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Back to Basics:
the science at CROI focuses the tough questions
that remain in HIV pathogenesis, Part 2 (focal points for new therapies.)
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CROI 2009 Montreal Feb -12
David Margolis MD, University of North Carolina
Even after 25 years of study of the mechanisms by which HIV replicates and causes immunodeficiency, many steps of the HIV lifecycle are only superficially understood. Several presentations at CROI focused on new insights into viral integration into the human genome, viral entry into cells, and the release of new viral particles from productively infected cells; all steps of replication that may be focal points for new therapies.
Leading the horse to water: chaperones for HIV DNA integration: The emergence of raltegravir, and the anticipation of other HIV integrase inhibitors such as elvitegravir, has been a significant advance for HIV therapeutics. However, the process of HIV integration into the host genome involves an array of host factors that are hijacked by the virus to guide the viral genome to a chromosomal target site, and to stitch viral DNA into human host DNA.
Christ, working in the Debyser group, described the role of Transportin-SR2 (TRN-SR2). HIV seems to have selectively adopted the use of this cellular factor to carry its DNA across or through host nuclear pores, to gain access to the nucleus (abstr. 23). Christ showed that TRN-SR2 was specifically bound by a domain of HIV integrase. Further, targeted depletion of TRN-SR2 within human cells by the technique of RNA interference (a research tool that allows the targeted destruction of a selected RNA and thereby the depletion within a cell of a single protein or factor under study) greatly blunted the ability of HIV to integrate, and increased the number of HIV genomes per infected cell that were trapped outside the nucleus. As the binding of HIV integrase to its TRN-SR2 shuttle appeared to involve a unique domain of TRN-SR2 that did not appear critical for the normal function of TRN-SR2, Christ suggested that a specific molecule designed to inhibit this interaction should serve as a new type of HIV integrase inhibitor, and perhaps would work in synergy with the current inhibitors that directly targetthe viral enzyme.
At a later plenary, Debyser (abstr. 74) discussed his group's overall program to identify and validate cellular co-factors of nuclear import and integration as novel targets for anti-HIV therapy. If small-molecule inhibitors of the interaction between HIV integrase and cellular co-factors can be developed, it might be difficult for HIV to develop resistance to these drugs as the cellular binding partner does not mutate, as the virus does. However, as with CCR5 inhibitors, the virus could still adapt to bind its required cellular partner at a different site, or in the presence of the inhibitor. Nevertheless, given the potency of HIV integrase inhibitors, the idea of a second antiviral that targets part of the integration process is an attractive one.
LEDGF/p75 is a cellular protein that was the first identified as a binding partner of HIV-1 integrase. LEDGF/p75 erves to tether the pre-integration complex -- the conglomeration of HIV DNA and cellular and viral proteins that enter the nucleus and home in on the host chromosome - to the chromosome.
Again using the RNA interference or "RNA knockdown" technique, depletion of LEDGF/p75 in human cells and molecular studies in mouse cells that lack LEDGF/p75 showed that the factor was required for HIV integrase function. Mutants of LEDGF/p75 that lack its integrase-binding domain (IBD) acted as strong HIV inhibitors in tissue culture replication assays.
The Debyser group then began designing inhibitors of the LEDGF/p75-to-HIV integrase interaction by computer modeling using available data on the crystal structure of these proteins. This "rational design" technique for new antivirals is not a sure thing but has had some success in recent years. The screening has thus far generated 50 compounds that behave has HIV inhibitors at micromolar concentrations. Resistance mutants to one of the lead compounds has been seen in selection experiments, indicating that a real antiviral effect is at work (that the virus selects a specific mutant against the drug suggests that the compound is acting on the virus and not non-specifically on the cell). As ever, drugs that target, even in part, host factors have the potential to lead to host toxicitiy, but these efforts are promising.
The nuts and bolts of viral entry and exit: As the viral particle attempts to enter a new cell, and as a newly created particle exits a productively infected cell, the virus must interact with a highly complex physical environment. The understanding of these interactions has already led to the potent and clinically useful antiviral strategies of chemokine receptor blockade, and fusion inhibition. Drugs, antibodies really, which block CD4 receptor binding are in advanced testing. As the complexity of these processes is incompletely understood, it seems likely that further therapeutic targets await discovery and investigation.
Several research groups have developed new techniques to watch the process of viral entry on a single cell level. Besides making a really cool presentation that you might have fun watching on the CROI website, these techniques may yield important insights. Ben Chen (abstr. 132LB) presented the findings of high-speed 3-D video scanning laser confocal microscopic observations of an infectious, fluorescent clone of HIV. These microscopy techniques allow cells to be microscopic movies to be taken of cells, using a laser to excite the fluorescently tagged virus to glow, and confocal techniques to scan through a depth-of-field so that 3-D images can be formed, rather than a single slice of the visible, focused microscopy field. Specifically, Chen and co-workers were interested in studying HIV entry via the "immunological synapse." This is a cell-to-cell viral transfer event that appears to occur between different cells of the immune system, including T cell-to-T cell interactions, and macrophage-to-T cell transfers.
Chen saw rapid formation of micron-sized "buttons" containing oligomerized HIV Gag core protein that appeared concentrated at the site of adhesion between two cells, with glowing Gag molecules gathering there by recruitment from nearby sites in the infected cell membrane. Electron microscopy showed that these buttons were packed with budding viral crescents.
As virus was transferred, virus-laden internal compartments formed within target cells. These compartments would migrate at times to the opposite pole of the cell, suggesting that they were latched to the internal structural actin skeleton of the infected cell. Of note, this sort of infection was blocked by anti-CD4 antibodies, but not by fusion inhibitors or chemokine antagonists.
Amy Hulme (abstr. 24) screened film of the next step of the viral lifecycle, the uncoating of the virion after it enters the cell. This step is required for the viral core to release its cargo of viral genomic RNA. The cast of characters in this movie featured viral particles with two colored labels. Virions were produced in which the envelope glycoprotein was labeled linked to the red-emitting Cherry fluorophore, and the Vpr capsid protein tagged with green-emitting GFP. This allows measurement of the uncoating process, as dually labeled red-green particles lose the red envelope and remain green after uncoating. This technique showed that about 50% of uncoating happens within the first hour of infection, but some particles have not disassembled up to 4 hours after infection.
Other studies in this work showed that inhibitors of reverse transcription also inhibited uncoating. It was previously known that RT can begin within the viral capsid, prior to uncoating, but is not completed until after uncoating. Perhaps the expanding DNA reverse transcript lend force that help drive the uncoating process, and perhaps inhibitors of uncoating (if developed) could work synergistically with RT inhibitors.
Jouvenet presented her work (abstr. 166) using virions in which envelope proteins were labeled with GFP or Cherry to study viral exit, or budding, from cells. She was able to observe the genesis of individual virions in real time using fluorescent microscopy imaging techniques in living cells. Viral particles appeared at the cell surface only one at a time over a period of about 5-7 minutes, and never appeared together as a burst. This presentation raised questions about how viremia could be perpetuated, and suggested that either an infected cell must be able to produced particles from many separate sites on the cell surface simultaneously, contrary to prior models, or must produce particles for longer periods of time that previously estimated.
In related studies, Gupta (abstr. 27) presented findings on the cellular protein tetherin. Tetherin is an interferon-responsive protein that interacts with the virion at the cell surface, and prevents the release of virion particles. The HIV "accessory" protein Vpu acts to degrade tetherin, allowing HIV virion release. Gupta andf coworkers performed genetic analysis of tetherin sequences in mammalian genomes, and identified several amino acids within tetherin that were positively selected, presumably by the pressure of retroviruses in the environment over evolutionary timescales. They discovered that several of these residues within tetherin were required for interaction with Vpu. This led them to suggest that this site of Vpu-tetherin interaction could be used to develop inhibitors that block Vpu binding to tetherin, and enhance the ability of tetherin to restrict HIV release and virion production.
How does HIV cause AIDS, anyway? Douek (abstr. 20) presented a sweeping vision of AIDS pathogenesis, beginning with depletion of CD4+ T cells from the gastrointestinal tract. He linked the systemic immune activation seen in the chronic phase of pathogenic HIV or SIV infection, to the sustained breakdown of the mucosal barrier in the GI tract leading to the translocation of immuno-stimulatory microbial products from the gut lumen into the systemic circulation. Recent findings have created further association between levels of plasma bacterial lipopolysaccharide (LPS), bacterial 16S ribosomal RNA, the CD38 activation marker, and levels of inflammatory interferon alpha. These level of these circulating bacterial products correlates with measures of immune activation and CD4 T cell decline, independent of the level of viral load.
He also pointed out that Th17 cells, unique CD4+ T effector cell type critical in the defense against microbes, particularly at mucosal surfaces, are depleted in HIV infection, regardless of antiretroviral therapy. This was contrasted with the state of affairs in SIV-infected sooty mangabeys, which do not develop AIDS despite SIV infection, and maintain healthy frequencies of Th17 cells in the blood and gastrointestinal tract. Douek postulated that mangabeys had learned to co-exist with SIV by evolving to display less CCR5 on the surface of their CD4+ T cells in the GALT. This avoided depletion of a non-renewable T cell pool, and so in these monkeys viremia was generated from only activated, effector T cells that are continuously renewable, and non-renewable central memory T cell populations were less susceptible to infection.
Dandekar (abstr. 54 and 398) similarly discussed the role of GALT as a site of ZHIV replication, cell depletion, and pathogenesis. In studies in pathogenic SIV infection, immediate antiretroviral therapy within one week of SIV infection led to TH17 cell preservation, GALT CD4 cell recovery, poly-functional anti-HIV T cell responses, and lower levels of plasma LPS leakage. Martinelli (abstr 133) also identified a novel subset of CD4+ cells in humans, those that express high levels of the cell membrane Integrin a4β7, as preferentially infected and depleted in mucosa.
HIV transmission: Advancing studies in patients identified in the first weeks of HIV infection, and in careful studies of non-human primates after SIV infection have recently highlighted some of the challenges that face the development of a protective HIV vaccine.
Keele (abstr. 53) presented the findings of single genome amplification (SGA) of plasma viral RNA at transmission. SGA is sequencing done on viral RNAs at limiting dilution, so that only a single viral genome is being sequenced. In studies in acutely infected humans, this group has recently shown that the transmitted/founder HIV-1 virus is a single viral species in ca. 75% of infections, and a few in the other 25%.
Keele reviewed this published data (PNAS 2008) and highlighted that within 8 days of infection escape mutants for envelope-directed CTLS appeared in the viral population, and within 33 days escape mutants for neutralizing antibodies also presented themselves. He then discussed the use of the rhesus macaques by simian immunodeficiency virus (SIV) to model intrarectal or intravaginal infection, to confirm that this system was a relevant comparison to human infection. They found that mucosal transmission of SIV was dose-dependent, and like HIV only a single or few SIVs were transmitted this way, contrasted with the swarm of viruses that can be transmitted intravenously. Similarly, transmitted viral species underwent rapid intra-host recombination, low-level mutation induced by the host protective APOBEC system, and random virus evolution, later followed by antibody- or CTL-mediated immune selection.
Salazar-Gonzalez (abstr. 45) also presented similar data from infected humans. Plasma HIV from 10 men and 2 women who were detected during acute infection was studied. Patients were HIV RNA + but Western blot negative at the time of diagnosis. The maximal diversity of viral species in all but one was less than 0.1%, suggesting infection with a single virus. In only one, diversity was 2.46%; this patient was infected with two viruses.
Transmitted/founder viruses, which were CD4 and CCR5 tropic, replicated efficiently in activated normal human CD4+ T lymphocytes but not in monocyte-derived macrophages from the same donors. This suggested that while macrophages or dendtritic cells may serve to present HIV, the lymphocytes are the primary initial site of HIV replication (abstr. 492).
Rapid immune escape was documented. In one patient, with no significant muational diversity seen in the circulating viral swarm on the day of HIV diagnosis, a single nef CTL escape mutation was found to have taken over the viral swarm by day 16. In another patient, evidence of both neutralizing antibody and CTL escape mutations was seen by day 14 and 32 after diagnosis of infection (abstr. 111). This presents a daunting challenge to CTL-based protective strategies.
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