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Multivalent interactions essential for lentiviral integrase function.

ABSTRACT: A multimer of retroviral integrase (IN) synapses viral DNA ends within a stable intasome nucleoprotein complex for integration into a host cell genome. Reconstitution of the intasome from the maedi-visna virus (MVV), an ovine lentivirus, revealed a large assembly containing sixteen IN subunits¹. Herein, we report cryo-EM structures of the lentiviral intasome prior to engagement of target DNA and following strand transfer, refined at 3.4 and 3.5 Å resolution, respectively. The structures elucidate details of the protein-protein and protein-DNA interfaces involved in lentiviral intasome formation. We show that the homomeric interfaces involved in IN hexadecamer formation and the α-helical configuration of the linker connecting the C-terminal and catalytic core domains are critical for MVV IN strand transfer activity in vitro and for virus infectivity. Single-molecule microscopy in conjunction with photobleaching reveals that the MVV intasome can bind a variable number, up to sixteen molecules, of the lentivirus-specific host factor LEDGF/p75. Concordantly, ablation of endogenous LEDGF/p75 results in gross redistribution of MVV integration sites in human and ovine cells. Our data confirm the importance of the expanded architecture observed in cryo-EM studies of lentiviral intasomes and suggest that this organization underlies multivalent interactions with chromatin for integration targeting to active genes.

SUBMITTER: Ballandras-Colas A

PROVIDER: S-EPMC9065133 | biostudies-literature | 2022 May

REPOSITORIES: biostudies-literature

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Multivalent interactions essential for lentiviral integrase function.

Ballandras-Colas Allison A Chivukula Vidya V Gruszka Dominika T DT Shan Zelin Z Singh Parmit K PK Pye Valerie E VE McLean Rebecca K RK Bedwell Gregory J GJ Li Wen W Nans Andrea A Cook Nicola J NJ Fadel Hind J HJ Poeschla Eric M EM Griffiths David J DJ Vargas Javier J Taylor Ian A IA Lyumkis Dmitry D Yardimci Hasan H Engelman Alan N AN Cherepanov Peter P

Nature communications 20220503 1

A multimer of retroviral integrase (IN) synapses viral DNA ends within a stable intasome nucleoprotein complex for integration into a host cell genome. Reconstitution of the intasome from the maedi-visna virus (MVV), an ovine lentivirus, revealed a large assembly containing sixteen IN subunits<sup>1</sup>. Herein, we report cryo-EM structures of the lentiviral intasome prior to engagement of target DNA and following strand transfer, refined at 3.4 and 3.5 Å resolution, respectively. The structur ...[more]

PMID: 35504909

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Project description:Viral vectors represent an attractive technology for vaccine delivery. We exploited the integrase defective lentiviral vector (IDLV) as a platform for delivering relevant antigens within the context of the ADITEC collaborative research program. In particular, Influenza virus hemagglutinin (HA) and nucleoprotein (NP) were delivered by IDLVs while H1N1 A/California/7/2009 subunit vaccine (HAp) with or without adjuvant was used to compare the immune response in a murine model of immunization. In order to maximize the antibody response against HA, both IDLVs were also pseudotyped with HA (IDLV-HA/HA and IDLV-NP/HA, respectively). Groups of CB6F1 mice were immunized intramuscularly with a single dose of IDLV-NP/HA, IDLV-HA/HA, HAp alone, or with HAp together with the systemic adjuvant MF59. Six months after the vaccine prime all groups were boosted with HAp alone. Cellular and antibody responses to influenza antigens were measured at different time points after the immunizations. Mice immunized with HA-pseudotyped IDLVs showed similar levels of anti-H1N1 IgG over time, evaluated by ELISA, which were comparable to those induced by HAp + MF59 vaccination, but significantly higher than those induced by HAp alone. The boost with HAp alone induced an increase of antibodies in all groups, and the responses were maintained at higher levels up to 18 weeks post-boost. The antibody response was functional and persistent overtime, capable of neutralizing virus infectivity, as evaluated by hemagglutination inhibition and microneutralization assays. Moreover, since neuraminidase (NA)-expressing plasmid was included during IDLV preparation, immunization with IDLV-NP/HA and IDLV-HA/HA also induced functional anti-NA antibodies, evaluated by enzyme-linked lectin assay. IFNγ-ELISPOT showed evidence of HA-specific response in IDLV-HA/HA immunized animals and persistent NP-specific CD8+ T cell response in IDLV-NP/HA immunized mice. Taken together our results indicate that IDLV can be harnessed for producing a vaccine able to induce a comprehensive immune response, including functional antibodies directed toward HA and NA proteins present on the vector particles in addition to a functional T cell response directed to the protein transcribed from the vector.

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Multivalent interactions essential for lentiviral integrase function.

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Multivalent interactions essential for lentiviral integrase function.

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