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.     

Nuclear exportin receptor CAS regulates the NPI-1-mediated nuclear import of HIV-1 Vpr

Vpr, an accessory protein of human immunodeficiency virus type 1, is a multifunctional protein that plays an important role in viral replication. We have previously shown that the region between residues 17 and 74 of Vpr (Vpr(N17C74)) contained a bona fide nuclear localization signal and it is targeted Vpr(N17C74) to the nuclear envelope and then imported into the nucleus by importin α (Impα) alone. The interaction between Impα and Vpr is important not only for the nuclear import of Vpr but also for HIV-1 replication in macrophages; however, it was unclear whether full-length Vpr enters the nucleus in a manner similar to Vpr(N17C74). This study investigated the nuclear import of full-length Vpr using the three typical Impα isoforms, Rch1, Qip1 and NPI-1, and revealed that full-length Vpr is selectively imported by NPI-1, but not Rch1 and Qip1, after it makes contact with the perinuclear region in digitonin-permeabilized cells. A binding assay using the three Impα isoforms showed that Vpr bound preferentially to the ninth armadillo repeat (ARM) region (which is also essential for the binding of CAS, the export receptor for Impα) in all three isoforms. Comparison of biochemical binding affinities between Vpr and the Impα isoforms using surface plasmon resonance analysis demonstrated almost identical values for the binding of Vpr to the full-length isoforms and to their C-terminal domains. By contrast, the data showed that, in the presence of CAS, Vpr was released from the Vpr/NPI-1 complex but was not released from Rch1 or Qip1. Finally, the NPI-1-mediated nuclear import of Vpr was greatly reduced in semi-intact CAS knocked-down cells and was recovered by the addition of exogenous CAS. This report is the first to show the requirement for and the regulation of CAS in the functioning of the Vpr-Impα complex.     

HIV-1 integrase is capable of targeting DNA to the nucleus via an importin alpha/beta-dependent mechanism

In addition to its well-documented role in integration of the viral genome, the HIV-1 enzyme IN (integrase) is thought to be involved in the preceding step of importing the viral cDNA into the nucleus. The ability of HIV to transport its cDNA through an intact nuclear envelope allows HIV-1 to infect non-dividing cells, which is thought to be crucial for the persistent nature of HIV/AIDS. Despite this, the mechanism utilized by HIV-1 to import its cDNA into the nucleus, and the viral proteins involved, remains ill-defined. In the present study we utilize in vitro techniques to assess the nuclear import properties of the IN protein, and show that IN interacts with members of the Imp (Importin) family of nuclear transport proteins with high affinity and exhibits rapid nuclear accumulation within an in vitro assay, indicating that IN possesses potent nucleophilic potential. IN nuclear import appears to be dependent on the Imp alpha/beta heterodimer and Ran GTP (Ran in its GTP-bound state), but does not require ATP. Importantly, we show that IN is capable of binding DNA and facilitating its import into the nucleus of semi-intact cells via a process that involves basic residues within amino acids 186-188 of IN. These results confirm IN as an efficient mediator of DNA nuclear import in vitro and imply the potential for IN to fulfil such a role in vivo. These results may not only aid in highlighting potential therapeutic targets for impeding the progression of HIV/AIDS, but may also be relevant for non-viral gene delivery.     

Host protein Ku70 binds and protects HIV-1 integrase from proteasomal degradation and is required for HIV replication

HIV-1 integrase (IN) is a key viral enzymatic protein acting in several viral replication steps, including integration. IN has been shown to be an unstable protein degraded by the N-end rule pathway through the host ubiquitin-proteasome machinery. However, it is still not fully understood how this viral protein is protected from the host ubiquitin-proteasome system within cells during HIV replication. In the present study, we provide evidence that the host protein Ku70 interacts with HIV-1 IN and protects it from the Lys(48)-linked polyubiquitination proteasomal pathway. Moreover, Ku70 is able to down-regulate the overall protein polyubiquitination level within the host cells and to specifically deubiquitinate IN through their interaction. Mutagenic studies revealed that the C terminus of IN (residues 230-288) is required for IN binding to the N-terminal part of Ku70 (Ku70(1-430)), and their interaction is independent of Ku70/80 heterodimerization. Finally, knockdown of Ku70 expression in both virus-producing and target CD4(+) T cells significantly disrupted HIV-1 replication and rendered two-long terminal repeat circles and integration undetectable, indicating that Ku70 is required for both the early and the late stages of the HIV-1 life cycle. Interestingly, Ku70 was incorporated into the progeny virus in an IN-dependent way. We proposed that Ku70 may interact with IN during viral assembly and accompany HIV-1 IN upon entry into the new target cells, acting to 1) protect IN from the host defense system and 2) assist IN integration activity. Overall, this report provides another example of how HIV-1 hijacks host cellular machinery to protect the virus itself and to facilitate its replication.     

The formation of cysteine-linked dimers of BST-2/tetherin is important for inhibition of HIV-1 virus release but not for sensitivity to Vpu

Background

The integrase (IN) of human immunodeficiency virus type 1 (HIV-1) has been implicated in different steps during viral replication, including nuclear import of the viral pre-integration complex. The exact mechanisms underlying the nuclear import of IN and especially the question of whether it bears a functional nuclear localization signal (NLS) remain controversial.

Results

Here, we studied the nuclear import pathway of IN by using multiple in vivo and in vitro systems. Nuclear import was not observed in an importin α temperature-sensitive yeast mutant, indicating an importin α-mediated process. Direct interaction between the full-length IN and importin α was demonstrated in vivo using bimolecular fluorescence complementation assay (BiFC). Nuclear import studies in yeast cells, with permeabilized mammalian cells, or microinjected cultured mammalian cells strongly suggest that the IN bears a NLS domain located between residues 161 and 173. A peptide bearing this sequence -NLS-IN peptide- inhibited nuclear accumulation of IN in transfected cell-cycle arrested cells. Integration of viral cDNA as well as HIV-1 replication in viral cell-cycle arrested infected cells were blocked by the NLS-IN peptide.

Conclusion

Our present findings support the view that nuclear import of IN occurs via the importin α pathway and is promoted by a specific NLS domain. This import could be blocked by NLS-IN peptide, resulting in inhibition of viral infection, confirming the view that nuclear import of the viral pre-integration complex is mediated by viral IN.     

Multiple importins function as nuclear transport receptors for the Rev protein of human immunodeficiency virus type 1

The Rev protein of human immunodeficiency virus type 1 is an RNA-binding protein that is required for nuclear export of unspliced and partially spliced viral mRNAs. Nuclear import of human immunodeficiency virus type 1 Rev has been suggested to depend on the classic nuclear transport receptor importin beta, but not on the adapter protein importin alpha. We now show that, similar to importin alpha, Rev is able to dissociate RanGTP from recycling importin beta, a reaction that leads to the formation of a novel import complex. Besides importin beta, the transport receptors transportin, importin 5, and importin 7 specifically interact with Rev and promote its nuclear import in digitonin-permeabilized cells. A single arginine-rich nuclear localization sequence of Rev is required for interaction with all importins tested so far. In contrast to the importin beta-binding domain of importin alpha, Rev interacts with an N-terminal fragment of importin beta. Transportin contains two independent binding sites for Rev. Hence, the mode of interaction of importin beta and transportin with Rev is clearly distinct from that with their classic import cargoes. Taken together, the viral protein takes advantage of multiple cellular transport pathways for its nuclear accumulation.     

Inhibition of early steps of HIV-1 replication by SNF5/Ini1

To replicate, human immunodeficiency virus, type 1 (HIV-1) needs to integrate a cDNA copy of its RNA genome into a chromosome of the host cell, a step controlled by the viral integrase (IN) protein. Viral integration involves the participation of several cellular proteins. SNF5/Ini1, a subunit of the SWI/SNF chromatin remodeling complex, was the first cofactor identified to interact with IN. We report here that SNF5/Ini1 interferes with early steps of HIV-1 replication. Inhibition of SNF5/Ini1 expression by RNA interference increases HIV-1 replication. Using quantitative PCR, we show that both the 2-long terminal repeat circle and integrated DNA forms accumulate upon SNF5/Ini1 knock down. By yeast two-hybrid assay, we screened a library of HIV-1 IN random mutants obtained by PCR random mutagenesis using SNF5/Ini1 as prey. Two different mutants of interaction, IN E69G and IN K71R, were impaired for SNF5/Ini1 interaction. The E69G substitution completely abolished integrase catalytic activity, leading to a replication-defective virus. On the contrary, IN K71R retained in vitro integrase activity. K71R substitution stimulates viral replication and results in higher infectious titers. Taken together, these results suggest that, by interacting with IN, SNF5/Ini1 interferes with early steps of HIV-1 infection.     

Role of the karyopherin pathway in human immunodeficiency virus type 1 nuclear import.

The interaction of the human immunodeficiency virus type 1 (HIV-1) nucleoprotein complex with the cell nuclear import machinery is necessary for viral replication in macrophages and for the establishment of infection in quiescent T lymphocytes. The karyophilic properties of two viral proteins, matrix (MA) and Vpr, are keys to this process. Here, we show that an early step of HIV-1 nuclear import is the recognition of the MA nuclear localization signal (NLS) by Rch1, a member of the karyopherin-alpha family. Furthermore, we demonstrate that an N-terminally truncated form of Rch1 which binds MA but fails to localize to the nucleus efficiently blocks MA- but not Vpr-mediated HIV-1 nuclear import. Correspondingly, NLS peptide inhibits the nuclear migration of MA but not that of Vpr and prevents the infection of terminally differentiated macrophages by vpr-defective virus but not wild-type virus. These results are consistent with a model in which Rch1 or another member of the karyopherin-alpha family, through the recognition of the MA NLS, participates in docking the HIV-1 nucleoprotein complex at the nuclear pore. In addition, our data suggest that Vpr governs HIV-1 nuclear import through a distinct pathway.     

Quantitative analysis of Tat peptide binding to import carriers reveals unconventional nuclear transport properties

A detailed study of nuclear import mediated by the HIV-1 Tat peptide (47YGRKKRRQRRR57, TatRRR) is reported. Fluorescence-based measurements, calibration of protein concentrations, and binding assays are exploited to address the physicochemical mechanisms of Tat peptide recognition by the classical importin α (Impα) and importin β (Impβ) receptors both in vitro and in intact cells. We show that TatRRR is an unconventional nuclear localization sequence that binds directly to both Impα and Impβ carriers in the absence of competitors (in vitro), whereas this property is silenced in the actual cellular environment. In the latter case, Impα/β-dependent nuclear import can be successfully restored by replacing the "RRR" stretch with "GGG". We apply a recently developed method to determine quantitatively TatGGG affinity for each receptor. Based on these results, we can rationalize previous controversial reports on the Tat peptide and provide coherent guidelines for the design of novel intracellular targeting sequences.     

Contribution of host nucleoporin 62 in HIV-1 integrase chromatin association and viral DNA integration

HIV-1 integration is promoted by viral integrase (IN) and its cellular cofactors. The lens epithelium-derived growth factor (LEDGF/p75), an IN interacting cellular cofactor, has been shown to play an important role in HIV-1 chromatin targeting and integration. However, whether other cellular cofactors are also involved in viral replication steps is still elusive. Here, we show that nucleoporin 62 (Nup62) is a chromatin-bound protein and can specifically interact with HIV-1 IN in both soluble nuclear extract and chromatin-bound fractions. The knockdown of Nup62 by shRNA reduced the association of IN with host chromatin and significantly impaired viral integration and replication in HIV-1-susceptible cells. Furthermore, the expression of the IN-binding region of Nup62 in CD4(+) T cells significantly inhibited HIV-1 infection. Taken together, these results indicate that the cellular Nup62 is specifically recruited by HIV-1 IN and contribute to an efficient viral DNA integration.     

Two nuclear localization signals in the HIV-1 matrix protein regulate nuclear import of the HIV-1 pre-integration complex

Replication of HIV-1 in non-dividing and slowly proliferating cell populations depends on active import of the viral pre-integration complex (PIC) into the cell nucleus. While it is commonly accepted that this process is mediated by an interaction between the HIV-1 PIC and the cellular nuclear import machinery, controversial results have been reported concerning the mechanisms of this interaction. Here, we demonstrate that a recently identified nuclear localization signal within the HIV-1 matrix protein (MA), MA NLS-2, together with previously described MA NLS-1, mediates nuclear import of the HIV-1 PIC. Inactivation of both MA NLSs precluded nuclear translocation of MA and rendered the virus defective in nuclear import and replication in non-dividing macrophage cultures, even when functional Vpr and integrase (IN), two more viral proteins implicated in HIV-1 nuclear import, were present. Taken together, these results indicate that Vpr does not function as an independent nuclear import factor and demonstrate that HIV-1 MA, by virtue of its two nuclear localization signals, regulates HIV-1 nuclear import.     

Characterization of the minimal DNA-binding domain of the HIV integrase protein.

The human immunodeficiency virus (HIV) integrase (IN) protein mediates an essential step in the retroviral lifecycle, the integration of viral DNA into human DNA. A DNA-binding domain of HIV IN has previously been identified in the C-terminal part of the protein. We tested truncated proteins of the C-terminal region of HIV-1 IN for DNA binding activity in two different assays: UV-crosslinking and southwestern blot analysis. We found that a polypeptide fragment of 50 amino acids (IN220-270) is sufficient for DNA binding. In contrast to full-length IN protein, this domain is soluble under low salt conditions. DNA binding of IN220-270 to both viral DNA and non-specific DNA occurs in an ion-independent fashion. Point mutations were introduced in 10 different amino acid residues of the DNA-binding domain of HIV-2 IN. Mutation of basic amino acid K264 results in strong reduction of DNA binding and of integrase activity.     

Insulin-like growth factor II mRNA binding protein 1 associates with Gag protein of human immunodeficiency virus type 1, and its overexpression affects virus assembly

The assembly of human immunodeficiency virus type 1 (HIV-1) particles is driven by viral Gag protein. This function of Gag not only benefits from its self-multimerization property but also depends on its interaction with a number of cellular factors such as TSG101 and ALIX/AIP1 that promote virus budding and release from cell surfaces. However, interaction with Gag also allows some cellular factors such as APOBEC3G and Trim5alpha to access viral replication machinery and block viral replication. In this study, we report a new HIV-1 Gag-binding factor named insulin-like growth factor II mRNA binding protein 1 (IMP1). Gag-IMP1 interaction requires the second zinc finger of the nucleocapsid (NC) domain of Gag and the KH3 and KH4 domains of IMP1. A fourfold reduction of HIV-1 infectivity was seen with overexpression of the wild-type IMP1 and its mutant that is able to interact with Gag but not with overexpression of IMP1 mutants exhibiting Gag-binding deficiency. The decreased viral infectivity was further shown as a result of diminished viral RNA packaging, abrogated Gag processing on the cellular membranes, and impeded maturation of virus particles. Together, these results demonstrate that IMP1 interacts with HIV-1 Gag protein and is able to block the formation of infectious HIV-1 particles.     

An AlphaScreen®-based assay for high-throughput screening for specific inhibitors of nuclear import

Specific viral proteins enter the nucleus of infected cells to perform essential functions, as part of the viral life cycle. The integrase (IN) molecule of human immunodeficiency virus (HIV)-1 is of particular interest in this context due to its integral role in integrating the HIV genome into that of the infected host cell. Most IN-based antiviral compounds target the IN/DNA interaction, but since IN must first enter the nucleus before it can perform these critical functions, nuclear transport of IN is also an attractive target for therapeutic intervention. Here the authors describe a novel high-throughput screening assay for identifying inhibitors of nuclear import, particularly IN, based on amplified luminescent proximity homogeneous assay (AlphaScreen(?)) technology, which is high throughput, requires low amounts of material, and is efficient and cost-effective. The authors use the assay to screen for specific inhibitors of the interaction between IN and its nuclear transport receptor importin α/β, successfully identifying several inhibitors of the IN/importin α/β interaction. Importantly, they demonstrate that one of the identified compounds, mifepristone, is effective in preventing active nuclear transport of IN in transfected cells and hence may represent a useful anti-HIV therapeutic. The screen also identified broad-spectrum importin α/β inhibitors such as ivermectin, which may represent useful tools for nuclear transport research in the future. The authors validate the activity and specificity of mifepristone and ivermectin in inhibiting nuclear protein import in living cells, underlining the utility of the screening approach.     

Simian immunodeficiency virus Vpx is imported into the nucleus via importin alpha-dependent and -independent pathways

Vpx protein of human immunodeficiency virus type 2/simian immunodeficiency virus (SIV) has been implicated in the transport of the viral genome into the nuclei of nondividing cells. The mechanism by which Vpx enters the nucleus remains unknown. Here we have identified two distinct noncanonical nuclear localization signals (NLSs) in Vpx of SIV(smPbj1.9) and defined the pathways for its nuclear import. Although nuclear targeting signals identified here are distinct from known nuclear import signals, translocation of Vpx into the nucleus involves the interaction of its N-terminal NLS (amino acids 20 to 40) or C-terminal NLS (amino acids 65 to 75) with importin alpha and, in the latter case, also with importin beta. Collectively, these results suggest that importins interact with Vpx and ensure the effective import of Vpx into the nucleus to support virus replication in nondividing cells.