The HIV Blog

A new story came out from Fauci’s lab at NIAID indicating that the HIV spike component gp120 can bind the integrin alpha4 beta7 (a4b7). The gp120 protein is responsible for HIV binding to CD4; it is also known that it can bind some other molecules, most notably galactoceramide. The physiologic ramifications of gp120 binding to proteins other than CD4 is not really known, although I think most feel that it is not a major contributor to HIV disease. 

The a4b7 story seems to have a little more importance, though. Integrins have been known to be important for HIV attachment, as they are found on the virus and studies have shown that if you block the integrin interactions (particularly the ICAM-1/LFA-1 interaction), you decrease HIV attachment. Fauci’s group, led by James Arthos, found the interaction while investigating CD4 T cell depletion in the gut associated lymphoid tissue (GALT). Early in HIV infection, CD4 cells undergo a massive depletion in the gut—the site of approximately 2/3 of CD4 cells in the body. It’s believed that this massive insult is important to establishing chronic infection and disease. One of the hypotheses for the massive infection and depletion in the gut is due to the high number of CD4+ CCR5+ T cells in the gut. However, the mechanisms for this depletion are not yet worked out—hence the new paper in Nature Immunology from this group. 

They started with natural killer (NK) cells, which do not express CD4 (although I believe NK T cells do), yet have disrupted activity by gp120. The researchers found that gp120 binds the NK cells, but only in the presence of calcium. Using binding studies with cell lysate and mass spectrometry, the authors found that HIV binds the integrin a4b7. Furthermore, they found that this binding activates the integrin (as measured by phosphorylation of p38 mitogen-activated kinase). These findings also hold for T cells. The authors note that T cells in the periphery have low levels of active a4b7, but that T cells in the gut have high levels of the activated integrin. This integrin is actually important in signaling T cells to migrate to and stay in the gut. Therefore, the authors used PBMCs (not from the gut), stimulating them with retinoic acid to achieve a “gut-like” phenotype. Now, it is difficult to differentiate whether the gp120 is binding to CD4 or to a4b7, so the authors conducted their binding studies in the presence of a monoclonal antibody that blocks the CD4-gp120 interaction. They noted gp120 binding to T cells in the presence of the antibody. This binding was not evident in the absence of calcium, suggesting that the binding was mediated by a molecule other than CD4. The binding was also abrogated when they added the a4b7-ligand MadCAM (sounds like the name of a heavy metal band, right?). The authors also blocked the interaction using three other antibodies that interact with a4b7. They observed similar results for CD8 cells. So, the binding studies seem pretty solid to me. 

The next order of business was to find the a4b7-gp120 binding sites. Since gp120 binding activates the integrin, it makes sense that gp120 might bind the same region as the natural ligands. A hexapeptide mimicking the MadCAM binding site inhibited gp120-a4b7 binding in a dose-dependent manner. The gp120 molecule has a region similar to the MadCAM binding site. One amino acid in this region is conserved in more than 98% of the gp120 sequences in the Los Alamos database; it’s in the V2 loop of gp120. Using site-directed mutagenesis and binding studies with surface plasmon resonance, the authors confirm that the binding site is in the HIV V2 loop. These gp120 mutants could still bind CD4, but had little CD4 independent binding. 

Apparently, not all gp120 molecules bind a4b7 with the same efficiency. The “V” in the “V2” loop stands for “variable”, which would explain differences in binding ability between gp120 molecules. Previous studies with the chimeric SHIV (a mix of HIV and SIV) produced two viral variants with differential ability to infect the GALT. One strain is CXCR4 oriented and the other is CCR5 oriented (see an earlier post for an explanation of this if you need it). As noted above CCR5+ CD4+ T cells are more highly expressed in the GALT than in the periphery. The authors tested the ability of gp120 from these viruses to attach to T cells with the gut phenotype. They found that in the presence of CD4-blocking antibody, the gp120 from virus that preferentially infects GALT has residual binding, while the other variant does not. They also discovered that the difference in biding is due to the differences in the V2 loop binding site, suggesting that the background binding in the absence of CD4 is a4b7 mediated and that the variant which infects GALT better has better CD4-independent binding. 

Finally, the authors wondered whether this binding has any functional significance. LFA-1 activation is linked to the a4 molecule. LFA-1 also happens to be a really important player in the immune response, forming part of the immunologic synapse (the pSMAC—peripheral supramolecular activation complex). Everyone knows that HIV needs activated T cells to replicate. As you might expect, gp120 that has the ability to bind a4b7 and this activates LFA-1, facilitating the clustering of LFA-1 and CD4. So, this could help the virus enter the cell. Of course, these sorts of things are also dependent on the stoichiometry of an interaction, so there is no telling whether this occurs under physiologic conditions. Bombarding a cell with recombinant gp120 is vastly different than having variable viral levels. So, you have to make that caveat. 

However, the group conducted some in vitro studies showing that the gp120-a4b7 binding is important for HIV replication. PBMCs cultured in retinoic acid had decreased viral replication when exposed to viral variants with mutated V2-loop binding sites; although this is not necessarily due to decreased a4b7 binding (it’s only suggestive). 

The authors make three arguments that this interaction is important:

·        “The highest frequencies of HIV-1 infection occur in the memory CD4+ T cell compartment in the gut, in which, unlike in almost all other tissue compartments, memory CD4+ T cells express activated a4b7.”

·        “Second, this interaction is well conserved. We have shown that gp120 proteins derived from HIV-1 subtypes A, B, C and D, as well a simian immunodeficiency virus (SIVsmm) gp120, bound to a4b7.”

·        “Finally, we have shown that gp120 activated LFA-1 in an a4b7-dependent way. The capacity of LFA-1 to increase the efficiency of HIV-1 infection is well established.” 

Is this an important observation? Well, it’s certainly an important basic science observation; the physiological relevance remains to be determined. If a4b7 is important in the establishment of infection, then this might offer a way to either prevent initial infection or lessen the impact of initial HIV pathogenesis in the GALT. At best, this could potentially prolong HIV disease progression. That’s still a long way off, though, so it will be interesting to see where this goes. It appears that there are a4b7-blocking agents in development, so studies in animal models may help clarify some of these questions. 

I would also like to point put that there are 21 authors on this paper. For those of you that haven’t worked in laboratory science, it often (but not always) takes this many people to produce a high-quality paper. It’s one of the reasons why PhD studies now take closer to 6 or 7 years, and not the traditional 4 years, and that post-doctoral fellowships have been extended by 2-3 years. It makes me think that the traditional endpoint of PhD candidacy should have less emphasis on publication, because—speaking from experience—it is very difficult to compete with large groups working on similar projects. But that’s another story.

M. Linde