Murine leukemia virus p12 tethers the capsid-containing pre-integration complex to chromatin by binding directly to host nucleosomes in mitosis

Wanaguru, Madushi; Barry, David J.; Benton, D.J.; O’Reilly, Nicola; Bishop, Kate N.

doi:10.1371/journal.ppat.1007117

“…We therefore think it likely that some sort of uncoating event begins at the nuclear pore and finishes at the site of integration. We have also previously shown that the murine leukaemia virus (MLV) p12 protein binds directly to both CA and nucleosomes, tethering the MLV core to chromatin during mitosis, and have proposed that p12 could be acting in a similar capacity to CPSF6 in HIV-1 infection [ 87 ]. Fig 9 includes our model of MLV uncoating.…”

Section: Discussionmentioning

confidence: 99%

HIV-1 requires capsid remodelling at the nuclear pore for nuclear entry and integration

Guedán

¹

,

Donaldson

²

,

Caroe

³

et al. 2021

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The capsid (CA) lattice of the HIV-1 core plays a key role during infection. From the moment the core is released into the cytoplasm, it interacts with a range of cellular factors that, ultimately, direct the pre-integration complex to the integration site. For integration to occur, the CA lattice must disassemble. Early uncoating or a failure to do so has detrimental effects on virus infectivity, indicating that an optimal stability of the viral core is crucial for infection. Here, we introduced cysteine residues into HIV-1 CA in order to induce disulphide bond formation and engineer hyper-stable mutants that are slower or unable to uncoat, and then followed their replication. From a panel of mutants, we identified three with increased capsid stability in cells and found that, whilst the M68C/E212C mutant had a 5-fold reduction in reverse transcription, two mutants, A14C/E45C and E180C, were able to reverse transcribe to approximately WT levels in cycling cells. Moreover, these mutants only had a 5-fold reduction in 2-LTR circle production, suggesting that not only could reverse transcription complete in hyper-stable cores, but that the nascent viral cDNA could enter the nuclear compartment. Furthermore, we observed A14C/E45C mutant capsid in nuclear and chromatin-associated fractions implying that the hyper-stable cores themselves entered the nucleus. Immunofluorescence studies revealed that although the A14C/E45C mutant capsid reached the nuclear pore with the same kinetics as wild type capsid, it was then retained at the pore in association with Nup153. Crucially, infection with the hyper-stable mutants did not promote CPSF6 re-localisation to nuclear speckles, despite the mutant capsids being competent for CPSF6 binding. These observations suggest that hyper-stable cores are not able to uncoat, or remodel, enough to pass through or dissociate from the nuclear pore and integrate successfully. This, is turn, highlights the importance of capsid lattice flexibility for nuclear entry. In conclusion, we hypothesise that during a productive infection, a capsid remodelling step takes place at the nuclear pore that releases the core complex from Nup153, and relays it to CPSF6, which then localises it to chromatin ready for integration.

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“…[ 26 ] The murine leukemia virus (MLV) GAG p12 fragment interaction with mitotic chromatin has also been characterized. [ 36 ] Chromatin binding by MLV‐p12 and PFV‐CBS appear mutually exclusive, as their binding sites are the same. The structure of the tetrameric PFV intasome bound to nucleosome reveals how the viral DNA gets integrated into host chromatin.…”

Section: Nucleosome Acidic Patch: a Docking Station For Viral Proteinmentioning

confidence: 99%

Structural Basis of Nucleosome Recognition and Modulation

Sundaram

¹

,

Vasudevan

²

2020

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Chromatin structure and dynamics regulate key cellular processes such as DNA replication, transcription, repair, remodeling, and gene expression, wherein different protein factors interact with the nucleosomes. In these events, DNA and RNA polymerases, chromatin remodeling enzymes and transcription factors interact with nucleosomes, either in a DNA‐sequence‐specific manner and/or by recognizing different structural features on the nucleosome. The molecular details of the recognition of a nucleosome by different viral proteins, remodeling enzymes, histone post‐translational modifiers, and RNA polymerase II, have been explored in the recent past. The present review puts forth critical insights into the basic mechanisms of nucleosome recognition by the various protein factors and the role of distinct surface epitopes on a nucleosome. These determinants of the underlying specificity include features such as the acidic patch, arginine anchor, histone post‐translational modifications, core DNA, DNA lesions, and linker DNA.

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“…We therefore think it likely that some sort of uncoating event begins at the nuclear pore and finishes at the site of integration. We have also previously shown that the murine leukaemia virus (MLV) p12 protein binds directly to both CA and nucleosomes, tethering the MLV core to chromatin during mitosis, and have proposed that p12 could be acting in a similar capacity to CPSF6 in HIV-1 infection [84]. Figure 9 includes our model of MLV uncoating.…”

Section: Discussionmentioning

confidence: 98%

HIV-1 requires capsid remodelling at the nuclear pore for nuclear entry and integration

Guedán

¹

,

Donaldson

²

,

Cosnefroy

³

et al. 2021

Preprint

Self Cite

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The capsid (CA) lattice of the HIV-1 core plays a key role during infection. From the moment the core is released into the cytoplasm, it interacts with a range of cellular factors that, ultimately, direct the pre-integration complex to the integration site. For integration to occur, the CA lattice must disassemble. Early uncoating or a failure to do so has detrimental effects on virus infectivity, indicating that an optimal stability of the viral core is crucial for infection. Here, we introduced cysteine residues into HIV-1 CA in order to induce disulphide bond formation and engineer hyper-stable mutants that are slower or unable to uncoat, and then followed their replication. From a panel of mutants, we identified three with increased capsid stability in cells and found that, whilst the M68C/E212C mutant had a 5-fold reduction in reverse transcription, two mutants, A14C/E45C and E180C, were able to reverse transcribe to approximately WT levels. Moreover, these mutants only had a 5-fold reduction in 2-LTR circle production, suggesting that not only could reverse transcription complete in hyper-stable cores, but that the nascent viral cDNA could enter the nuclear compartment. Furthermore, we observed significant levels of A14C/E45C mutant capsid in nuclear and chromatin-associated fractions implying that the hyper-stable cores themselves entered the nucleus. Immunofluorescence studies revealed that although the A14C/E45C mutant capsid reached the nuclear pore with the same kinetics as wild type capsid, it was then retained at the pore in association with Nup153. Crucially, infection with the hyper-stable mutants did not promote CPSF6 re-localisation to nuclear speckles, despite the mutant capsids being competent for CPSF6 binding. These observations suggest that hyper-stable cores are not able to uncoat, or remodel, enough to pass through or dissociate from the nuclear pore and integrate successfully. This, is turn, highlights the importance of capsid lattice flexibility for nuclear entry. In conclusion, we hypothesise that during a productive infection, a capsid remodelling step takes place at the nuclear pore that releases the core complex from Nup153, and relays it to CPSF6, which then localises it to chromatin ready for integration.AUTHOR SUMMARYThe mature viral core of human immunodeficiency virus (HIV) consists of a highly organised lattice formed by capsid molecules that encloses the viral RNA and viral enzymes. This lattice is crucial during the early stages of viral replication, as it has to break down – uncoat – at the right time and place in order for the viral DNA to integrate successfully. Lentiviruses, like HIV, can infect non-dividing cells and are able to access the host cell DNA by entering the nucleus through nuclear pores. Until recently, uncoating was thought to occur in the cytoplasm as the whole core was thought too large to pass through the nuclear pore. However, lately it has been suggested that uncoating might occur at the nuclear pore or even inside the nucleus and the site of uncoating is currently hotly debated. By investigating HIV mutants with an increased lattice stability, we have shown that lattice flexibility is crucial for nuclear entry. Provocatively, we observed hyper-stable mutant capsid in nuclear and chromatin-associated fractions suggesting that uncoating is not required for nuclear entry. Nonetheless, microscopy experiments suggested that these hyper-stable mutants were retained on the inner side of the nuclear pore, and were impaired for downstream events in the nucleus, leading to a severe infectivity defect. Therefore, we believe that an essential uncoating, or capsid lattice remodelling event normally takes place at the nuclear pore.

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Murine leukemia virus p12 tethers the capsid-containing pre-integration complex to chromatin by binding directly to host nucleosomes in mitosis

Cited by 24 publications

References 54 publications

HIV-1 requires capsid remodelling at the nuclear pore for nuclear entry and integration

HIV-1 requires capsid remodelling at the nuclear pore for nuclear entry and integration

Structural Basis of Nucleosome Recognition and Modulation

HIV-1 requires capsid remodelling at the nuclear pore for nuclear entry and integration

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