- Open Access
MxB binds to the HIV-1 core and prevents the uncoating process of HIV-1
© Fricke et al.; licensee BioMed Central Ltd. 2014
- Received: 25 July 2014
- Accepted: 30 July 2014
- Published: 14 August 2014
The IFN-α-inducible restriction factor MxB blocks HIV-1 infection after reverse transcription but prior to integration. Genetic evidence suggested that capsid is the viral determinant for restriction by MxB. This work explores the ability of MxB to bind to the HIV-1 core, and the role of capsid-binding in restriction.
We showed that MxB binds to the HIV-1 core and that this interaction leads to inhibition of the uncoating process of HIV-1. These results identify MxB as an endogenously expressed protein with the ability to inhibit HIV-1 uncoating. In addition, we found that a benzimidazole-based compound known to have a binding pocket on the surface of the HIV-1 capsid prevents the binding of MxB to capsid. The use of this small-molecule identified the MxB binding region on the surface of the HIV-1 core. Domain mapping experiments revealed the following requirements for restriction: 1) MxB binding to the HIV-1 capsid, which requires the 20 N-terminal amino acids, and 2) oligomerization of MxB, which is mediated by the C-terminal domain provides the avidity for the interaction of MxB with the HIV-1 core.
Overall our work establishes that MxB binds to the HIV-1 core and inhibits the uncoating process of HIV-1. Moreover, we demonstrated that HIV-1 restriction by MxB requires capsid binding and oligomerization.
The myxovirus resistance proteins (Mxs) represent a family of interferon-inducible factors with a wide range of antiviral activities ,. The MxB gene was originally cloned from a human glioblastoma cell line treated with interferon-α (IFN-α) ,. MxB as well as the related protein MxA belongs to the dynamin-like family of proteins, which have diverse functions ranging from vesicle transport to antiviral activity ,-. The most studied dynamin-like protein that exhibits antiviral activity is MxA ,. Contrary to MxB, the antiviral role of MxA has been extensively studied for viruses including influenza ,-, tick-born Thogoto , African swine fever , hepatitis B , and La Crosse ,. Only recently the antiviral activity of the long form of MxB was described ,-; these investigations lead to the discovery that the IFN-α-inducible protein MxB blocks HIV-1 infection.
Genetic evidence suggested that HIV-1 capsid is the determinant for the ability of MxB to block HIV-1 infection ,,. HIV-1 viruses bearing capsid changes such as P90A, G89V and N57S escape MxB restriction suggesting that capsid is the determinant for the block imposed by MxB. These experiments imply that MxB is directly interacting with the HIV-1 core early during infection. However, the ability of MxB to associate with HIV-1 cores has not been explored.
MxB blocks HIV-1 infection after the occurrence of reverse transcription but before integration ,,. This evidence suggested that MxB might be interfering with one or more of the following processes: 1) HIV-1 uncoating, 2) nuclear import of the HIV-1 pre-integration complex, or 3) nuclear maturation of the pre-integration complex. However, the mechanism by which MxB interferes with early steps of HIV-1 infection is not understood.
This work explores the ability of MxB to bind to the HIV-1 core in vitro and during infection. We showed that MxB interacts with in vitro assembled HIV-1 capsid-nucleocapsid (CA-NC) complexes, which recapitulate the surface of the HIV-1 core. In agreement, we found that MxB associates with HIV-1 cores during infection using the fate of the capsid assay. Remarkably, the binding of MxB to the HIV-1 core inhibits the uncoating process of HIV-1 defining MxB as an endogenously expressed protein that prevents HIV-1 uncoating. To find small-molecule inhibitors that prevent the binding of MxB to the HIV-1 core, we screened a battery of structurally well-known HIV-1 capsid inhibitors for their ability to prevent the binding of MxB to the HIV-1 core. Interestingly, a benzimidazole-based compound known to have a binding pocket on the surface of the HIV-1 capsid prevents the binding of MxB to the capsid. These experiments suggested an overlap between the capsid binding sites for MxB and the benzimidazole-based compound. Assaying the contribution of the different MxB protein domains to capsid binding and restriction revealed that the 20 N-terminal amino acids are responsible for the ability of MxB to bind to the HIV-1 core. In addition, we provide evidence that the C-terminal leucine zipper domain of MxB provides the necessary avidity for the interaction of MxB with the HIV-1 core. Overall, our studies showed that MxB binds to the HIV-1 core and inhibits the uncoating process of HIV-1 leading to an infection block.
MxB binds in vitro assembled HIV-1 CA-NC complexes
To further test the role of MxB binding to HIV-1 capsid in restriction, we tested the ability of MxB to bind in vitro assembled HIV-1 CA-NC complexes bearing the capsid changes P90A, G89V or N57S, which when incorporated into the virus allow HIV-1 to partially escape MxB restriction ,,. As shown in Figure 1B, MxB binds to in vitro assembled HIV-1 CA-NC complexes bearing the capsid change P90A, suggesting that binding is necessary but not sufficient for restriction. Interestingly, MxB bound less to in vitro assembled HIV-1 CA-NC complexes bearing changes G89V and N57S when compared to wild type complexes (Figure 1B). As previously shown, HIV-1 bearing the capsid change P90A, G89V or N57S partially escaped MxB restriction (Additional file 2B). Notwithstanding HIV-1 viruses bearing the capsid change P90A, G89V or N57S partially escape MxB restriction, we found that P90A is the only capsid change that did not result in a virus with an infectivity defect (Additional file 2C). To confirm the bona fide origin and functionality of in vitro assembled HIV-1 CA-NC complexes tubes bearing capsid mutants P90A, G89V and N57S, we tested the binding of cleavage and polyadenylation specific factor 6 (CPSF6) to the different capsid mutants. CPSF6 was able to bind in vitro assembled HIV-1 CA-NC complexes bearing changes P90A and G89V, but not N57S (Additional file 2D). In addition, we showed the ability of TRIM5αrh to bind in vitro assembled HIV-1 CA-NC complexes bearing the change N57S (Additional file 2D).
Furthermore, we performed electron microscopy to show that the in vitro assembled HIV-1 CA-NC mutants form similar tubular structures when compared to wild type (Additional file 2E).
Next we tested whether MxA, which is the closest human homolog to MxB that contains antiviral activity , binds to in vitro assembled HIV-1 CA-NC complexes. MxB is composed of the same domains when compared to MxA with the exception that MxB exhibits a longer N-terminal domain (Figure 1C, bottom panel) . Interestingly, MxA was not able to interact with in vitro assembled HIV-1 CA-NC complexes when compared to MxB (Figure 1C, upper left panel). In agreement, MxA was not able to block HIV-1 infection when compared to MxB (Figure 1C, upper right panel) . Next we tested the capsid binding ability of a protein chimera containing residues 1–90 of MxB fused to residues 43–662 of MxA [MxB (1–90)-MxA (43–662)]. Contrary to MxA, the protein chimera MxB (1–90)-MxA (43–662) gained the ability to bind in vitro assembled HIV-1 CA-NC complexes (Figure 1C, left panel). These results suggested that the N-terminal region of MxB is involved in its ability to bind capsid. In addition, MxB (1–90)-MxA (43–662) gained the ability to block HIV-1 infection (Figure 1C, right panel). Furthermore, these results correlated the ability of MxB to bind capsid with HIV-1 restriction.
Next we measured the ability of HIV-1 cores to colocalize with MxB in living cells using fluorescence microscopy over time, as described . For this purpose, we infected Cf2Th cells stably expressing MxB with VSV-G pseudotyped R7∆Env HIV-1 viruses in the presence or absence of Bafilomycin A1, which inhibits the entry of pH-dependent viruses . At 6 hours post-infection, samples were fixed and MxB colocalization with HIV-1 cores was quantified (Figure 1D). The percentage of p24 that colocalizes with MxB was calculated as the number of p24 positive virions that are associated with MxB as described in Methods (Figure 1D, lower panel). As a control, we performed similar experiments in cells expressing MxB-∆(1–20), which is a deletion mutant that no longer binds capsid or restrict HIV-1 (see below). Colocalization between MxB-∆(1–20) and p24 was not observed. These results showed an increase in p24 colocalization with MxB over time suggesting that MxB directly associates with HIV-1 cores in living cells.
In agreement with the fact that MxB modestly blocks SIVmac and HIV-2 infection (Additional file 1C), we found that MxB binds to in vitro assembled SIVmac and HIV-2 CA-NC complexes (Additional file 1D).
Overall, these results showed that MxB binds to HIV-1 capsid, and that binding to capsid is necessary for the ability of MxB to block HIV-1 infection. By the use of the protein chimera MxB (1–90)-MxA (43–662), we have correlated binding with restriction. Additionally, we showed that the N-terminal 90 amino acids of MxB are important for the ability of MxB to bind the HIV-1 capsid and restriction of HIV-1 infection.
MxB binds to the HIV-1 core and inhibits the uncoating process during infection
Consequently, we tested the ability of MxB to inhibit the uncoating process of HIV-1 mutants that partially overcome restriction. For this purpose, we challenged Cf2Th cells stably expressing MxB or containing the empty vector LPCX with similar amounts of HIV-1-P90A, HIV-1-G89V or HIV-1-N57S viruses (Figure 2B), which partially escape MxB restriction ,,. MxB only minimally inhibits the uncoating process of HIV-1 viruses bearing capsid changes P90A, G89V and N57S (Figure 2B). Accordingly, MxB partially affected the infection of HIV-1-P90A, HIV-1-G89V and HIV-1-N57S viruses. In agreement with our binding assays, we also observed that HIV-1 cores bearing the different capsid mutations were able to recruit MxB.
We and others have previously observed that cells expressing the CPSF6 protein fused to a nuclear export signal (NES-CPSF6) inhibits the uncoating process of HIV-1 ,; therefore, we compared the abilities of MxB and NES-CPSF6 to inhibit the uncoating process of HIV-1. As shown in Figure 2C, MxB is as potent as NES-CPSF6 in inhibiting the uncoating process of HIV-1. As expected, MxB, NES-CPSF6 and TRIM5αrh potently restricted HIV-1 infection (Additional file 2F).
Next we tested the ability of IFN-α-treated human HT1080 cells to inhibit the uncoating process of HIV-1. For this purpose, we challenged human HT1080 cells that were treated or not with IFN-α with similar amounts of HIV-1-GFP and performed the fate of the capsid assay. As shown in Figure 2D, IFN-α-treated HT1080 cells inhibit the uncoating process of HIV-1. These findings suggested that IFN-α-treated HT1080 cells prevent the uncoating process of HIV-1, which could be mediated by any of the IFN-α inducible genes. In agreement with our previous findings, endogenously expressed MxB co-sedimented with pelletable capsids suggesting that endogenously expressed MxB interacts with the HIV-1 core in living cells. As expected, IFN-α-treated human HT1080 expressed endogenous MxB and potently blocked HIV-1 infection (Figure 2D, right panels).
Subsequently, we tested whether MxB is able to retain HIV-1 cores in the cytoplasm of infected living cells by fluorescence microscopy. For this purpose, we challenged Cf2Th cells stably expressing MxB, MxB-∆(1–20), or containing the empty vector LPCX, with dually labeled HIV-1 particles containing Vpr-GFP and the S15-mCherry, as described . Infected cells were fixed at the indicated time points, and the number of fused virions (S15-mCherry negative particles) was counted in 10 different visual fields. As shown on Figure 2E, infected Cf2Th cells expressing MxB allowed the accumulation of viral cores over time when compared to infected Cf2Th stably cells expressing MxB-∆(1–20) or containing the empty vector LPCX. These results suggested that expression of MxB allows the accumulation of HIV-1 cores in the cytoplasm over time.
The interaction between MxB and the HIV-1 core is prevented by a benzimidazole-based inhibitor that binds to a specific pocket in the HIV-1 capsid
Next we tested the ability of CPIPB to release HIV-1 restriction in mammalian cells expressing MxB. However, we could not measure release of restriction since we found that CPIPB is highly toxic for mammalian cells above 50 μM.
Contribution of the different protein domains of MxB to HIV-1 capsid-binding and restriction
Phenotypes of MxB variants
Restriction of HIV-1a
Binding to HIV-1
MxB (1–90)-MxA (43–662)
Contribution of oligomerization to the ability of MxB to bind to the HIV-1 core
The present work explores the ability of MxB to bind to the HIV-1 core, and the contribution of binding to HIV-1 restriction. Our findings provided novel insights into the mechanism used by MxB to block HIV-1 infection: 1) MxB binds to the HIV-1 capsid, 2) MxB colocalizes with HIV-1 cores in infected cells as determined by fluorescence microscopy, 3) MxB is associated biochemically with pelletable cores during infection, 4) MxB inhibits the uncoating process of HIV-1 during infection as measured by the fate of the capsid assay and a microscopy-based assay, 5) the small-molecule inhibitor CPIPB, which binds into a pocket between the bases of the Cyp A binding loop and the loop that connects helices 6 and 7 of HIV-1 capsid (Figure 3C) ,, interfered with the ability of MxB to bind HIV-1 capsid, 6) domain mapping experiments and the use of a protein chimera showed that capsid binding correlates with restriction, and 6) disruption of MxB’s leucine zipper motif abrogated the ability of MxB to oligomerize, to bind capsid and to block HIV-1 infection.
We tested the ability of MxB to bind in vitro assembled HIV-1 CA-NC complexes, and showed that MxB binds to the HIV-1 capsid in a similar manner when compared to the restriction factor TRIM5αrh,. The testing of MxB binding to capsid mutants P90A, G89V and N57S revealed that binding is necessary but not sufficient for restriction. By using the fate of the capsid assay, we also showed that MxB is contained in pelletable fractions suggesting that MxB associates with the HIV-1 core during infection. In agreement, fluorescence microscopy experiments showed that colocalization of HIV-1 cores with MxB increased over time in infected cells suggesting that MxB associates with the HIV-1 core.
We also compared the capsid-binding abilities of MxB with MxA, which does not restrict HIV-1 infection ,. Our experiments showed that MxA does not bind in vitro assembled HIV-1 CA-NC complexes. MxA and MxB exhibit similar sequences and domains suggesting analogous functions ,-. Interestingly, the difference between MxA and MxB is that the latter contains an additional 60 amino acids on the N-terminal end, which might be directly involved in the binding of MxB to capsid. In agreement with this hypothesis, we showed that the protein chimera MxB (1–90)-MxA (43–662) gains the ability to bind HIV-1 capsid and to restrict HIV-1 when compared to MxA. These results suggested that the N-terminal residues of MxB are involved in the ability of MxB to bind capsid and restrict HIV-1.
This work shows that MxB inhibits the HIV-1 uncoating process during infection. Using the fate of the capsid assay, which analyses the HIV-1 uncoating process during infection ,,, we showed that MxB stabilizes the HIV-1 core during infection. In agreement with these observations, we also showed that MxB facilitates the accumulation of HIV-1 cores over time by fluorescence microscopy in living cells. Overall our results showed that the IFN-α inducible MxB protein is a naturally expressed protein that inhibits the uncoating process of HIV-1.
To explore MxB’s binding site on the surface of the HIV-1 capsid, we tested a battery of capsid inhibitors that are well studied with respect to their binding sites on the HIV-1 capsid protein. From these investigations, we found that the compound CPIPB was able to compete with MxB for binding to capsid suggesting that the binding pocket of CPIPB overlaps with the binding site for MxB ,. The binding pocket for CPIPB is located between the base of the Cyp A binding loop and the loop that connects helices 6 and 7 of HIV-1 capsid (Figure 3C). Remarkably, CPIPB binding to capsid facilitates crystallization of capsid by decreasing the mobility of the Cyp A binding loop and the loop that connects helices 6 and 7 of HIV-1 capsid ,. In agreement, the binding of MxB to the HIV-1 capsid stabilizes the HIV-1 core suggesting that this particular capsid region modulates stability of the HIV-1 core.
Domain mapping studies showed that the 20 N-terminal amino acids are essential for the ability of MxB to bind HIV-1 capsid and restrict infection. Although MxB-∆(1–20) did not bind capsid or restrict HIV-1, it retained its oligomerization ability. These experiments suggested that the N-terminal domain is important for the ability of MxB to restrict HIV-1 infection. Future experiments destined to understand the structure of this region will shed light on the domain used by MxB to interact with the HIV-1 core.
Finally, we established that MxB’s oligomerization by its leucine zipper domain is important for its ability to bind capsid and restrict infection. Deletion and point mutations of MxB’s leucine zipper motif suggested that oligomerization contributes to MxB’s binding avidity for the HIV-1 core.
Overall, our work showed that MxB binds to the HIV-1 core and prevents the uncoating process of HIV-1. In addition, we demonstrated that MxB requires capsid binding and oligomerization for restriction.
Protocols for each methodology used in this manuscript are explained in detail in Additional file 5.
Infection with viruses expressing green fluorescent protein (GFP)
Recombinant HIV-1 expressing GFP was prepared as described . Recombinant viruses were pseudotyped with the VSV-G glycoprotein. For infections, 3 × 104 HeLa, U937 or Cf2Th cells seeded in 24-well plates were incubated at 37°C with virus for 24 h. Cells were washed and returned to culture for 48 h, and then GFP-positive cells were analyzed using a flow cytometer (Becton Dickinson).
Binding of MxB variants to in vitro assembled HIV-1 capsid-nucleocapsid (CA-NC) complexes
The binding of different proteins to in vitro assembled HIV-1 CA-NC complexes that do or do not contain capsid changes was carried out as previously described ,. Input and bound fractions were analyzed by Western blotting using the indicated antibodies.
Fate of the capsid assay
The fate of the capsid assay was performed as previously described ,,. Cells infected for 16 hours were lysed using 15 strokes in a 7.0 ml Dounce homogenizer with pestle B. Cellular debris was cleared by centrifugation for 3 minutes at 3000 rpm. The cleared lysate was layered onto a 50% sucrose (weight: volume) cushion in 1× PBS and centrifuged at 125,000 × g for 2 hours at 4°C in a Beckman SW41 rotor. Input, soluble and pellet fractions were analyzed by Western blotting using the indicated antibodies.
Creation of cells stably expressing wild type and mutant MxB proteins
Cell lines stably expressing wild type or mucbltant MxB proteins were created using the LPCX vector system (Clontech), as previously described ,,. The MxB proteins contained either an influenza hemagglutinin (HA) epitope tag or a FLAG epitope tag at the C terminus.
MxB oligomerization assay
The ability of MxB to oligomerize was determined by measuring the interaction of an MxB-FLAG protein with an MxB-HA protein. Immunoprecipitates were analyzed by Western blotting using either anti-HA or anti-FLAG antibodies (Sigma).
We would like to thank Dmitri Ivanov for providing reagents and Aime Lopez Aguilar for the chemdraw structure. We are thankful to the NIH/AIDS repository program for providing valuable reagents such as antibodies and drugs. This work was funded by NIH R01 AI087390 and R21 AI102824 grants to F.D.-G.
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