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.

Unraveling the Palindromic and Non-Palindromic Motifs of Retroviral Integration Site Sequences by Statistical Mixture Models

“…To uncover sequence motifs at retroviral integration sites (IS), we estimated the mixture models of IS data sets utilizing the expectation-maximization (EM) algorithm. We collected data representing IS of human immunodeficiency virus type 1 (HIV) 47 , human T-cell leukemia virus type 1 (HTLV) 46 , maedivisna virus (MVV) 25 , Moloney murine leukemia virus (MLV) 3 , prototype foamy virus (PFV) 48 and avian sarcoma and leukosis virus (ASLV) 49 (Extended Data Table 1).…”

“…For each variant, data from the transduction of different cell lines were mixed. Coordinates of MVV IS from HEK293T cells 25 and PFV IS sets 48 were obtained from the Gene Expression Omnibus database. MLV 3 set was obtained from raw sequencing reads that were mapped to hg38 human genome assembly using bowtie2 54 .…”

“…Typically, the STR sites on both strands of tDNA are 4 to 6 nucleotides apart depending on the retrovirus. Structural studies demonstrated that IN assembles into symmetric multimeric complexes that bend the tDNA enabling the STR inside a widened major groove of tDNA [18][19][20][21][22][23][24][25][26][27] .…”

Modulation of the functional interfaces between retroviral intasomes and the human nucleosome

“…The developed model and the selected drugs will help in better dissecting the molecular interactions within the intasome/nucleosome functional complex. In particular, this first monitoring of the retroviral/intasome complex paves the way for multiple applications, including the analysis of the influence of cellular partners and the study of additional retroviral intasomes, allowing us to determine the possible specific interfaces previously suggested (7, 8, 25). Our work also provides the technical basis for larger-scale screening of drugs specifically targeting these functional nucleocomplexes or additional nucleosome-partner complexes.…”

B-to-A transition in target DNA during retroviral integration

“…The lentiviruses, such as human immunodeficiency virus 1 (HIV-1) and maedi-visna virus (MVV), use 12 or 16 IN molecules (15)(16)(17)(18) to form the largest intasomes characterized to date. Prior to this work, intasome STC structures were reported for PFV (8), Rous sarcoma virus (RSV) (12), HIV-1 (17), HTLV-1 (10) and MVV (19). While the PFV and RSV structures were determined by X-ray diffraction, the HIV-1, HTLV-1 and MVV structures were determined by single particle cryogenic electron microscopy (cryo-EM).…”