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Prototype Foamy Virus (PFV) Intasome

Clara Fischman '13 and Katie Adlam '13


Contents:


I. Biological Motivation

Human Immunodeficiency Virus (HIV), considered a pandemic by the World Health Organization, is a retrovirus known to cause acquired immunodeficiency syndrome (AIDS).  Retroviruses have the disturbing ability to stay dormant in the host cell, allowing infected individuals to live symptom free for prolonged periods of time.  Due the silent transmission of HIV, AIDS exploded onto the scene from near anonymity, and has killed over 25 million people worldwide since its discovery in 19811. While treatments such as HAART, highly active antiretroviral therapy, have prolonged the lifespan of infected individuals, a clear remedy for HIV has yet to be discovered.  The mechanism by which retroviruses infect and propagate within cells still marks a wide area of research, with the hope that further understanding will yield a cure to HIV and other viruses of its kind.  

Due to its availability, the prototype foamy virus (PFV) has been used as a model of retroviral activity.  All retroviruses are single stranded RNA viruses that contain reverse transcriptase and integrase enzymes.  Reverse transcriptase produces viral DNA (vDNA) from an RNA genome, which is then inserted into the host cell chromosome by integrase (IN).  The intasome, vDNA-IN complex, engages with target chromosomal DNA (tDNA) and participates in strand transfer, irreversibly joining the viral and target cellular DNA. Figure 2a The mechanism of retroviral integration   

1"Global Statistics." AIDS.gov. Globalhealthfacts.org, 20 June 2011. Web. 27 Nov. 2011. <http://aids.gov/hiv-aids-basics/hiv-aids-101/overview/global-statistics/>.


II. General Structure 

The PFV forms an intasome composed of a tetrameric nucleoprotein complex of integrase proteins around the ends of replica viral DNA.   The PFV contains both alpha helices and beta sheets .

The interface between symmetrical dimers each comprised of four subunits, creates the active site of DNA insertion.  Upon interaction with the target DNA, the intasome retains its shape, forcing the tDNA into a severely bent conformation within the cleft between the IN dimers.  This allows the IN active sites to access the phosphodiester bonds necessary for vDNA insertion. 

The subunits, amino-terminal domain (NTD) the NTD extension domain (NED) , carboxy-terminal domain (CTD), and inner (CCDi) and outer (CCDii) catalytic core domains associate to bind the transfer and non-transfer strands of the viral DNA and the tDNA to form the tetramer . The  inner CCD, NTD, and CTD subunits are involved in the binding of the viral DNA and in tetramerization, while the outer CCD subunits only provide support to the intasome.  In addition, intermolecular interactions between the NTD and CCD domains stabilize the dimer, causing rigidity of the intasome.  The NED, smallest of the domains, contacts viral DNA, and is a concerted element in other spumaviral and potentially gammaretroviral integrases.


III. DNA Binding

Each of the subunits participates in protein-protein and protein-DNA interactions. The closest protein-DNA contacts occur within the terminal six nucleotides of the viral DNA, where the most deviation from the ideal B form is seen.  NTD and NED subunits interact with the vDNA that is located on the opposing CCD domain. The NTD, CTD, CCD, as well as the NTD-CCD and CCD-CTD linkers interact with the DNA bases, while the NED interacts with the phosphodiester backbone. 

Target DNA

The pre-catalytic target capture complex (TCC) and the post-catalytic strand transfer complex (STC) are stabilized by hydrogen bonds between the amide groups of Thr163, Gln186, Ser193, and Tyr212 and oxygen atoms from the tDNA phosphodiester backbone . These rigid bonds stabilize  the tDNA to prevent its movement in the active site of the intasome.  The target DNA phosphodiester backbone also has a pair of salt bridges formed by Arg362 within the inner chain CTD. 

Due to IN not being site sequence specific, interactions between IN and target DNA bases are rare. One occurs with Arg329, which forms a hydrogen bond to guanine 3, guanine -1, and thymine -2 in the major groove of the target DNA . This intercalation of Arg329 widens the major groove, which severely bends the tDNA, making it more accessible for viral genome invasion.  In a second sequence specific interaction, the methyl of Ala188 within the inner chain CCDi  contacts the oxygen of cytosine 6 in the minor groove through Van der Waals attractions . 

Viral DNA

More sequence specific interactions occur between IN and the viral DNA than do between IN and the target DNA. The carbonyl group of Gly218 forms a hydrogen bond with the fourth guanine of the non-transferred viral strand .  The protein-DNA interactions continue into the CCD α4 helix and into the minor grove at the terminal end of the viral DNA.  The side chain of Arg222 forms a hydrogen bond with the bases of the fifth thymine and sixth cytosine of the non-transferred strand . The side chain of Asn106 interacts with thymine 8 of the non-transferred strand, which widens the minor groove of the viral DNA allowing easier access to the phosphodiester bond .  

Not only are the subunits themselves involved in sequence specific interactions, but the NTD-CCD and CCD-CTD linkers also contact bases in the viral genome.  Within the minor groove, the side chain of Arg313 intercalates its guanidinium group, base stacking it against adenonsine 12 from the reactive strand to form a hydrogen bond with cytosine 11 Together, these interactions primarily aid in the stabilization of the  viral DNA within the protein subunits. 

The active-site loop, which includes residues Pro214 and Gln215, separates the viral DNA2 by a mechanism highly similar to HIV-1. Gln215 dislodges thymine 3 of the non-transferred strand, which causes it to turn away from the inside of the DNA Figure 3 Retroviral intasome assembly .  

2 For successful crystallization of the pre-catalytic target complex (TCC), a viral DNA mimic lacking the reactive 3'-OH was used. Therefore, separation of the viral DNA strands at the catalytic site cannot be shown.


IV. Active Site 


The active site, located deep within the dimer-dimer interface, contains Asp128, Asp185, and Glu221 , which engage with the reactive 3’ termini of the viral DNA2.

Two magnesium or manganese atoms promote catalysis in the active site3. Metal A, coordinated by Asp128 and Asp185, destabilizes the phosphodiester bond in the tDNA, while metal B , coordinated by Asp128 and Glu221, activates the 3' hydroxyl of the viral DNA for strand transfer. Figure 2b The mechanism of retroviral integration  Both ions facilitate the SN2 nucleophilic substitution reaction by which the viral strand invades the host genome.  Upon integration of the nucleic acids, the intasome shifts from the pre-catalytic target capture complex (TCC) to the post-catalytic strand transfer complex (STC), ejecting the newly-joined DNA from the active site, thus preventing a reversal of the strand transfer.

3For successful crystallization of the pre-catalytic target complex (TCC), a viral DNA mimic lacking the reactive 3'-OH was used with only one Mg2+ ion. This Mg2+ corresponds to metal B in Fig. 2b.  


V. References

"Global Statistics." AIDS.gov. Globalhealtfacts.org, 20 June 2011. Web. 27 Nov. 2011. <https://aids.gov/hiv-aids-basics/hiv-aids-101/overview/global-statisctics/>.

Hare, Stephen, Saumya Shree Gupta, Eugene Valkov, Alan Engelman, and Peter Cherepanov. "Retroviral Intasome Assembly and Inhibition of DNA Strand Transfer." Nature 464.7286 (2010): 232-36.

Maertens, Goedele N., Stephen Hare, and Peter Cherepanov. "The Mechanism of Retroviral Integration from X-ray Structures of Its Key Intermediates." Nature 468.7321 (2010): 326-29.

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