Abstract:Integration of the reverse-transcribed viral DNA into host chromosomes is a critical step in the life-cycle of retroviruses, including an oncogenic delta(δ)-retrovirus human T-cell leukemia virus type-1 (HTLV-1). Retroviral integrase forms a higher order nucleoprotein assembly (intasome) to catalyze the integration reaction, in which the roles of host factors remain poorly understood. Here, we use cryo-electron microscopy to visualize the HTLV-1 intasome at 3.7-Å resolution. The structure together with functio… Show more
“…We next wanted to understand the interactions between the CTD and the “tail” region of IN to promote the assembly of intasomes and to foster virus infectivity. CTDs play critical roles in stabilizing functional retrovirus intasome CICs 1 , 3 , 4 , 6 , 8 . The RSV IN “tail” region (defined as residues from Ile269 to Ala286; Supplementary Fig.…”
Section: Resultsmentioning
confidence: 99%
“…The structures of different retroviral IN-DNA complexes capable of concerted integration show diverse organizations but possess significant mechanistic similarities. These structures, generally termed intasomes, contain multimers of IN subunits ranging from 4 for the simiispumavirus prototype foamy virus (PFV) 1,2 and delta-retroviruses human T-cell leukemia virus type 1 (HTLV-1) 3 and simian T-lymphotropic virus type 1 (STLV-1) 4 to 8 for alpha-retrovirus Rous sarcoma virus (RSV) 5 and betaretrovirus mouse mammary tumor virus (MMTV) 6 . For lentiviral intasomes, there are variable numbers of IN subunits ranging from 4 to 12 for HIV-1 and simian immunodeficiency virus (SIV) [7][8][9] and up to 16 subunits for maedi-visna virus (MVV) 10 .…”
Despite conserved catalytic integration mechanisms, retroviral intasomes composed of integrase (IN) and viral DNA possess diverse structures with variable numbers of IN subunits. To investigate intasome assembly mechanisms, we employed the Rous sarcoma virus (RSV) IN dimer that assembles a precursor tetrameric structure in transit to the mature octameric intasome. We determined the structure of RSV octameric intasome stabilized by a HIV-1 IN strand transfer inhibitor using single particle cryo-electron microscopy. The structure revealed significant flexibility of the two non-catalytic distal IN dimers along with previously unrecognized movement of the conserved intasome core, suggesting ordered conformational transitions between intermediates that may be important to capture the target DNA. Single amino acid substitutions within the IN C-terminal domain affected intasome assembly and function in vitro and infectivity of pseudotyped RSV virions. Unexpectedly, 17 C-terminal amino acids of IN were dispensable for virus infection despite regulating the transition of the tetrameric intasome to the octameric form in vitro. We speculate that this region may regulate the binding of highly flexible distal IN dimers to the intasome core to form the octameric complex. Our studies reveal key steps in the assembly of RSV intasomes.
“…We next wanted to understand the interactions between the CTD and the “tail” region of IN to promote the assembly of intasomes and to foster virus infectivity. CTDs play critical roles in stabilizing functional retrovirus intasome CICs 1 , 3 , 4 , 6 , 8 . The RSV IN “tail” region (defined as residues from Ile269 to Ala286; Supplementary Fig.…”
Section: Resultsmentioning
confidence: 99%
“…The structures of different retroviral IN-DNA complexes capable of concerted integration show diverse organizations but possess significant mechanistic similarities. These structures, generally termed intasomes, contain multimers of IN subunits ranging from 4 for the simiispumavirus prototype foamy virus (PFV) 1,2 and delta-retroviruses human T-cell leukemia virus type 1 (HTLV-1) 3 and simian T-lymphotropic virus type 1 (STLV-1) 4 to 8 for alpha-retrovirus Rous sarcoma virus (RSV) 5 and betaretrovirus mouse mammary tumor virus (MMTV) 6 . For lentiviral intasomes, there are variable numbers of IN subunits ranging from 4 to 12 for HIV-1 and simian immunodeficiency virus (SIV) [7][8][9] and up to 16 subunits for maedi-visna virus (MVV) 10 .…”
Despite conserved catalytic integration mechanisms, retroviral intasomes composed of integrase (IN) and viral DNA possess diverse structures with variable numbers of IN subunits. To investigate intasome assembly mechanisms, we employed the Rous sarcoma virus (RSV) IN dimer that assembles a precursor tetrameric structure in transit to the mature octameric intasome. We determined the structure of RSV octameric intasome stabilized by a HIV-1 IN strand transfer inhibitor using single particle cryo-electron microscopy. The structure revealed significant flexibility of the two non-catalytic distal IN dimers along with previously unrecognized movement of the conserved intasome core, suggesting ordered conformational transitions between intermediates that may be important to capture the target DNA. Single amino acid substitutions within the IN C-terminal domain affected intasome assembly and function in vitro and infectivity of pseudotyped RSV virions. Unexpectedly, 17 C-terminal amino acids of IN were dispensable for virus infection despite regulating the transition of the tetrameric intasome to the octameric form in vitro. We speculate that this region may regulate the binding of highly flexible distal IN dimers to the intasome core to form the octameric complex. Our studies reveal key steps in the assembly of RSV intasomes.
“…While this paper was under review, Aihara and colleagues reported the cryo-EM structure of the HTLV-1 strand-transfer complex, representing the post-catalytic state of the deltaretroviral integration process. Using IN-Sso7d chimera, they obtained stable nucleoprotein complexes, which, as in our structures, are comprised of four IN and two B56γ molecules 38 . Fig.…”
Human T-cell lymphotropic virus type 1 (HTLV-1) is a deltaretrovirus and the most oncogenic pathogen. Many of the ~20 million HTLV-1 infected people will develop severe leukaemia or an ALS-like motor disease, unless a therapy becomes available. A key step in the establishment of infection is the integration of viral genetic material into the host genome, catalysed by the retroviral integrase (IN) enzyme. Here, we use X-ray crystallography and single-particle cryo-electron microscopy to determine the structure of the functional deltaretroviral IN assembled on viral DNA ends and bound to the B56γ subunit of its human host factor, protein phosphatase 2 A. The structure reveals a tetrameric IN assembly bound to two molecules of the phosphatase via a conserved short linear motif. Insight into the deltaretroviral intasome and its interaction with the host will be crucial for understanding the pattern of integration events in infected individuals and therefore bears important clinical implications.
“…Additionally, HTLV requires PPP2R5C for efficient strand-transfer activity and target integration ( Maertens, 2016 ). HTLV integrase uses a LxxIxE motif to recruit PPP2R5C to the viral integration complex ( Bhatt et al, 2020 ). Taken together, these observations support a model in which PPP2R5 antagonism, and global changes in the cellular phospho-proteome, are likely to be advantageous for the pathogenesis of HIV-1 as well as other prominent viruses.…”
Accessory proteins are a key feature that distinguishes primate immunodeficiency viruses such as human immunodeficiency virus type I (HIV-1) from other retroviruses. A prime example is the virion infectivity factor, Vif, which hijacks a cellular co-transcription factor (CBF-β) to recruit a ubiquitin ligase complex (CRL5) to bind and degrade antiviral APOBEC3 enzymes including APOBEC3D (A3D), APOBEC3F (A3F), APOBEC3G (A3G), and APOBEC3H (A3H). Although APOBEC3 antagonism is essential for viral pathogenesis, and a more than sufficient functional justification for Vif’s evolution, most viral proteins have evolved multiple functions. Indeed, Vif has long been known to trigger cell cycle arrest and recent studies have shed light on the underlying molecular mechanism. Vif accomplishes this function using the same CBF-β/CRL5 ubiquitin ligase complex to degrade a family of PPP2R5 phospho-regulatory proteins. These advances have helped usher in a new era of accessory protein research and fresh opportunities for drug development.
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