HIV-1 Virions Enter The Nucleus Via Nuclear Envelope Rupture And Restoration

Objective: The entry of pre-integration complex(PIC)into the nucleus is a prerequisite for the integration of HIV-1 genome and the generation of progeny virus,as well as a key factor for the successful replication of HIV-1 in cells.According to the classical theory,virus capsid(CA)disassembles in cytoplasm and releases genome into nucleus through nuclear pore.However,recent reports provide evidence that virus CA can exist in nucleus and play a functional role in it.Whether or not the viral CA enters the nucleus and how it passes through the undersized pore barrier into the nucleus are questions that remain to be clarified.Methods: Live cell real-time imaging strategy is an important method to analyze the behavior of virus entering the nucleus.For this purpose,we used fluorescent dyes and fluorescent proteins to label the viral and cell components differentially,followed up the nuclear entry process of the virus in real time and explored the related mechanism.Result: Fluorescence imaging showed that virions could be transported into the nucleus by means of nuclear envelope(NE)rupture and reconstruction.The process is as follows: after the virions enter the cells,they selectively gathered at the microtubule organization center(MTOC),causing the adjacent NE to deform,and the virions enter the deformed area;with the participation of the endosomal sorting complexes required for transport 3(ESCRT-?)the NE at both ends of the deformation extends to the middle to form a new NE.The NE is reconstructed to form a nuclear vesicle with virions being wrapped;then the inner membrane of the vesicle ruptured to release the virions into the nuclear.These findings were confirmed by transmission electron microscopy(TEM),including aggregation of virions,deformations of NE,encapsulation of virions into nuclear vesicles and rupture of the inner NE of nuclear vesicles.Biochemical experiments further confirmed that viral CA,integrase(IN)and viral genome could be co-located in the nucleus.Drug inhibition experiments showed that dissolved microtubules(MT)would stop the deformation of NE and disperse the accumulation of virus particles.At the same time,knocking down SUN protein can reduce the occurrence of nuclear membrane depression,indicating that the linker of nucleoskeleton and cytoskeleton complex(LINC)system is involved in the formation of NE deformation mediated by virus.Conclusions: This is the first time that the dynamic process of virion CA crossing the NE has been visualized,not through the nuclear pore into the nucleus,but through the formation and rupture pathway of the NE vesicles into the nucleus.We use a most intuitive way to give a reasonable explanation for this long-standing controversial and contradictory issue.Furthermore,it can be explained that virions can have different fates in certain key links after entering the cells,thus explaining many inconsistencies reported by the predecessors.This ingenious NE rupture repair method minimizes the long-term and large-area contact between nuclear matter and cytoplasm while achieving nuclear material exchange,and can effectively control nucleoplasm loss and cytoplasmic damage to the cell genome.