Cryo-EM structure of the Rous sarcoma virus octameric cleaved synaptic complex intasome

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

The HIV-1 Integrase Monomer Induces a Specific Interaction with LTR DNA for Concerted Integration

The assembly mechanism for the human immunodeficiency virus type 1 (HIV) synaptic complex (SC) capable of concerted integration is unknown. Molecular and structural studies have established that the HIV SC and prototype foamy virus (PFV) intasome contain a tetramer of integrase (IN) that catalyzes concerted integration. HIV IN purified in the presence of 1 mM EDTA and 10 mM MgSO₄ was predominately a monomer. IN efficiently promoted concerted integration of micromolar concentrations of 3′-OH recessed and blunt-ended U5 long terminal repeat (LTR) oligonucleotide (ODN) substrates (19–42 bp) into circular target DNA. Varying HIV IN to U5 DNA showed that an IN dimer:DNA end molar ratio of 1 was optimal for concerted integration. Integration activities decreased with an increasing length of the ODN, starting from the recessed 18/20 or 19/21 bp set to the 31/33 and 40/42 bp set. Under these conditions, the average fidelity for the HIV 5 bp host site duplication with recessed and blunt-ended substrates was 56%. Modifications of U5 LTR sequences beyond 21 bp from the terminus on longer DNA (1.6 kb) did not alter the ∼32 bp DNaseI protective footprint, suggesting viral sequences beyond 21 bp were not essential for IN binding. The results suggest IN binds differentially to an 18/20 bp than to a 40/42 bp ODN substrate for concerted integration. The HIV IN monomer may be a suitable candidate for attempting crystallization of an IN–DNA complex in the absence or presence of strand transfer inhibitors

Functional analyses of RSV IN (1–270) with point mutations at residues stabilizing the CTD dimer.

<p>A) In the left panel, the RSV IN (1–270) construct without (lanes 3 and 4) or with different amino acid substitutions indicated at the top (lanes 5 to 12) were assayed for strand transfer activities at 300 mM NaCl. IN concentration was 10 nM or 20 nM and the 3.6 kb GU3 donor DNA was used. The percentage of donor incorporated into the CHS and concerted integration products at 10 nM IN was 4% and 53%, respectively (lane 3). In the right panel, the same proteins were assayed as indicated above except that the NaCl concentration was 100 mM. Lanes 1 and 15 have molecular weight markers, lanes 2 and 16 have control reactions without protein. Lanes 13, 14, 27, and 28 contain wild type RSV IN (1–286) at 10 nM or 15 nM. B) IN (1–270) and (1–270)•W213A were assayed at two different NaCl concentrations as in (A). C) The 3′ OH processing activities of IN (1–270) and all of the above mutants for the 1–270 construct were analyzed at 20 nM IN with either MgCl₂ or MnCl₂ at 100 mM NaCl. Wild type IN (1–286) was also assayed at 20 nM.</p

A Possible Role for the Asymmetric C-Terminal Domain Dimer of Rous Sarcoma Virus Integrase in Viral DNA Binding

<div><p>Integration of the retrovirus linear DNA genome into the host chromosome is an essential step in the viral replication cycle, and is catalyzed by the viral integrase (IN). Evidence suggests that IN functions as a dimer that cleaves a dinucleotide from the 3′ DNA blunt ends while a dimer of dimers (tetramer) promotes concerted integration of the two processed ends into opposite strands of a target DNA. However, it remains unclear why a dimer rather than a monomer of IN is required for the insertion of each recessed DNA end. To help address this question, we have analyzed crystal structures of the Rous sarcoma virus (RSV) IN mutants complete with all three structural domains as well as its two-domain fragment in a new crystal form at an improved resolution. Combined with earlier structural studies, our results suggest that the RSV IN dimer consists of highly flexible N-terminal domains and a rigid entity formed by the catalytic and C-terminal domains stabilized by the well-conserved catalytic domain dimerization interaction. Biochemical and mutational analyses confirm earlier observations that the catalytic and the C-terminal domains of an RSV IN dimer efficiently integrates one viral DNA end into target DNA. We also show that the asymmetric dimeric interaction between the two C-terminal domains is important for viral DNA binding and subsequent catalysis, including concerted integration. We propose that the asymmetric C-terminal domain dimer serves as a viral DNA binding surface for RSV IN.</p> </div

The minimal RSV IN constructs functional in concerted integration.

<p>A) <i>In vitro</i> concerted integration activities of the wild type RSV IN (1–286), the C-terminally truncated IN (1–270), and IN 1–270•C23S. The proteins were assayed for strand transfer activities using a 1.1 kb GU3 DNA substrate and with a 2.7 kb supercoiled DNA target. The concentrations (nM) of each IN are indicated at the top. The circular half-site (CHS) and concerted integration products as well as the donor substrate are indicated on the left. Lane 1 contains no IN, and in lane 11(marked M) are molecular weight markers as indicated on the right. At 18 nM IN, the percentage of donor incorporated into the concerted integration products for RSV IN 1–286, 1–270, and 1–270•C23S were 41%, 37%, and 50%, respectively. The CHS products were 10%, 9%, and 13%, respectively. The NaCl concentration in the reaction condition was 300 mM. B) Size-exclusion chromatography profiles of purified RSV IN (1–270) and IN 1–270•C23S. The proteins at 1 mg/ml were injected into a Superdex-200 column (10/300) operating with a running buffer containing 1 M NaCl. The elution positions for the molecular weight standards are indicated by arrows.</p