Day 259: Trojan exosome hypothesis debunked?

Let me start by saying that I’m a product of the Hildreth lab, so I am fairly well-versed in the trojan exosome hypothesis. Basically, the hypothesis, as presented by Drs. Hildreth and Gould, is that HIV particles are basically glorified exosomes. They postulate that the HIV budding machinery usurps the normal exosome budding machinery to produce virions. Accordingly, HIV virions are pretty similar to exosomes, except that they carry the viral payload. There are a number of lines of evidence that suggest this, most of them outlined in the PNAS hypothesis paper published about 5 years ago.

 

Now, there are a number of people who don’t believe this is the case—some of them pretty prominent. I’m not writing to either support or refute the exosome hypothesis, but rather to discuss a paper that came out of David Ott’s lab claiming to refute the trojan exosome hypothesis.

 

A number of groups have noticed that CD45 is missing in viral particles. This observation is part of the evidence for HIV budding from lipid rafts—although I don’t really believe lipid rafts exist; I am much more partial to tetraspanin enriched membranes. In any event, one way to get highly purified viral particles is to run supernatant from infected cells through a series of density gradients, take the pellet, and then deplete for CD45. This gets rid of contaminating vesicles that contain CD45, leaving purified viral particles. A number of groups claim that exosomes also do not contain CD45, which would mean that CD45 depletion would leave both exosomes and HIV particles. Lori Coren reposts in Retrovirology that depleting supernatant from Jurkat and SupT1 cells of CD45 removes exosomes, suggesting that HIV particles and exosomes differ in CD45 composition and, therefore, likely do not share the same budding process.

 

They really state their case with one figure, although it’s split into six parts. They isolated exosomes from the two cell lines, depleted half for CD45 and then blotted for CD45, actin and did silver stains for total protein. After depleting they observed no CD45, no actin, and I think no protein (I think they mislabeled part of the figure). They did the same thing for infected  cell lines, except that they also blotted for p24. They found pretty much the same thing, although actin shows up in the Jurkat/HIV supernatant that was CD45 depleted and you can see protein (mostly viral proteins) in the CD45 depleted samples. They claim that CD45 depletion leaves virus (we know that), but not exosomes. They claim that the reason other groups didn’t find CD45 in exosomes was because they were using anti-CD45 antibodies that don’t recognize the cytoplasmic domain of the protein, while they use a pan-CD45 antibody that will recognize more isoforms. I’m not sure how immunoprecipitating with antibodies that recognize the cytoplasmic domain of a protein will pull out intact vesicles, but with this paper, it’s beside the point.

 

Now, their findings may be true, but this paper has several flaws preventing me from accepting their conclusions. Anyone who has tried to isolate exosomes knows that it is a total pain in the ass. They are hard to define biochemically, which is why in almost every paper you see on exosomes, exosome presence is confirmed by electron microscopy. Based on this paper, we have no idea what this group is actually working with. You can’t show the depletion of exosomes if you don’t show that the exosomes are there in the first place. And there is no control for depletion using markers we know are on exosomes (like CD81) and markers that we know are excluded. I would be much more inclined to believe the results if they showed depletion using some unrelated antibody, like an anti-GFP, didn’t produce the same effect. How do we know that their immunoprecipitation itself didn’t affect the outcome? There’s no protein left after immunoprecipitation and it’s not like Jurkats and SupT1s pump out exosomes; they may just not have enough protein in their sample following depletion to detect by Western blot. And why don’t you blot for exosome markers while you are at it? If you are going to do biochemical analyses, at least do it thoroughly.

 

So, I think if they added some controls, this could be an interesting observation. As it is, it doesn’t clarify anything.

 

M. Linde

Day 224: Looking for keys to a preventative vaccine

A recent PNAS paper with a huge group of authors from UAB and about half a dozen other universities (Shaw and Hahn are in the grandfather spots), attempts to discern whether there are conserved features of the HIV envelope (Env) gene at the time of transmission. I believe that this type of study is probably our best shot at developing a preventative vaccine.

 

I’ll explain. There is always interplay between viral diversity (quasispecies) and the host immune response. With less immune pressure, virus can replicate readily and the most fit viral quasispecies will dominate—that is, there is no selective pressure to drive viral evolution and, therefore, less viral diversity in the host. With heavy immune pressure, there is selection for whichever viral quasispecies can evade this pressure—again leading to less viral diversity in the host. Somewhere in the middle you get a scenario where the immune system exerts enough pressure to drive viral diversity, yet not enough pressure to control replication. So, you get a high number of quasispecies. Why is this important? Well, quasispecies and viral diversity are some of the problems for developing a vaccine; it’s difficult to effectively target many slightly different viral sequences. However, when HIV is transmitted, it may go through a “bottleneck”; there may be constraints on which viruses are fit to establish infection, so only a few viral quasispecies may be involved in the establishment of infection. So, if we can figure out the envelope sequences for these viral variants that are transmitted, we could potentially develop a vaccine to target these sequences.

 

The problem is that it’s not terribly easy to find these sequences. By the time most people reach treatment, the virus has long since passed this bottleneck and they have a large number of viral variants. Finding those patients who have been recently infected is difficult. On top of that, there are technical issues in figuring out the envelope sequences of the viral variants which established infection. Lastly, it’s entirely possible that a person may be infected with several different viral variants during transmission.

 

This may be the reason why there are about 30 authors on this paper—this is hard stuff. Keene and colleagues had access to samples from 102 acutely infected patients. They had stored blood on which they could sequence the patient’s envelope gene. Each patient had their HIV Env genes sequences an average of 25 times. As there are multiple sequences (HIV often mutates quite rapidly, as you may have heard), the authors used mathematical modeling to try and determine which sequence likely established infection (the founder sequence). Now, I am certainly no mathematician (one of the reasons why my lab work was less than stellar), so I can’t comment on the model they used. As usual, the validity of this sort of study depends in large part on the validity of the model, so just remember that these findings may be colored by their choice of model.

 

The authors found that 78 of the subjects had Env gene sequences that suggest infection with a single virus (homogeneous infection). The other 24 subjects showed evidence of infection with multiple quasispecies: around 2-5 different variants (heterogeneous infection). This is important because, in conjunction with similar studies, it appears that infection frequently occurs from establishment of a single or a few viruses. If these viruses have common features, then we can design a vaccine to target these features.

 

The authors then tested 55 of these envelope proteins for some common features. They found that 54 of these Env proteins were CCR5 dependent—something we’ve known for a while, but it always good to see further validated. The other envelope protein was dual-tropic. All of the envelopes were susceptible to several broadly neutralizing monoclonal antibodies (mAbs), while most were resistant mAbs targeting the V3 loop of the viral envelope. It appears that the V3 loops of these proteins are hidden from the antibodies, and not absent. They also found that the viral Env gene does not appear to mutate very rapidly in the period up to peak viremia, which you might expect if the patient does not mount a robust immune response to the virus (see above). The authors comment that “studies aimed at further analyzing Env glycoproteins of transmitted or early founder viruses may help to identify unique features and potential vulnerabilities relevant to vaccine design. Beyond this, the present study illustrates a strategy for identifying and characterizing full-length genomes and proteomes of transmitted or early founder viruses including, but not limited to, HIV-1. Such analyses may facilitate a better understanding of virus natural history and virus-specific cellular and humoral immune responses in naive and vaccinated individuals.”

 

The authors also note that different modes of transmission may lead to different founder Env sequences. So, it may be that there may eventually (hopefully?) be a vaccine that has sequences designed to target sexually transmitted HIV and sequences designed to block HIV transmitted through needle-use. The authors are currently looking at whether there are differences in the Env genes based on mode of transmission.

 

For those of you that follow this stuff, there are some other interesting observations in this paper, including 13 subjects who had G-to-A hypermutation in the HIV genome, indicative of APOBEC deamination, and the longitudinal evolution of Env followed in 10 subjects.

 

Of note, this study followed acute infection with clade B virus, which is likely necessary for studies conducted among US and European patients. Clades C and E virus dominate in Africa and Southeast Asia, so these sorts of studies need to be conducted among those populations to help elucidate the differences in transmitted virus among the different clades. I believe that some of this work is also being conducted, so it will be interesting to see if there are any conserved features of the virus during transmission and if these features are observed across clades. I hope so, because that would be the ideal scenario for vaccines designed based on these findings.

 

M. Linde

Day 93: Integrins again show importance in HIV infection

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

Day 81: What to do with maraviroc?

I read in the Wall Street Journal today that Pfizer is going to try and reformulate maraviroc (Selzentry) as a microbicide—something I have been saying they should do ever since I saw the efficacy data. Maraviroc is exciting because it is a new class of drug. It’s an entry inhibitor that works by blocking HIV’s interaction with one of the co-receptors, called CCR5. 

For those of you who don’t know, HIV predominantly uses two different co-receptors. The CD4 molecule is the primary receptor. HIV binds CD4, which causes a change in the viral entry proteins, allowing them to bind the co-receptor and enter the cell. Along with CCR5, CXCR4 acts as a major co-receptor (there are other minor co-receptors that HIV can use, but I am not sure how important these players are). So, any cells that HIV is going to infect need to have CD4 and either CXCR4 or CCR5. These are mainly CD4 cells, which carry CXCR4 and sometimes CCR5, and macrophages, which have CD4 and CCR5. 

A virus can be CCR5-trophic, CXCR4-trophic, or dual trophic. These used to be called M-trophic and T-trophic (for macrophage and T-cell, respectively), but these names changes when the co-receptors were discovered. As you might have figured out, M-trophic virus is also CCR5-trophic and T-trophic is CXCR4-trophic. When someone is infected with HIV via sexual contact (I am not sure about intravenous transmission), the virus that is almost always transmitted is CCR5-trophic. (One day I need to write about the viral genetic shift to baseline during transmission).  For some reason, the predominant strains in the body shift to CXCR4 as disease progresses. I don’t know this for fact, but I had always assumed that the reason why the transmitted virus is CCR5-trophic is because of virus-macrophage interaction in the genital tract. This could be totally wrong, though, so please don’t take this as fact. 

Getting back to Maraviroc, you might understand how a CCR5-inhibitor might have some limitations. First, it would only be effective for patients that have predominantly CCR5-trophic virus. This means you have to test the patients for their predominant strain, which can be expensive. Second, viral trophism changes and patients at later stages of disease progression tend to have the CXCR4 virus. Therefore, maraviroc would probably be useful for patients at earlier stages of disease progression, like first- or maybe second-line. The problem is that we already have good regimens for first line therapy, so maraviroc would have to be pretty efficacious to crack the line-up. The other problem is that most medications start as salvage therapy (multiple failures); maraviroc is probably not going to help these patients because it likely won’t be active against their viral strains. So you can see where Pfizer might have a problem with this drug. It is approved for human use, though, so why not try to use it in another manner? 

It seems to me like a good candidate for a microbicide. It’s a small molecule, so it should be fairly stable. Formulation is always an issue, but the real test is going to be what it does to the genital tract. We know it has antiviral activity—that’s not the issue. But there are a lot of compounds that have antiviral activity, which doesn’t mean they’ll work as microbicides. One of the more famous cases is nonoxinol-9, the spermicide. It can kill virus and was thought to be a potential microbicide. The problem was that it increased the infection rate, instead of decreasing it (sound familiar?). It turns out that N-9 also causes inflammation, and HIV loves inflammation. So, any compound that works as a microbicide cannot be inflammatory. Another issue is that microbicide trials are essentially like vaccine trials; that is, they are big and expensive. Last year a promising microbicide was halted early during Phase III trials because it increased the transmission rate. This trial had over 3,000 patients and was at something like eight trial centers on three or four continents. Lastly, it may be that a microbicide is going to need to be used in combination with another microbicide, similar to HAART. This would be to prevent the development of strains that are resistant to the microbicide. Unfortunately, we don’t have any microbicides right now, so that would be a problem. 

I hate to end on a pessimistic note, though. I think it is great that Pfizer is taking my advice and doing this (well, they never consulted me, but they should have). Not only do I hope it is successful, I hope maraviroc makes oodles of money as a microbicide and spurs other pharmaceutical companies to look at microbicide development as a viable business plan. 

M. Linde

Day 72: What’s on the virus and what’s needed to make the virus

I’ve just spent the last hour comparing what’s found in HIV virions with what host factors the virus needs to produce virions. Chertova et al. published a great paper a few years ago in the Journal of Virology where they used mass spectrometry to determine what proteins are found in HIV particles produced in macrophages (primary cells). Brass and colleagues recently published a study in Science in which they used an RNAi knockdown screen to determine what host proteins are needed for viral production in HeLa and TZM-Bl cells (cell lines). It is very important to note that HeLas and TZM-Bls (which I believe are derived from HeLas, if memory serves) are very different cells from primary macrophages, so you might expect that HIV might have different requirements for each cell type and that some of the proteins found in one cell type may not even be present in the other types. Just to give you an idea of how different they are, macrophages are monocytic cells that are isolated from donors and can remain in culture for maybe a month (longer maybe?) and HeLa cells are epithelial cells originally derived from a cervical cancer tumor in 1951 at Hopkins. They’ve been around for over 50 years and they grow like weeds. That’s my caveat.

What I noticed between the two studies is that there is virtually no overlap in the proteins found. Now, some of this is because of the assays used—the Brass study failed to find some of the proteins that we know are needed for HIV production, so we know that the screen didn’t identify all the proteins involved in HIV production. Also, Brass ruled out any knockdowns that severely limited cell viability or growth; some of those proteins might be found in the virus. I won’t rule out the human factor either, since the Brass article uses protein abbreviations and the Chertova article uses full names (my eyes are only so good, folks). Still, the amazing lack of concordance is really striking. Of all the proteins found in each of the studies (~200 each), I could only find one host protein that is both necessary for viral production and is also found in the viral particle: KIFC3 kinesin. I don’t know anything about this protein specifically, except that it is a motor and probably is involved in viral protein trafficking. It’s also interesting that the mass spec paper identified a bunch of Rab and VPS proteins that are incorporated in the virions and the RNAi paper found members of these classes that are needed for production—just not the same proteins. This could be due to trafficking differences between the cell types, but I found it odd that none of the proteins from these classes overlapped in the two studies. 

I am still not sure what this means. I have always felt that the host proteins found in the virion are probably important or necessary for viral production. However, after studying this in the lab for four years and having little-to-no success, maybe I need to reevaluate this stance. Certainly, the lack of correlation between these studies can be due to a number of factors. Also, the fact that host proteins in the virion may not be necessary for viral production may not mean they are not important in HIV disease. I would really like to see someone use these two assays in one paper to try and make some of these correlations. Even better, I wish someone would do an RNAi screen and produce virus, testing whether virus produced is less infectious. This might be a great way to produce a therapeutic vaccine. 

M. Linde

Day 67: Is it time to coformulate Viread with Viagra?

Ok, so I am sure the title of this entry will be the standard joke among HIV docs for a bit. Actually, it has probably been made far too many times already. The joke is based on recent data indicating that Viread (FTC/TDF) was shown to decrease the rate of vaginal infection among humanized mice. The mice essentially have human immune systems, which is why the can acquire HIV. Mice that received pre-exposure prophylaxis (PrEP) with Viread did not get infected when exposed vaginally, while 88% of the mice that did not receive PrEP acquired infection. The mice that received PrEP were given Viread 48 hrs and 24 hrs prior to HIV exposure and every 24 hrs for 5 days after exposure. The study was published by Denton and colleagues in PLoS Medicine this month. 

So, these data are kind of exciting. It’s humanized mice, so you can’t get too excited yet. I would assume a monkey study would be next and then a large-scale human trial. You have to take animal studies as they are—they don’t always translate to the same results in humans. But the data could be a boon for high-risk populations, especially women. One of issues with condoms is that it requires the cooperation of your partner. If there is an alternate method to block infection—one that does not require the partner’s cooperation—hopefully the transmission rate might be reduced. This could also be really helpful for serodiscordant couples who want to conceive. Granted, sperm-washing techniques (the process of eliminating virions from semen) are very successful, but this might be considerably less expensive and a lot easier. Viread PrEP could also be helpful for intravenous drug users, but it has yet to be established (as far as I know) if this method would protect against HIV acquired via needles.

Of course, there are also concerns. While I don’t think anyone would advocate using Viread PrEP instead of condoms (especially at this point), we have to establish whether Viread PrEP is as efficient in preventing infection as condom use. If PrEP with Viread is not as efficient as condom use for blocking infection, then you have to question the common utility of Viread PrEP. Additionally, condom use is one time and Viread PrEP might require a weeks worth of adherence, which might be difficult for some. Finally, Viread is not without side effects, although I doubt this would be a major concern considering Viread’s toxicity profile and the fact that the dosing would be intermittent instead of chronic. And cost is always an issue, especially in underdeveloped nations where HIV transmission is rampant. 

All in all, the study is good news. We desperately need more ways to prevent infection. A pill is a good start as it would help circumvent some of the social and political problems faced with condoms. We still need a barrier microbicide, but the data for PReP are encouraging. So, assuming this does work, does Viread go over the counter?

M. Linde

Day 58: SOCS1 and gag trafficking

A Japanese group recently reported some interesting findings on a new player involved in HIV trafficking. The report comes in the Proceedings of the National Academy of Science (PNAS), which usually means the article is pretty good, although because of the PNAS submissions process, you have to be careful. Occasionally articles get through that belong in a lower impact journal. This article, by Ryu et al. turns out to be well done. The authors implicate the protein Suppressor of Cytokine Signaling 1 (SOCS1) in gag protein trafficking. They start by doing gene arrays in the T-cell line MOLT-4. Now, cell lines and gene arrays can be deceiving because cell lines are so genetically messed-up to begin with. Ryu and colleagues found that SOCS1 is upregulated in these cells and, importantly, they confirm these findings using RT-PCR and western blots in primary PBMCs from two different donors. 

The authors then go on to show that over-expressing SOCS1 in T cells increases release of viral particles. These viral particles look normal (at least with the one electron micrograph shown). Further, SOCS1 has a dose-dependent increase on intracellular gag cleavage products without decreasing the amount of uncleaved gag (p55) and also increases gag localization at the plasma membrane of the cell. Now, this means that SOCS1 is either increasing the transcription of gag or it is decreasing the degradation. Using a reporter construct with the HIV LTR, the authors show that SOCS1 over-expression does not increase gag transcription. Later, they show evidence suggesting that SOCS1 expression may decrease degradation. 

Using various biochemical assays, Ryu and colleagues show that SOCS1 binds to the matrix (p24) and nucleocapsid (NC) portions of gag and that the p17 binding is the important interaction for increasing HIV release. SOCS1 also has a domain—an SH2 domain—that is necessary for binding. This SH2 domain is important for the function of SOCS1, which is involved in protein ubiquitination. 

The findings are confirmed by SOCS1 knock-down using siRNAs targeting SOCS1. Knocking down the cellular protein causes an increase in gag perinuclear localization. When knocking down SOCS1, gag colocalizes with lysosomal markers, but not late endosome markers. The data suggest that in the absence of SOCS1, gag is potentially shifted to lysosomal degradation. Blocking lysosomal degradation and silencing SOCS1 causes an increase in gag compared with just SOCS1 silencing. Notably, the authors comment on another paper which found that silencing SOCS1 in dendritic cells increases env-specific CD8 responses, CD4 response, and antibody production. My first thought is that in the absence of SOCS1, gag (and possibly env—based on the env CD8 response increase) is not getting to the plasma membrane and is entering into the antigen presentation pathway. 

Gag ubiquitination is known to be important for gag trafficking and SOCS1 is known to be a player in ubiquitination. From this report, it appears that SOCS1 is potentially decreasing gag degradation and increasing the trafficking to the plasma membrane, possibly through SOCS1 modulation of gag ubiquitination. The fact that silencing SOCS1 increases CD8 responses against HIV makes this protein an interesting therapeutic candidate in two fronts: blocking it will decrease viral release and may also increase the cell mediated response, hopefully helping people control existing infection.

M. Linde

Day 55: Maintaning resistance

One of the fascinating aspects of HIV (and really all virology and microbiology) is the relationship between viral evolution and the selective pressures on the virus. HIV, like living creatures, adapts to its environment. If conditions are very harsh or not harsh at all, the viral population tends to be very uniform. In a very harsh environment, only viral particles with a very specific make-up may be able to spread; in the absence of these pressures, the viral particles that can replicate the fastest end up dominating the population. Somewhere in the middle, the viral population becomes diverse, with a number of different quasispecies or genetic variants. This is a key concept for antiviral therapy. Antiviral therapy needs to be stringent enough to place extremely harsh conditions on the virus. That’s why anti-HIV therapy needs to include three active drugs. 

Now, if the selective pressure on the virus during antiviral therapy lessens, which can happen for several reasons (not the least of which is non-adherence to a regimen), the virus can replicate and a viral variant will emerge that can replicate in the less harsh environment. In common terms, this is called resistance development. For useful drugs, there are usually only a few viral variants that can survive in these conditions. These viral variants may have to sacrifice certain advantages they would normally have to replicate under a moderate selective pressure. Often, they don’t replicate as well as normal virus (called wild-type) does in the absence of selective pressure. So, you get a scenario where it is believed that a resistant virus replicates alright when the drug is around, but not quite as well as a wild-type virus does in the absence of drug. 

This may have clinical importance. If the rate a viral variant replicates (called the replicative capacity) is slower, does that mean that a person carrying this variant as the predominant quasispecies will have a slower disease progression? Well, several studies have suggested that this is the case, but unfortunately it is difficult to determine how fast a viral variant replicates in the body. Outside of the body, yes, you can determine this rate, but no one knows if what happens outside the body is true in the body. Now, if the answer to the above question is “yes”, it would argue that for certain antiretrovirals, the development of resistance should not necessarily mean that drug is no longer a useful component of ant-HIV therapy. The idea is to maintain selective pressure on the virus and keep the predominant variant a person carries less fit than the wild-type virus, hopefully delaying disease progression. 

Recently in the Journal of Medical Virology, Gianotti and colleagues looked at the replicative capacity of HIV from people on lamivudine (3TC) monotherapy who have 3TC-associated resistance mutations (the methionine-to-valine switch at position 184 in reverse transcriptase, noted as M184V). Now, since you can’t measure the replicative capacity in the body, the authors looked at ex vivo samples from patients with the M184V mutation, comparing variants at 24 and 48 weeks from patients on 3TC monotherapy to patients who had stopped all therapy. What the authors found was that virus from patients who maintained their resistance mutations had reduced replicative capacity compared with those who “lost” these mutations (note: you don’t really lose these mutations, they just become a minority population while another variant takes over the majority role). Furthermore, those patients who lost the mutations had greater reductions in their CD4/CD8 T cell ratio. 

The study is important because it helps tie the reduced fitness hypothesis to what is observed in the clinic. This strategy of maintaining drugs to keep the viral replicative capacity down may help extend the options for patients who have been through several treatment options or those who have difficulty with adherence (provided they already have the M184V mutation). Of course drug companies also love this stuff because it suggests that some patients should stay on their meds, even after the development of resistance. There are necessary questions to answer; such as, is it really worth staying on these drugs for the reduced replicative capacity? Antiviral therapy is not without side effects and cost. However, now that there has been at least a preliminary link between reduced viral fitness and an immunologic parameter, the answer appears to be headed in the affirmative. Whether this holds for drugs other than 3TC remains to be determined. 

M. Linde

Day 34: HIV infectivity in semen

In a rather unexpected paper, Frank Kirchoff’s group and others (about 20 authors or so) show that amyloid fibers in semen enhance the infectivity of HIV. According to the Cell paper, HIV piggybacks on the fibers. Now, the issue of HIV attachment to spermatozoa has always been a controversial one. You see data for and against, much of it terrible. There are a couple of papers out there that claim that HIV actually enters (and possible infects) spermatozoa. I’ve seen papers with electron micrographs of spermatozoa with enveloped cytoplasmic particles that the authors claim are virions. Never mind that this seems virtually impossible—I might buy it if the particles were in a vesicle or there was just a viral core, but I wish they would explain how the virus manages to pass the cell membrane and retain its envelope. There are also data with pull-downs of spermatozoa/virus that show by RT-PCR that no virus attaches to the spermatozoa. The only problem is that the process used to pull-down the spermatozoa in these papers is the same process used to “wash” HIV from sperm, so the authors never actually show that the pull-down process doesn’t shear or detach HIV from the spermatozoa surface. My own (unpublished) data indicate that HIV does attach to spermatozoa, in a ratio of approximately 12 virions per cell.

 

How attachment is mediated is also controversial. Some people claim that spermatozoa carry CD4 (which I do not believe); while others claim it is mediated by gp120 affinity for glycosphingolipids. I am not certain whether this is the whole story. Semen contains a lot of what are called “prostasomes”. Prostasomes are exosomes made by the prostate. If you look at the prostasome protein composition and compare it to HIV composition, they look very, very similar. Prostasome, electron micrographs look almost identical to immature HIV particles. It’s known that prostasomes are important for spermatozoa viability, although it is not entirely clear how. If I remember correctly, prostasomes attach to spermatozoa and some believe that they may dampen an immune response targeted against the sperm (sexual reproduction is an evolutionary war waged by the immune system, but that is another story). So, it stands to reason that HIV may use the same methods as prostasomes to attach to spermatozoa.

 

Why is this important? I believe the key to creating a viable preventative vaccine is by preventing the key steps that aid in the establishment of infection (not a shocking statement—kind of like saying the key to driving is starting the car). As my old advisor was fond of saying, once the puck gets past the goalie, the game is over (only true in overtime). Preventative vaccines right now focus on priming the adaptive immune response so it will occur before the viral replication gets out of control. I think a better response is to target conserved aspects of the virus that allow it to infect those first few cells. So, what happens to the billions spermatozoa that don’t fertilize an egg? I imagine some of them are taken up and cleared by macrophages (I am not sure, but it seems reasonable). Any virions attached to these sperm would love to be exposed to macrophages.

 

This brings us back to the Cell paper by Munch and colleagues. They show that prostatic acid phosphatase (PAP) fragments forms amyloid fibers capture HIV and present virus to target cells. They call these fibers SEVI, for semen-derived enhancer of viral infectivity. They suggest that the by piggybacking on the SEVI fibers, the virus is presented to target cells that are normally protected by the mucosal barrier. The fibers increased infectivity by at least 1000-fold. This is important, because it has been observed that sexual transmission of HIV is actually not that efficient—although it is obviously efficient enough to result in a massive epidemic. If you could target these fibers in some capacity (like a microbicide) and decrease the sexual transmissibility by 1000-fold, that would be a very good thing.

 

One aspect of the findings that the group does not discuss, which I would like to see, is the effect of these fibers on the immune cell activation. It is hypothesized that the adaptive immune system is primed by “danger signals”. Do these amyloid fibers activate immune cells? Do macrophage toll-like receptors recognize them? It could be that one of the reasons HIV attached to these fibers is more infectious is because these virions are presented to cells in that are in a state of initial activation, which would be the perfect target. This might also be true for dendritic cells, which may travel to the lymph nodes following interaction with SEVI fibers. If that is the case, than a small molecule which blocks this activation could also be a reasonable microbicide. That’s all conjecture, though.

 

M. Linde

Day 31: Another player for infection in trans?

You would think that after 25+ years of research, we would have some sort of handle on how HIV infection is actually established. But as far as I can tell, there is still a considerable amount of debate on the subject. Quick, which is more infectious—cell-associated virus or free virus? Which is more important in establishing infection—genital tract infection or spread in the lymph nodes?

 

One of the widely regarded hypotheses is that HIV “hitchhikes” its way to the lymph nodes, where it gains easy access to CD4 cells. It has been shown that viral particles can attach to dendritic cells (and possibly infect, but that’s another story), which make their way to the lymph nodes when activated. Dendritic cells are professional antigen presenting cells, meaning that they do a lot of T-cell activation. They are designed to interact and stimulate CD4 cells. So, you can easily see how a virus attached to or hiding in a dendritic cell would be able to successfully infect an activated CD4 cell. The DCs pick up the virus, the DCs travel to the lymph nodes, stimulate some CD4 cells and, at the same time, HIV infects these cells.

 

The main protein on DCs that has been identified in this trans-infection is DC-SIGN, an adhesion molecule. A new paper in PNAS has also identified another player, syndecan-3. Most of the figures in the paper are supportive that syndecan-3 is a player, but they are not conclusive. However, the authors use siRNA to knockdown syndecan-3 and show that this results in less HIV attachment, which is fairly strong evidence. The authors showed that syndecan-3 knockdown did not affect DC-SIGN expression by flow cytometry, but it did not say in the paper whether this was surface expression or intracellular expression. They also did not show whether DC-SIGN trafficking was altered by syndecan-3 knockdown. Obviously, if syndecan-3 knockdown prevents DC-SIGN from reaching the cell surface or changes the molecule’s conformation, then knockdown of syndecan-3 would affect binding mediated through DC-SIGN. It would have been nice if they had incubated virus (both wild-type and virus without gp120) with DC lysate, pelleted the virus, and then done a western blot for syndecan-3. Still, the paper shows that syndecan-3 knockdown affects both HIV binding and also transmission of the virus to T cells, so it has to be doing something.

If syndecan-3 and DC-SIGN are important players for infection in trans, then small molecules that target these interactions may be good candidates for microbicides.

M. Linde