Cryo-EM structural studies of avian retrovirus intasomes

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Retroviruses are ancient infectious elements that are ubiquitous in nature. Retroviruses had many evolutionary roles in different biological species by integrating their viral DNA, produced by reverse transcription of their RNA genome, into cellular chromosomes. The viral encoded integrase (IN) mediates the integration of the viral DNA.

As a microbiology student at the University of Iowa in 1970, I attended a virology class which sparked my interest in this field. This course initiated my career studying avian retroviruses at Saint Louis University, leading to the discovery of the avian myeloblastosis virus IN by my lab in 1978. By 1983, the retrovirus research field was primed to unravel the mysterious and harmful human immunodeficiency virus type 1 (HIV-1) that caused acquired immunodeficiency syndrome (AIDS).

Besides our biological approaches using Rous sarcoma virus (RSV), biochemistry played an important role in understanding IN functions. Numerous other labs studied IN in the vast retroviridae family including the genera lentivirus, betaretrovirus, and simiispumavirus among others. Near-atomic resolution structural studies of various IN/viral DNA complexes generally termed intasomes, including the presence of target DNA in these complexes, were sure to play an important role for understanding integration. We determined the structure of the RSV intasome containing viral DNA by single-particle cryo-EM (1). The intasome showed significant conformational flexibility in its overall architecture as well as the interactions of an HIV-1 IN strand transfer inhibitor (INSTI) in the active site. The availability of the state of the art cryo-EM facility at Washington University in St. Louis as well as our long collaboration with the structural biologist Dr. Hideki Aihara (University of Minnesota) made this project possible. Our work complements earlier pioneering x-ray crystallography studies of simiispumavirus prototype foamy virus (PFV) IN in complex with viral DNA, in the absence and presence of HIV-1 INSTIs (2). Structural studies of intasomes revealed different number of IN subunits ranging from 4 for the PFV and delta-retroviruses human T-cell leukemia virus type 1 (HTLV-1) (3) and simian T-lymphotropic virus type 1 (4), 8 for alpha-retrovirus RSV (5) and beta-retrovirus mouse mammary tumor virus (MMTV) (6). For lentiviral intasomes, variable number of IN subunits ranging from 4 to 12 for HIV-1 and simian immunodeficiency virus (SIV) (7-9) and up to 16 subunits for maedi visna virus (MVV) (10) were revealed. These studies provided a highly needed understanding of INSTIs interactions within the conserved intasome core (CIC) containing the active site. The CIC structure is maintained across all retrovirus intasomes.

Fig. 1. Cryo-EM structure of the RSV octameric intasome trapped with MK-2048. Ribbon model representation of RSV octameric intasome viewed down its pseudo 2-fold axis. The two proximal IN dimers (green/cyan and pink/gray) plus the distal IN CTDs (magenta, slate blue) bridging between them constitute the core of the complex, referred as ‘conserved intasome core (CIC)’. Disordered CCD-CTD linkers for the distal IN dimers are shown as dashed lines.
Fig. 2. Assembly pathway of RSV octameric and tetrameric intasomes. Octameric intasome can be assembled with IN 1-278 while a tetrameric intasome is formed with IN 1-269. INSTI stabilizes the intasomes.

What are the basic assembly mechanisms associated with these architecturally diverse IN intasome structures that carry out integration? We approached this question using the RSV octameric intasome containing 8 IN subunits with viral DNA as a model (1) (Fig. 1). Earlier studies demonstrated that a tetrameric RSV intasome, the precursor to the octameric form, was preferentially assembled by a C-terminal truncated RSV IN (1-269) that lacks the last 17 amino acids (11,12) (Fig. 2). We utilized HIV-1 INSTIs as “effective tools” to stabilize the tetrameric intasome, a necessary step for isolation by size-exclusion chromatography and, future structural determination by cryo-EM. RSV octameric intasome is produced and isolated in a similar fashion with IN 1-278, the optimum size truncation for its assembly in comparison to wt IN 1-286. Single-particle cryo-EM analysis produced surprising results. It revealed significant flexibility of the two non-catalytic distal IN dimers along with previously unrecognized CIC movement, suggesting ordered conformational transitions between intermediates that may be important to capture the target DNA. In collaboration with Dr. Alan Engelman (Harvard Medical School), we investigated how C-terminal IN truncations and structure-based missense mutations affected assembly in vitro and infectivity of pseudotyped RSV virions. Truncations of the C-terminal region up to Ile269 did not affect viral infectivity despite regulating the transition of the tetrameric intasome to the octameric form in vitro. Missense mutations helped identify critical residues that allowed the identification of amino acids affecting the assembly of either the tetrameric or octameric intasomes in vitro and under physiological conditions. Hopefully, these studies will provide further insights into investigating the assembly of different retrovirus intasomes including HIV-1.

References

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Duane Grandgenett

Professor, Saint Louis University