Cryo-EM reveals a novel octameric integrase structure for betaretroviral intasome function

Abstract

Retroviral integrase (IN) catalyzes the integration of viral DNA (vDNA) into host target (tDNA), which is an essential step in the lifecycle of all retroviruses1. Prior structural characterization of IN-vDNA complexes, or intasomes, from the spumavirus prototype foamy virus (PFV) revealed a functional IN tetramer2–5, and it is generally believed that intasomes derived from other retroviral genera will employ tetrameric IN6–9. However, the intasomes of orthoretroviruses, which include all known pathogenic species, have not been characterized structurally. Using single-particle cryo-electron microscopy (cryo-EM) and X-ray crystallography, we determine here an unexpected octameric IN architecture for the β-retrovirus mouse mammary tumor virus (MMTV) intasome. The structure is composed of two core IN dimers, which interact with the vDNA ends and structurally mimic the PFV IN tetramer, and two flanking IN dimers that engage the core structure via their IN C-terminal domains (CTDs). Contrary to the belief that tetrameric IN components are sufficient to catalyze integration, the flanking IN dimers were necessary for MMTV IN activity. The IN octamer solves a conundrum for the β- as well as α-retroviruses by providing critical CTDs to the intasome core that cannot be provided in cis due to evolutionarily restrictive catalytic core domain (CCD)-CTD linker regions. The octameric architecture of the MMTV intasome provides a new paradigm for the structural basis of retroviral DNA integration.

Critical Contribution of Tyr15 in the HIV-1 Integrase (IN) in Facilitating IN Assembly and Nonenzymatic Function through the IN Precursor Form with Reverse Transcriptase

“…vDNAs play a key role in intasome assembly, which is required for HIV-1 IN to exert its enzymatic function (33)(34)(35). In line with this finding, vRNA might play a critical role in vRNP complex assembly.…”

“…Structure analyses have revealed that each HIV-1 IN domain (28)(29)(30) and the two domains composing the NTD-CCD (31) or the CCD-CTD (32) assemble into either a dimer or a tetramer form. Recently, the entire structures of other retroviral INs in a complex with their cognate viral DNA ends, referred to as intasomes, have been successfully resolved (33)(34)(35). These structure analyses of intasomes revealed the unprecedented assembly of INs into a tetramer form for the prototype foamy virus (33) and octamer forms for the Rous sarcoma virus (34) and mouse mammary tumor virus (35), providing new insights into the enzymatic role for IN during retroviral DNA integration and the inhibitory results of INSTI (36).…”

Retroviral intasomes arising

“…However, recent intasome structures from four orthoretroviruses revealed both striking complexity and diversity. Reported from February 2016 to January 2017, the new structures elucidated intasomes from the β-retrovirus mouse mammary tumor virus (MMTV) [19], α-retrovirus Rous sarcoma virus (RSV) [20], lentivirus maedi-visna virus (MVV) [7], and lentivirus human immunodeficiency virus 1 (HIV-1) [21]. As three of the studies utilized single particle cryo-electron microscopy [7,19,21], much of the work benefited from the ongoing revolution in this structural biology platform (reviewed in [22]).…”

The conformational feasibility for the formation of reaching dimer in ASV and HIV integrase: a molecular dynamics study

“…In contrast to this, both NTD and CTD of HIV A reaching dimer have to undergo sterically unfavorable rotation, translation and closure motions to attain the core dimer conformation. The flexibility of CCD-CTD linker due to its disordered nature is reported to be important for the proper functioning of IN by positioning the CTD in cis and trans to the neighboring monomer (Ballandras-Colas et al 2016; Yin et al 2016). The area and volume of the dimer interface is also higher in core dimer when compared to the reaching dimers (Table 1).…”

“…In addition to DNA binding, the inner monomeric subunits of PFV intasome are associated through the interactions between NTD of one monomer and CCD of another monomer, while the two outer monomeric units interact with the inner monomers by CCD-CCD interactions. The cryo-EM and X-ray studies of Mouse mammary tumour virus (MMTV) and Rous sarcoma virus (RSV) intasomes have reported an octameric state of IN bound to both viral and target DNA (Ballandras-Colas et al 2016; Yin et al 2016). Similar to the PFV intasome, the inner dimers of both MMTV and RSV express domain-swapping interactions, while the outer dimers provide CTDs to the inner dimers for integration in a trans fashion.…”