Antiviral Mechanism Of MxB Against HIV-1

As a member of the interferon-induced dynein-like GTPase superfamily,the human myxovirus resistance 2（MxB/Mx2）protein plays a role in regulating nucleocytoplasmic transport and cell cycle progression.It is later discovered that MxB has antiviral activity against a wide variety of viruses,including HIV-1,HTNV,Herpesviruses,HCV and HBV.MxB usually interferes with the viral replication after entering the target cell,but the mechanism of inhibition remains unclear.MxB can directly target HIV-1 capsid protein（CA）to suppress HIV-1 uncoating,nuclear import and integration.The restriction of MxB to HIV-1 is affected by various factors.In addition to HIV-1 CA,MxB-mediated HIV-1 inhibition also depends on other cofactors,such as Cyp A,and the interaction between multiple cofactors and HIV-1CA.Moreover,the anti-HIV-1 activity of MxB is also affected by itself,including its nuclear localization signal（NLS）.However,the role of GTPase activity,including GTP binding and GTP hydrolysis,in recognition of viral targets and inhibition of viral replication by MxB is not well understood.Our study focuses on the mechanism of MxB-mediated HIV-1 inhibition and the role of MxB GTPase activity in viral restriction.For a start,He La cells and HIV-1 target cells,including primary macrophages and primary CD4⁺T cells,were stably transduced with lentiviruses to overexpress MxB,then the cells were infected with VSV-G pseudotyped HIV-1 luciferase reporter virus,HIV-1_Bal and HIV-1_NL4-3respectively,we confirmed that MxB can effectively inhibit HIV-1 replication in these cells.We used real-time fluorescent quantitative PCR（q PCR）and Alu-PCR to measure viral reverse transcripts representing three phases of replication,namely late reverse transcription product（Late RT）,nuclear circular DNA（2-LTR circles）and provirus DNA（Integrated DNA）.We found that MxB can significantly reduced the accumulation of 2-LTR circles and Integrated DNA in infected cells,confirming that MxB can inhibit HIV-1 nuclear import and integration in these cells.HIV-1 pre-integration complex（PIC）needs to enter the nucleus through the nuclear pore complex（NPC）for integration.As an important component of PIC,HIV-1 CA will participate in this process.Through immunofluorescence confocal experiments in He La cells and co-immunoprecipitation experiments in 293T cells,we found that MxB localized at the nuclear pore and bound to nucleoporin NUP358,disrupted HIV-1 CA-NUP358 interaction,thereby inhibiting PIC nuclear import.Next,RNA interference（RNAi）was used to knock down the nucleoporins（NUPs）in He La cells,the cells were then infected with VSV-G pseudotyped HIV-1 bearing either the wild-type（WT）CA(HIV-1_WT)or N74D CA mutant(HIV-1_N74D),we found that MxB inhibited NUP358-mediated replication of HIV-1_WT,while HIV-1_N74D,which was independent on NUP358,could escape the restriction of MxB.Immunofluorescence confocal experiments revealed that HIV-1_N74D PIC did not rely on NUP358 to entry into the nucleus,nor did MxB inhibit its nuclear import,further confirming that MxB inhibits NUP358-mediated HIV-1 PIC nuclear import.The N74D mutation abolished HIV-1 CA binding to the splicing and polyadenylation specificity factor subunit 6（CPSF6）.We used three methods to disrupt the binding of HIV-1 CA to CPSF6:CA N74D mutation,CPSF6 RNAi and PF-3450074（PF74）compound treatment,and identified a new cofactor CPSF6,which was required for the inhibition of MxB to HIV-1.Next,we detected the amount of HIV-1 DNA using q PCR and Alu-PCR and found CPSF6 was involved in restrction of MxB to HIV-1 nuclear import and integration.Immunofluorescence confocal experiments in He La cells further confirmed that the binding of CPSF6 to HIV-1 CA facilitated inhibition of MxB to HIV-1 PIC nuclear import.The co-immunoprecipitation experiment in 293T cells indicated that CPSF6 and MxB did not bind directly,both CPSF6 and MxB bound with HIV-1 CA,importantly,neither CPSF6 affect interaction of MxB and HIV-1 CA,nor MxB affect interaction of CPSF6 and HIV-1 CA.These data showed that MxB impedes the NUP358-mediated HIV-1 PIC nuclear import and viral replication cooperatively with CPSF6.To understand the role of MxB GTPase activity,including GTP binding and GTP hydrolysis,in restriction of HIV-1 replication,we genetically separated these two functions and evaluated their contributions to restriction.We constructed a series of MxB mutant plasmids,including NLS truncation mutation(MxB_ΔNLS),GTP binding defective K131A mutation(MxB_K131A)and GTP hydrolysis defective T151A mutation(MxB_T151A).The immunofluorescence confocal experiment in He La cells and co-immunoprecipitation experiment in 293T cells showed that in addition to the NLS domain,GTP binding function of MxB also contributed to its accumulation on the nuclear membrane,and interaction with the cytoplasmic face of nuclear pore（NUP358）;while the GTP binding and hydrolysis facilitated MxB interaction with the inner side of nuclear pore（NUP98）.Next,we examined the effect of MxB and the mutants on HIV-1 replication and HIV-1 DNA accumulation in He La cells.It was found that except MxBΔNLS,both MxB_K131A and MxB_T151A retained the same antiviral ability as MxB.The difference was that MxB_K131A and MxB_T151A inhibited HIV-1nuclear import and integration less strong compared to MxB.Interestingly,we found that only MxB_K131A inhibited HIV-1 reverse transcription,indicating that based on GTP binding and GTP hydrolysis,MxB inhibited HIV-1 replication by different mechanisms.MxB interacts with the HIV-1 CA via its N-terminal and GTPase（G）domains.Here we explored the role of MxB GTPase activity in viral target recognition.Both immunofluorescence confocal experiments and co-immunoprecipitation experiments found that in addition to NLS,the GTP binding function of MxB also facilitated viral target recognition.Furthermore,we investigated the effect of MxB and the mutants on HIV-1 PIC nuclear import in He La cells by immunofluorescence confocal analysis.It was found that MxB hindered the binding of HIV-1 CA to NUP358 and NUP98.In contrast,neither MxB_ΔNLS nor MxB_K131A affected the binding of HIV-1 CA to NUP358 and NUP98.MxB_T151A increased the binding of HIV-1 CA to NUP358 but reduced the binding to NUP98,which is different from MxB.These data confirmed that MxB relied on its GTPase activity to disrupt the binding of HIV-1 CA and NUPs.Therefore,the role of MxB GTPase activity in inhibiting HIV-1 replication is multifaceted.MxB and GTPase-defective mutants inhibit HIV-1 replication using completely different mechanisms.In summary,this study found that MxB can bind to nucleoporins NUP358 and NUP98,preventing HIV-1 CA binding to NUP358 and NUP98,thereby inhibiting the nuclear import of HIV-1 PIC and viral replication.In addition,CPSF6 is a cofactor required for MxB restriction of HIV-1.Moreover,MxB exploits different mechanisms to inhibit HIV-1 replication based on GTP binding and GTP hydrolysis.These findings provide important evidence for explaining the exact mechanism of MxB MxB-mediated HIV-1 inhibition.