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HIV-1 capsid-cyclophilin interactions determine nuclear import pathway, integration targeting and replication efficiency.

ABSTRACT: Lentiviruses such as HIV-1 traverse nuclear pore complexes (NPC) and infect terminally differentiated non-dividing cells, but how they do this is unclear. The cytoplasmic NPC protein Nup358/RanBP2 was identified as an HIV-1 co-factor in previous studies. Here we report that HIV-1 capsid (CA) binds directly to the cyclophilin domain of Nup358/RanBP2. Fusion of the Nup358/RanBP2 cyclophilin (Cyp) domain to the tripartite motif of TRIM5 created a novel inhibitor of HIV-1 replication, consistent with an interaction in vivo. In contrast to CypA binding to HIV-1 CA, Nup358 binding is insensitive to inhibition with cyclosporine, allowing contributions from CypA and Nup358 to be distinguished. Inhibition of CypA reduced dependence on Nup358 and the nuclear basket protein Nup153, suggesting that CypA regulates the choice of the nuclear import machinery that is engaged by the virus. HIV-1 cyclophilin-binding mutants CA G89V and P90A favored integration in genomic regions with a higher density of transcription units and associated features than wild type virus. Integration preference of wild type virus in the presence of cyclosporine was similarly altered to regions of higher transcription density. In contrast, HIV-1 CA alterations in another patch on the capsid surface that render the virus less sensitive to Nup358 or TRN-SR2 depletion (CA N74D, N57A) resulted in integration in genomic regions sparse in transcription units. Both groups of CA mutants are impaired in replication in HeLa cells and human monocyte derived macrophages. Our findings link HIV-1 engagement of cyclophilins with both integration targeting and replication efficiency and provide insight into the conservation of viral cyclophilin recruitment.

SUBMITTER: Schaller T

PROVIDER: S-EPMC3234246 | biostudies-literature | 2011 Dec

REPOSITORIES: biostudies-literature

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HIV-1 capsid-cyclophilin interactions determine nuclear import pathway, integration targeting and replication efficiency.

Schaller Torsten T Ocwieja Karen E KE Rasaiyaah Jane J Price Amanda J AJ Brady Troy L TL Roth Shoshannah L SL Hué Stéphane S Fletcher Adam J AJ Lee KyeongEun K KewalRamani Vineet N VN Noursadeghi Mahdad M Jenner Richard G RG James Leo C LC Bushman Frederic D FD Towers Greg J GJ

PLoS pathogens 20111208 12

Lentiviruses such as HIV-1 traverse nuclear pore complexes (NPC) and infect terminally differentiated non-dividing cells, but how they do this is unclear. The cytoplasmic NPC protein Nup358/RanBP2 was identified as an HIV-1 co-factor in previous studies. Here we report that HIV-1 capsid (CA) binds directly to the cyclophilin domain of Nup358/RanBP2. Fusion of the Nup358/RanBP2 cyclophilin (Cyp) domain to the tripartite motif of TRIM5 created a novel inhibitor of HIV-1 replication, consistent wit ...[more]

PMID: 22174692

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Project description:The HIV-1 capsid protein performs multiple roles in virus replication both during assembly and particle release and during virus trafficking into the nucleus. In order to decipher the roles of capsid protein during early replication, a reliable method to follow its intracellular distribution is required. To complement existing approaches to track HIV-1 capsid during early infection, we developed an HIV-1 imaging strategy, relying on viruses incorporating enhanced green fluorescent protein (eGFP)-tagged capsid (CA-eGFP) protein and mCherry-tagged integrase (IN-mCherry). Wild-type infectivity and sensitivity to inhibition by PF74 point to the functionality of CA-eGFP-containing complexes. Low numbers of CA-eGFP molecules were located inside the viral core and imported into the nucleus without significant loss in intensity. Less than 5% of particles carrying both CA-eGFP and IN-mCherry retained both labelled proteins after nuclear entry, implying a major uncoating event at the nuclear envelope dissociating IN and CA. Still, 20% of all CA-eGFP-containing complexes were detected in the nucleus. Unlike for IN-mCherry complexes, addition of the integrase inhibitor raltegravir had no effect on CA-eGFP-containing complexes, suggesting that these may be not (yet) competent for integration. Our imaging strategy offers alternative visualization of viral capsid trafficking and helps clarify its potential role during integration.IMPORTANCE HIV-1 capsid protein (CA) builds a conical shell protecting viral genomic RNA inside the virus particles. Upon entry into host cells, this shell disassembles in a process of uncoating, which is coordinated with reverse transcription of viral RNA into DNA. After uncoating, a portion of CA remains associated with the viral DNA and mediates its nuclear import and, potentially, integration into host DNA. In this study, we tagged CA with eGFP to follow its trafficking in host cells and address potential CA roles in the nucleus. We found that while functional viruses import the tagged CA into the nucleus, this capsid protein is not part of integration-competent complexes. The roles of nuclear CA thus remain to be established.

Project description:Human immunodeficiency virus type 1 (HIV-1) can infect nondividing cells via passing through the nuclear pore complex. The nuclear membrane-imbedded protein SUN2 was recently reported to be involved in the nuclear import of HIV-1. Whether SUN1, which shares many functional similarities with SUN2, is involved in this process remained to be explored. Here we report that overexpression of SUN1 specifically inhibited infection by HIV-1 but not that by simian immunodeficiency virus (SIV) or murine leukemia virus (MLV). Overexpression of SUN1 did not affect reverse transcription but led to reduced accumulation of the 2-long-terminal-repeat (2-LTR) circular DNA and integrated viral DNA, suggesting a block in the process of nuclear import. HIV-1 CA was mapped as a determinant for viral sensitivity to SUN1. Treatment of SUN1-expressing cells with cyclosporine (CsA) significantly reduced the sensitivity of the virus to SUN1, and an HIV-1 mutant containing CA-G89A, which does not interact with cyclophilin A (CypA), was resistant to SUN1 overexpression. Downregulation of endogenous SUN1 inhibited the nuclear entry of the wild-type virus but not that of the G89A mutant. These results indicate that SUN1 participates in the HIV-1 nuclear entry process in a manner dependent on the interaction of CA with CypA.IMPORTANCE HIV-1 infects both dividing and nondividing cells. The viral preintegration complex (PIC) can enter the nucleus through the nuclear pore complex. It has been well known that the viral protein CA plays an important role in determining the pathways by which the PIC enters the nucleus. In addition, the interaction between CA and the cellular protein CypA has been reported to be important in the selection of nuclear entry pathways, though the underlying mechanisms are not very clear. Here we show that both SUN1 overexpression and downregulation inhibited HIV-1 nuclear entry. CA played an important role in determining the sensitivity of the virus to SUN1: the regulatory activity of SUN1 toward HIV-1 relied on the interaction between CA and CypA. These results help to explain how SUN1 is involved in the HIV-1 nuclear entry process.

Project description:BACKGROUND: Chikungunya virus (CHIKV) is an alphavirus of the Togaviridae family. After autoproteolytic cleavage, the CHIKV capsid protein (CP) is involved in RNA binding and assembly of the viral particle. The monomeric CP is approximately 30 kDa in size and is small enough for passive transport through nuclear pores. Some alphaviruses are found to harbor nuclear localization signals (NLS) and transport of these proteins between cellular compartments was shown to be energy dependent. The active nuclear import of cytoplasmic proteins is mediated by karyopherins and their export by exportins. As nuclear and cytoplasmic trafficking may play a role in the life cycle of CHIKV, we have sought to identify nuclear localization and nuclear export signals in CHIKV CP in a virus-free system. METHODS: EGFP-fusion proteins of CHIKV CP and mutants thereof were created and used to monitor their intracellular localization. Binding of cellular proteins was confirmed in pull-down assays with purified CP using co-immuoprecipitation. Nuclear localization was demonstrated in a virus-free system using fluorescence microscopy. RESULTS: Here we show that CHIKV CP is a nuclear-cytoplasmic shuttling protein with an active NLS that binds to karyopherin ? (Kar?) for its nuclear translocation. We also found that the Kar?4 C-terminal NLS binding site is sufficient for this interaction. We further demonstrate that CHIKV CP interacts directly with the export receptor CRM1 to transport this viral protein out of the nucleus via a nuclear export signal (NES). The CHIKV CP NES was mapped between amino acids 143 and 155 of CP. Deduced from in silico analyses we found that the NES has a mode of binding similar to the snurportin-1 CRM1 complex. CONCLUSIONS: We were able to show that in a virus-free system that the CHIKV capsid protein contains both, a NLS and a NES, and that it is actively transported between the cytoplasma and the nucleus. We conclude that CHIKV CP has the ability to shuttle via interaction with karyopherins for its nuclear import and, vice versa, by CRM1-dependent nuclear export.

Project description:BackgroundHIV-1 is inhibited early after entry into cells expressing some simian orthologues of the tripartite motif protein family member TRIM5α. Mutants of the human orthologue (TRIM5αhu) can also provide protection against HIV-1. The host protein cyclophilin A (CypA) binds incoming HIV-1 capsid (CA) proteins and enhances early stages of HIV-1 replication by unknown mechanisms. On the other hand, the CA-CypA interaction is known to increase HIV-1 susceptibility to restriction by TRIM5α. Previously, the mutation V86M in the CypA-binding loop of HIV-1 CA was found to be selected upon serial passaging of HIV-1 in cells expressing Rhesus macaque TRIM5α (TRIM5αrh). The objectives of this study were (i) to analyze whether V86M CA allows HIV-1 to escape mutants of TRIM5αhu, and (ii) to characterize the role of CypA in the resistance to TRIM5α conferred by V86M.ResultsWe find that in single-cycle HIV-1 vector transduction experiments, V86M confers partial resistance against R332G-R335G TRIM5αhu and other TRIM5αhu variable 1 region mutants previously isolated in mutagenic screens. However, V86M HIV-1 does not seem to be resistant to R332G-R335G TRIM5αhu in a spreading infection context. Strikingly, restriction of V86M HIV-1 vectors by TRIM5αhu mutants is mostly insensitive to the presence of CypA in infected cells. NMR experiments reveal that V86M alters CypA interactions with, and isomerisation of CA. On the other hand, V86M does not affect the CypA-mediated enhancement of HIV-1 replication in permissive human cells. Finally, qPCR experiments show that V86M increases HIV-1 transport to the nucleus of cells expressing restrictive TRIM5α.ConclusionsOur study shows that V86M de-couples the two functions associated with CA-CypA binding, i.e. the enhancement of restriction by TRIM5α and the enhancement of HIV-1 replication in permissive human cells. V86M enhances the early stages of HIV-1 replication in restrictive cells by improving nuclear import. In summary, our data suggest that HIV-1 escapes restriction by TRIM5α through the selective disruption of CypA-dependent, TRIM5α-mediated inhibition of nuclear import. However, V86M does not seem to relieve restriction of a spreading HIV-1 infection by TRIM5αhu mutants, underscoring context-specific restriction mechanisms.

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HIV-1 capsid-cyclophilin interactions determine nuclear import pathway, integration targeting and replication efficiency.

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HIV-1 capsid-cyclophilin interactions determine nuclear import pathway, integration targeting and replication efficiency.

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