues. Surprisingly, when the leucine-rich Vpr(1-71) fragment, previously shown to harbor an NLS, or full-length Vpr is fused to the C terminus of a green fluorescent protein-pyruvate kinase (GFP-PK) chimera, the resultant protein is almost exclusively detected in the cytoplasm. However, the addition of leptomycin B (LMB), a potent inhibitor of CRM1-dependent nuclear export, produces a shift from a cytoplasmic localization to a nuclear pattern, suggesting that these Vpr fusion proteins shuttle into and out of the nucleus. Studies of nuclear import with GFP-PK-Vpr fusion proteins in the presence of LMB reveals that both of the leucine-rich alpha-helices are required for effective nuclear uptake and thus define a unique NLS. Using a modified heterokaryon analysis, we have localized the Vpr nuclear export signal to the second leucine-rich helix, overlapping a portion of the amino-terminal nuclear import signal. These studies thus define HIV-1 Vpr as a nucleocytoplasmic shuttling protein.     

A hard way to the nucleus

As a member of the Retrovirus family, human immunodeficiency virus (HIV), a causative agent of AIDS, replicates by integrating its genome into the host cell's nuclear DNA. However, in contrast to most retroviruses that depend on mitotic dissolution of the nuclear envelope to gain access to the host cell's genome, the HIV pre-integration complex can enter the nucleus of the target cell during the interphase. Such capacity greatly enhances HIV replication and allows the virus to productively infect terminally differentiated nonproliferating cells, such as macrophages. Infection of macrophages is a critical factor in the pathogenesis of diseases caused by HIV-1 and other lentiviruses. The mechanisms responsible for this unusual feature of HIV have enticed researchers since the early 90s, when the first characterization of the HIV-1 pre-integration complex was reported. Several viral factors, including matrix protein, integrase, viral protein R, and central DNA flap, have been proposed as regulators of HIV-1 nuclear import, only to be later shown as nonessential for this process. As a result, after more than a decade of intense research, there is still no consensus on which HIV-1 and cellular proteins control this critical step in HIV-1 replication. In this review, we will discuss recent advances and suggest possible solutions to the controversial issue of HIV-1 nuclear import.     

Requirement of active human immunodeficiency virus type 1 integrase enzyme for productive infection of human T-lymphoid cells.

The human immunodeficiency virus type 1 (HIV-1) integrase enzyme exhibits significant amino acid sequence conservation with integrase proteins of other retroviruses. We introduced specific amino acid substitutions at a number of the conserved residue positions of recombinant HIV-1 integrase. Some of these substitutions resulted in proteins which were not able to be purified in the same manner as the wild-type enzyme, and these were not studied further. The remaining mutant enzymes were assessed for their abilities to perform functions characteristic of the integrase protein. These included specific removal of the terminal dinucleotides from oligonucleotide substrates representative of the viral U5-long terminal repeat, nonspecific cleavage of oligonucleotide substrates, and mediation of the strand transfer (integration) reaction. Substitution at position 43, within the protein's zinc finger motif region, resulted in an enzyme with reduced specificity for cleavage of the terminal dinucleotide. In addition, a double substitution of aspartic acid and glutamine for valine and glutamic acid, respectively, at positions 151 and 152 within the D,D(35)E motif region rendered the integrase protein inactive for all of its functions. The introduction of this double substitution into an infectious HIV-1 provirus yielded a mutant virus that was incapable of productively infecting human T-lymphoid cells in culture.     

Identification and characterization of a functional nuclear localization signal in the HIV-1 integrase interactor LEDGF/p75

Human lens epithelium-derived growth factor (LEDGF)/p75 protein forms a specific nuclear complex with human immunodeficiency virus type 1 (HIV-1) integrase and is essential for nuclear localization and chromosomal association of the viral protein. We now studied nuclear import of LEDGF/p75 in live and semipermeabilized cells. We show that nuclear import of LEDGF/p75 is GTP-, Ran-, importin-alpha/beta-, and energy-dependent and that the protein competes with the canonical SV40 large T antigen nuclear localization signal (NLS) for nuclear import receptors. We identified the NLS of LEDGF/p75 through deletion analysis and site-directed mutagenesis. The LEDGF/p75 NLS, 148GRKRKAEKQ156, belongs to the canonical SV40-like family. Fusion of this short peptide to the amino terminus of Escherichia coli beta-galactosidase rendered the fusion protein nuclear, confirming that the LEDGF/p75 NLS is transferable. Moreover, a single amino acid change in the NLS was sufficient to exclude the mutant LEDGF/p75 protein from the nucleus and abolish nuclear import of HIV-1 integrase.     

Cellular distribution and karyophilic properties of matrix, integrase, and Vpr proteins from the human and simian immunodeficiency viruses

Infections by human and simian immunodeficiency viruses (HIV and SIV) are independent of host cell division since the preintegration complex (PIC), containing the viral DNA, is able to undergo active nuclear import after viral entry. In order to clarify the mechanisms responsible for nuclear import of the PIC, we have analyzed the subcellular distribution and the karyophilic properties of its viral components, matrix protein (MA), integrase (IN), Vpr, and Vpx. Although MA has been reported to contain a nuclear localization signal, the MA/GFP fusions are excluded from the nucleus and associated with cellular membranes. In contrast, both HIV-1 and SIV IN and Vpr localize in the nucleus of transfected cells. Interestingly, only Vpx from SIVsm virus accumulate in the nucleus while SIVsm Vpr is uniformly distributed throughout nucleus and cytoplasm. Coexpression of MA, Vpr, and IN does not induce any change in their respective intracellular localizations. Finally, we confirm the karyophilic properties of HIV-1 IN and Vpr using an in vitro nuclear import assay. These results indicate that the viral proteins IN and Vpr, which are strongly associated with the viral DNA within PIC, may participate in the nuclear import of the HIV PIC.