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.

A C-terminal "Tail" Region in the Rous Sarcoma Virus Integrase Provides High Plasticity of Functional Integrase Oligomerization during Intasome Assembly.

The retrovirus integrase (IN) inserts the viral cDNA into the host DNA genome. Atomic structures of five different retrovirus INs complexed with their respective viral DNA or branched viral/target DNA substrates have indicated these intasomes are composed of IN subunits ranging from tetramers, to octamers, or to hexadecamers. IN precursors are monomers, dimers, or tetramers in solution. But how intasome assembly is controlled remains unclear. Therefore, we sought to unravel the functional mechanisms in different intasomes. We produced kinetically stabilized Rous sarcoma virus (RSV) intasomes with human immunodeficiency virus type 1 strand transfer inhibitors that interact simultaneously with IN and viral DNA within intasomes. We examined the ability of RSV IN dimers to assemble two viral DNA molecules into intasomes containing IN tetramers in contrast to one possessing IN octamers. We observed that the last 18 residues of the C terminus ("tail" region) of IN (residues 1-286) determined whether an IN tetramer or octamer assembled with viral DNA. A series of truncations of the tail region indicated that these 18 residues are critical for the assembly of an intasome containing IN octamers but not for an intasome containing IN tetramers. The C-terminally truncated IN (residues 1-269) produced an intasome that contained tetramers but failed to produce an intasome with octamers. Both intasomes have similar catalytic activities. The results suggest a high degree of plasticity for functional multimerization and reveal a critical role of the C-terminal tail region of IN in higher order oligomerization of intasomes, potentially informing future strategies to prevent retroviral integration.

Crystal structure of the Rous sarcoma virus intasome.

Integration of the reverse-transcribed viral DNA into the host genome is an essential step in the life cycle of retroviruses. Retrovirus integrase catalyses insertions of both ends of the linear viral DNA into a host chromosome. Integrase from HIV-1 and closely related retroviruses share the three-domain organization, consisting of a catalytic core domain flanked by amino- and carboxy-terminal domains essential for the concerted integration reaction. Although structures of the tetrameric integrase-DNA complexes have been reported for integrase from prototype foamy virus featuring an additional DNA-binding domain and longer interdomain linkers, the architecture of a canonical three-domain integrase bound to DNA remained elusive. Here we report a crystal structure of the three-domain integrase from Rous sarcoma virus in complex with viral and target DNAs. The structure shows an octameric assembly of integrase, in which a pair of integrase dimers engage viral DNA ends for catalysis while another pair of non-catalytic integrase dimers bridge between the two viral DNA molecules and help capture target DNA. The individual domains of the eight integrase molecules play varying roles to hold the complex together, making an extensive network of protein-DNA and protein-protein contacts that show both conserved and distinct features compared with those observed for prototype foamy virus integrase. Our work highlights the diversity of retrovirus intasome assembly and provides insights into the mechanisms of integration by HIV-1 and related retroviruses.

A possible role for the asymmetric C-terminal domain dimer of Rous sarcoma virus integrase in viral DNA binding.

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.

Professor Hidesaburo Hanafusa: a 50-year quest for the molecular basis of cancer.

A pioneer of oncogene research, Hidesaburo Hanafusa, Emeritus Director of the Osaka Bioscience Institute and Emeritus Professor of the Rockefeller University in New York, passed away on 15 March of this year in Japan. For his seminal works in cancer research during six decades (1950s until now), he received a number of awards including the Lasker Award, the Asahi Prize, the Ricketts Award, the Clowes Award, The Order of Culture (Bunka Kunsho in Japan) and the General Motors Sloan Award. I would like to provide a summary of his many contributions to research on viral and cellular oncogenes in order to appreciate the significance of his work on our understanding of the molecular basis of cancer and the implications for diagnosis and treatment.

Identifying amino acid residues that contribute to the cellular-DNA binding site on retroviral integrase.

Although retroviral integrase specifically trims the ends of viral DNA and inserts these ends into any sequence in cellular DNA, little information is available to explain how integrase distinguishes between its two DNA substrates. We recently described novel integrase mutants that were improved for specific nicking of viral DNA but impaired at joining these ends into nonviral DNA. An acidic or bulky substitution at one particular residue was critical for this activity profile, and the prototypic protein--Rous sarcoma virus integrase with an S124D substitution--was defective at nonspecifically binding DNA. We have now characterized 19 (including 16 new) mutants that contain one or more aspartic acid substitutions at residues that extend over the surface of the protein and might participate with residue 124 in binding cellular DNA. In particular, every mutant with an aspartate substitution at residue 98 or 128, similar to the original S124D protein, showed improved specific nicking of viral DNA but disturbed nonspecific nicking of nonviral DNA. These data describe a probable cellular-DNA binding platform that involves at least 5 amino acids, in the following order of importance: 124>128>(98, 125)>123. These experimental data are vital for new models of integrase and will contribute to identifying targets for the next generation of integrase inhibitors.

Effects of varying the spacing within the D,D-35-E motif in the catalytic region of retroviral integrase.

The catalytic domain of all retroviral integrases contains an Asp,Asp-35-Glu (D,D-35-E) motif with precisely 35 amino acids between the second aspartate and the glutamate. We have now made several mutations designed to alter the length or flexibility of a mobile loop within this 35-amino-acid spacer region in full-length Rous sarcoma virus integrase. Surprisingly, most of the mutants had enzymatic activity, including ones that shortened or lengthened the loop by up to 6 amino acids. Several size mutants exhibited the two biologically relevant activities of integrase in reactions with Mn(2+), although they were inactive with Mg(2+). No viruses containing integrase with an altered length, however, replicated in cell culture, and these viruses were blocked at the integration step. Thus, the conserved 35-amino-acid spacing is not absolutely required for enzymatic activity, but the correlation between infectivity and Mg(2+)-dependent activity supports magnesium as the metal cofactor used by integrase in vivo.

Retroviral integrases that are improved for processing but impaired for joining.

Retroviral integrase specifically trims (or processes) the ends of retroviral DNA, then inserts (or joins) these ends into cellular DNA nonspecifically. We previously showed that Rous sarcoma virus integrase with a serine-to-aspartate substitution at amino acid 124 was markedly improved for processing but dramatically impaired for joining, making it the first mutant to separate the activities of integrase in this way. We now show that placing glutamic acid at this residue has the same effect, whereas asparagine or glutamine, which resemble aspartate and glutamate but without the negatively charged acid group, improved processing and impaired joining to a lesser extent. Placing aspartic acid at either of the adjacent residues 123 or 125 also had an intermediate effect. Thus, the charge, structure, and position of the substitution all contribute to the properties of the S124D protein. Infectivity of virions containing these mutations paralleled the in vitro findings, with substitutions having the greatest effect on joining completely blocking replication. Additional studies indicated the replication-defective viruses were blocked at integration and that the S124D protein is impaired at binding nonviral DNA. These functional, biochemical, and genetic data implicate this particular integrase residue as a key part of the binding site for cellular DNA.