The amount of HIV diversity within a single infected individual can exceed the variability generated over the course of a global influenza epidemic, the latter of which results in the need for a new vaccine each year.
–Walker BD, Burton DR (2008) Toward an AIDS vaccine. Science 320:760–764.
(See my previous posts here and here for more explanation.)
I’ve talked several times about Charlie Janeway’s “dirty little secrets“, and the insights into fundamental immunity that arose from the concept. I’ve also mentioned a couple of potential clinical advances arising from it. Here’s another one, that I find particularly elegant for its use of the weak to conquer the powerful. 1
As a very quick reminder: Janeway’s insight2 was that an immune response wouldn’t start unless there were signals present, indicating that a hazardous situation was at hand. Janeway proposed that the immune system would be on the alert for molecular patterns that are generic to many pathogens. Without such patterns the immune system would ignore “foreign” antigen; when pathogen-associated molecular patterns (”PAMPs”) appear, the immune system kicks on and starts looking for trouble. (By the way, sorry about all the acronyms in this. I usually try to avoid using too many, but it’s unavoidable this time. There’s a glossary in the footnote here if you need it.)3
Janeway, and subsequently many others, went on to identify some of the PAMP receptors; first the toll-like receptors (TLRs) and then several other types. There are quite a few — maybe a dozen TLRs, maybe a couple dozen other types, in mice or humans. The different PAMP receptors recognize different subsets of PAMPs, and we have relatively recently reached the point where we understand enough about the receptors to make occasional predictions: Researchers can analyze a virus, say, and say with some confidence that a certain PAMP receptor is likely to recognize it.
Immune recognition of mousepox virus
Hubertus Hochrein’s group is interested in smallpox, the archetypal poxvirus, and they’re using ectromelia (mousepox) as their model for smallpox. Poxviruses are large DNA viruses that are remarkably versatile in their dealings with the immune system; as a group, and as individual viruses, they have evolved molecules that evade multiple components of the immune system. One of those components is the TLR system, apparently, because at least some poxviruses encode molecules that block TLR signalling. 4
There’s an interesting general question, by the way, about how to interpret immune evasion molecules in viruses. If we find that vaccinia virus encodes blockers of TLR signaling, do we argue that TLRs must be important in protecting against vaccinia virus? Or do we instead say that TLRs must not be important, because the virus has defenses against them? In this case, at any rate, Hochrein’s group guessed that TLRs are important, and further guessed that TLR9 might be important.
TLR9 recognizes DNA, both viral and bacterial, but until now there haven’t been any instances of virus recognition that’s strictly dependent on TLR9. Ectromelia, however, turned out to be the first; immune activation by ectromelia is almost entirely dependent on TLR9 signaling, and mice lacking TLR9 were highly sensitive to ectromelia infection:
The in vivo relevance of this TLR9-only dependence for ECTV5 recognition was clearly illustrated by our in vivo studies that revealed that the lack of TLR9 rendered mice more than 100-fold more susceptible to infection with ECTV. … We calculated an LD506 of 19 TCID507 for the TLR9-deficient mice and an LD50 of about 2,120 TCID50 for the WT mice.
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| Cells (actin cytoskeleton in green) infected with vaccinia virus (red) |
Broader recognition of a weakened poxvirus
Does TLR9, and only TLR9, recognize poxviruses in general? Ectromelia is a highly virulent virus even as poxviruses go. There are plenty of more benign viruses, such as vaccinia virus; and even within vaccinia viruses there is a wide range of virulence. Probably the least virulent vaccinia virus is a semi-artificial version of it called “Modified vaccinia Ankara” (MVA). 8 MVA has lost about 13% of its genome compared to its more virulent ancestor, and many of its remaining genes are damaged as well.9
Like ectromelia, TLR9 drove an immune response to MVA. Unlike ectromelia, that isn’t the whole story; even without TLR9, the immune system recognizes MVA.
This is almost certainly an immune evasion function that has been lost in MVA. That is, both wild-type vaccinia virus and ectromelia virus seem to have a gene (or genes) that blocks recognition by PAMP receptors other than TLR9, whereas the massively defective MVA has lost this gene and is recognized by both TLR9 and this other, unknown, receptor.
Overriding blindness
So if immune activation by ectromelia is partially blocked by its immune evasion function, would we reduce its virulence by artificially activating the immune system after ectromelia infection? Ideally, of course, we’d want to only activate the components that are involved in protecting against poxviruses. Like, for example, the aspects that the poxvirus MVA activates.
You see where this is going. Can MVA act almost like an adjuvant, turning on the immune components that ectromelia virus has blinded? And the answer is yes. If you infect mice with a lethal dose of ectromelia, and then superinfect them with MVA, they survive:
MVA given at the same time or immediately after challenge with a high lethal dose of ECTV of 1 × 105 TCID50 completely protected WT mice against death, whereas all control mice died with the 10-fold-lower dose of 1 × 104 TCID50.
You wouldn’t normally think that two viruses would be better than one; and you wouldn’t normally think that the dainty little MVA could override its brutally virulent cousin’s lethality. But at least in mice, it seems that therapeutic infection worked.
CD1 is a fascinating molecule, but it hasn’t traditionally been associated with antiviral protection. Viruses, however, seem to disagree.
CD1 is (actually, CD1 are, since these are a family of related molecules) members of the MHC class I family, with many of the traditional MHC class I features — binding to β2-microglobulin, a “groove” made of two alpha-helices on top of a beta-pleated sheet ( (in classical MHC, the peptide-binding groove: the “bun” of the peptide’s “hot dog”).
I previously wrote a field guide to the MHC family that shows these features across a wide range of the MHC family, but here are some comparisons of CD1d (PDB number 2GAZ) to classical MHC (HLA-A2; PDB number 2GTW). Here I’m showing the “heavy chain” of the complex in red, the ligand that fits in the binding groove is green, and β2-microglobulin in blue. (The “top view” is looking “down” at the molecule more or less as a T cell would “see” it; the ribbon view lets you see the ligand interactions a little more easily; the “ligand only” view shows the thing that goes in the groove with all the MHC or CD1 resides removed.) (Click on a molecule for a larger version.)
| Side view | Top view | Top view (ribbon) | Ligand only | |
| CD1d |  |
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| HLA-A2 |  |
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The similarities are pretty obvious, but it’s the difference that makes CD1 particularly interesting. Classical MHC molecules present peptide ligands; CD1d presents, instead, very hydrophobic molecules: lipids, glycolipids, and lipopeptides. These sorts of things are typically found in mycobacteria (as with phosphatidylinositol mannoside, shown in the images here), and I’ve thought of CD1 as an example of how our physiology has been shaped by pathogens — this whole branch of the immune system, devoted to detection and elimination of tuberculosis and leprosy.
My view started to slip in around 2000, with Frank Chisari’s observation that NKT cells may be involved in control of hepatitis B virus in his transgenic mouse model.1 (NKT cells are the main lymphocytes that recognize CD1 molecules.) I’ve talked about Chiasri’s HBV mouse model before — it’s so artificial that I always am hesitant to extrapolate from it. That said, his findings in that model have all (as far as I know) held up in more natural systems, and the NKT observation is no exception; several other groups have seen similar things. 2
What really confirmed to me that CD1 can be antiviral, though, was the virus’s side of the story. Viruses employ an arsenal of anti-immune molecules, presumably targeting whichever immune components that are especially dangerous to the particular virus. Over the past few years, there’s been an increasing number of sightings of viruses that block CD1-mediated presentation. The first (that I know of) was HIV,3 and since then vaccinia virus4 and herpes simplex5 have also been shown to block CD1-mediated antigen presentation. The latest addition to the list is human cytomegalovirus.6 These viruses (HIV, poxviruses, and herpesviruses) are particularly good at blocking classical MHC class I presentation as well; I don’t know if this dual blockade is typical, or if people have mainly looked in those viruses most renowned for immune evasion — in other words, maybe we’re seeing this double action because people are looking under the streetlamps.
It’s interesting that HSV and HCMV (though not HIV, which blocks both classical MHC class I and CD1 with the same protein, nef) have apparently developed separate systems to block CD1 and classical MHC. The molecules responsible for their CD1 blockade are not yet identified, but they don’t seem to be the same as the ones that block MHC class I. If CD1 blockade is the main function of these genes (and not a side-effect of blocking some other aspect of immunity, say), the implication is that CD1 is an important-enough player in controlling these viruses that they have had to maintain distinct pathways to escape from it.
I wonder what it’s doing.
Natural killer cells were originally identified as cells that spontaneously killed cancer cells. It’s been a bit surprising, then, that there’s been relatively little direct evidence that NK cells protect against spontaneous cancer.
For example, there was the study I talked about some time ago, looking at tumors in equilibrium with the immune system. There, the authors treated mice with a carcinogen, waited until tumors stopped arising, and immunosuppressed the tumor-free survivors. 1 In most of these apparently cancer-free animals, immune suppression resulted in tumors appearing within a few weeks. This showed that the immune system can control tumors.
The interesting part (at least, the interesting part as far as NK cells are concerned) was that treatment with anti-NKG2D did not let tumors grow out; whereas shutting down T cells (with anti-CD4/anti-CD8) or interferon (with anti-IFN) did let the tumors reappear. NKG2D is an important receptor for NK cells, so the implication was that NK cells were not keeping the tumor in check, but T cells were.
Now, however, a similar set of experiments has shown that’s not necessarily true — NK cells probably are important in controlling some tumors. The paper is
Guerra, N., Tan, Y. X., Joncker, N. T., Choy, A., Gallardo, F., Xiong, N., Knoblaugh, S., Cado, D., Greenberg, N. R., and Raulet, D. H. (2008). NKG2D-Deficient Mice Are Defective in Tumor Surveillance in Models of Spontaneous Malignancy. Immunity 28, 571-580. DOI: 10.1016/j.immuni.2008.02.016
David Raulet’s group has been one of the leaders in NK cell research, and as far as I know they are the first to have made a knockout mouse lacking NKG2D. The mice are normal and happy and actually have normal numbers of NK cells that are functional. NK cells notoriously have many ligands, which is one of the reasons it took longer to figure out NK receptor/ligand interactions than for T cells, and presumably there are enough alternatives to NKG2D that NK cells can get whatever signals they need during development. However, of course, none of the ligands for NKG2D triggered NK cells.
Rather than wait for truly spontaneous tumors (which are rare enough even in mice that you need very large numbers of mice to figure out what’s going on) they crossed the NKG2D -/-mice with a couple of transgenic lines that are highly cancer-prone. They also tried treating the mice with carcinogens, as was done in the Koebel et al study I mentioned earlier.
The carcinogen-treated NKG2D knockout mice got no more tumors than did wild-type mice — so that’s exactly consistent with the previous experiment. However, the “spontaneous” tumor transgenic models showed a big difference. The NKG2D knockouts had much earlier, more aggressive tumors than did the wild-type mice.
As well as evidence for cancer equilibrium, the Koebel et al paper showed evidence for immunoediting. 2 That is, tumors that grow in the presence of a healthy immune system are resistant to the immune response — they have been selected for immune invisibility or resistance. By comparison, tumors that grow in immune deficient mice are much more immunogenic — they haven’t had to develop immune resistance.
In one of the two transgenic systems, the NKG2D knockout mice showed the same thing: Their tumors were more likely to have NKG2D ligands than the wild-type mice. “These data suggest that NKG2D-dependent immunoselection (or editing) favors loss of NKG2D ligands on early-arising, aggressive tumors.”
But in the other tumor system no such evidence was seen; NKG2D ligands were just as prevalent:
There was no indication in this survey that expression of NKG2D ligands was selected against in Klrk1+/+ mice, despite the clear evidence that NKG2D-mediated surveillance is operative for these lymphomas. These data suggest that evasion of NKG2D-mediated surveillance by Eμ-myc-induced lymphomas occurs by mechanisms that do not depend on loss of NKG2D ligands.
So it seems that NK immune surveillance is much more complicated than the (already very complicated) T cell immune surveillance of tumors:
Taken together, these data suggest that the role of NKG2D-dependent surveillance differs in the three types of tumors studied here. In the case of early-arising prostate carcinomas in TRAMP mice, many of the tumors are eliminated, and the few that are not eliminated evade surveillance by extinguishing expression of NKG2D ligands. In the case of Eμ-myc lymphomas, it appears that the emerging tumors are mostly NKG2D sensitive, but a fraction of tumors escape NKG2D surveillance without losing NKG2D ligands. … The final category is represented by late-arising prostate carcinomas, which appear to be generally refractory to NKG2D-dependent surveillance.
But the bottom line is that NK cells do control some tumors. That’s not a surprise, because it’s pretty much been the assumption for a long time, but it’s reassuring to get evidence for it.
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| Sand rat, Psammomys obesus |
Although a lot of viruses have ways of blocking recognition by T cells and NK cells, there’s not much known about the importance of these mechanisms in actual infections. That’s because the best-studied viruses in this class tend to be highly species-specific. So, for example, we don’t have good animal models for the human herpesviruses human cytomegalovirus, Epstein-Barr virus, varicella-zoster virus, or Kaposi’s sarcoma herpesvirus. Herpes simplex virus does infect mice, but its immune evasion molecule ICP47 doesn’t work well in mice, so we’re no further ahead.1
Immune evasion by all these viruses has been studied pretty extensively in cultured cells, but because they essentially only infect humans we only have circumstantial evidence for a role in vivo. Similarly, porcine, bovine, and equine herpesviruses encode immune evasion molecules, but pigs, cattle, and horses are not very convenient models for basic research either.Another major group of viruses, besides the herpesviruses, that are noted for immune evasion are the adenoviruses. However, only the human (and primate) adenoviruses contain the classical E3gp19k immune evasion molecule. There’s an animal model for human adenoviruses (the cotton rat) but as I pointed out the other day, there’s little evidence for an important function of CTL immune evasion in this model.
| Virus | Host | Family | Genome |
| Mouse CMV | Mouse | β | NC_004065 |
| Rat CMV | Rat | β | NC_002512 |
| Mouse thymic HV | Mouse | ||
| MHV68 | Mouse | γ | NC_001826 |
| Field mouse HV | Microtus pennsylvanicus |
||
| Sand rat nuclear inclusion agent |
Sand rat |
So what we need are small animal, and preferably lab mouse, models for infection with adenoviruses or herpesviruses that include immune evasion molecules. As far as we know, mouse adenoviruses don’t have T cell or NK cell immune evasion properties. That leaves us with mouse herpesviruses. Of the hundreds of known herpesviruses, six are known to be murid-specific (see the table at right), and three of those infect lab mice. One of those is totally obscure (there’s very little known about mouse thymic herpesvirus), leaving us with mouse cytomegalovirus and mouse herpesvirus 68 (MHV68). I’ve already commented on immune evasion by mouse CMV. The bottom line is that removing all known T cell evasion molecules from MCMV makes almost no difference to infection or latency; the one difference is that the virus persists longer and at higher levels in salivary glands. That may be important in virus transmission, but lacks a little oomph.
That leaves MHV68, and I’m pleased to say that there is actually some evidence that T cell immune evasion is important for this guy. (I’ve mentioned this in passing earlier, but it deserves its own post.) MHV68 uses a gene “mK3″ to attack MHC class I (MHC class I is recognized by cytotoxic T lymphocytes). 2 In 2002, Philip Stevenson and Stacey Efstathiou made a mutant of MHV68 lacking mK3,3 and tested its ability to infect mice:
Stevenson, P., May, J., Smith, X., Marques, S., Adler, H., Koszinowski, U., Simas, J., Efstathiou, S. (2002). K3-mediated evasion of CD8+ T cells aids amplification of a latent γ-herpesvirus. Nature Immunology DOI: 10.1038/ni818
In cultured cells, where there’s no immune system, the mutant virus grows exactly as well as wild-type MHV68. As well (more surprisingly) there was no difference in the initial virus clearance; the mutant virus and the parent were both cleared from the lungs of infected mice at the same rate, and were undetectable after about 13 days. However (finally!) there was a big difference in the amount of latency. The figure to the right shows latent wild-type (left panel) and mutant (right panel) MHV68, the black dots, in spleens of mice infected 13 days previously. What’s more, during the latent phase there was a better immune response to mutant MHV68; mice infected with the mutant virus had about twice as many CTL specific for MHV68.
The association of higher virus-specific CTL frequencies with lower viral loads suggested that CTLs were responsible for the elimination of DeltaK3 viruses during latency amplification.
Eliminating CTL from the mice removed the difference; in the absense of CTL, the mK3 knockout virus established latency just as well as the wild-type. This shows that effects on CTL, and probably not some other unknown function of mK3, are responsible for the difference.
So with the two and a half authentic models of infection (I count the herpes simplex virus one as a half because it’s more contrived than a natural infection) we have immune evasion molecules helping to establish latency (MHV68), helping to reactivate from latency (herpes simplex) and helping with persistence (MCMV) . In no case is there much effect on acute infection.
I’ve lost an old friend. Apparently it’s been dead for quite a while, but I just found out about it:1 E3gp19k doesn’t protect against pulmonary inflammation in cotton rats. A moment of silence, please.
The “friend” in question is the paper
Ginsberg, H., S, Lundholm-Beauchamp, U., Horswood, R., L, Pernis, B., Wold, W., S, Chanock, R., M, and Prince, G., A (1989). Role of early region 3 (E3) in pathogenesis of adenovirus disease. Proceedings of the National Academy of Sciences of the United States of America Proc Natl Acad Sci U S A 86, 3823-3827.
I’ve been using it for years to illustrate a particular point. Unfortunately, while grinding through the literature a couple of weeks ago, I discovered that the paper’s main conclusion was undercut in 1994, and then again in 2002. No one else seems to have noticed this either, so at least I have lots of company.
Viruses have immune evasion genes that allow them to escape or resist the immune response; they target all aspects of the immune system, from interferon to antibodies to cytotoxic T lymphocytes (CTL) to natural killer (NK) cells. I’m particularly interested in the class of viral immune evasion molecules that target CTL recognition by blocking MHC class I antigen presentation. (A summary of antigen presentation is here.) However, as I’ve pointed out before, there is surprisingly little evidence that these genes are important in actual infections, as opposed to their effect in cultured cells. This contrasts with the clear and striking evidence that cytokine escape is a critical virulence factor for several viruses.
CTL evasion in vivo
One big stumbling block in analyzing the importance of antigen presentation blockade in viral infection is that the viruses that have developed this approach tend to be highly species-specific: they’re herpesviruses and adenoviruses. (And HIV; same deal as far as species specificity.) Because, for obvious reasons, research has focused on human pathogens, it’s been hard to move cell culture results into in vivo studies. You don’t see volunteers lining up to be infected with mutant herpes simplex virus, and even where lab animals can be infected with one of these viruses (e.g. herpes simplex) the immune evasion may not work in the lab animal, or there may be some other difference in the infection that makes interpretation difficult.
There are a couple of herpesvirus models. Mouse cytomegalovirus and mouse herpesvirus 68 are both natural mouse pathogens with authentic CTL immune evasion systems. In these cases there isn’t a whole lot of effect from the MCMV CTL evasion molecules (reduced virus titer in salivary glands), and there’s a moderate effect from MHV68 CTL evasion (reduced establishment of latency).
There are in fact a bunch of animal adenoviruses, but — strangely — none of these seem to have CTL evasion molecules. At any rate, none of the dozen or so whose genomes I’ve looked at share the human E3gp19k protein that’s long been shown to block MHC class I antigen processing. In particular, mouse adenovirus 1 does NOT block antigen presentation, at least as determined in one careful study. And while human adenoviruses will just about infect mice, it’s not a productive infection. The human adenoviruses do express some genes in mice, but they don’t efficiently go all the way through replication. It’s not a good model for the natural infection, and so there was some interest, 25-odd years ago, when an animal model for human adenovirus infection was identified.
This is the cotton rat, Sigmodon hispidus. It turns out that at least some human adenoviruses, including the popular type 5, replicate quite well in cotton rats and establish a pneumonia that is vaguely reminiscent of the human disease.2 The system has never really become very popular, probably because cotton rats are vicious, evil little bastards that are as much wolverine as rodent. You handle them with steel-mesh gloves, and it’s still a sporting proposition as to whether the researcher or the rat draws first blood. Subsequent experiments showed that in fact the disease in mice looked quite similar to that in cotton rats, suggesting that mice were an adequate model after all,3 and there was an audible sigh of relief as researchers went back to peaceful little mice again.
Adenovirus immune evasion in cotton rats
But before the boom was over, Harry Ginsberg’s group tested what happened when you infect cotton rats with adenoviruses with, or without, the CTL evasion gene E3gp19k. The Ad5 E3 region is mostly involved in immune evasion of various kinds — cytokines as well as CTL — and as such it’s non-essential in cultured cells, where there’s no immune system. It’s not unusual for spontaneous E3 deletions to pop up in virus stocks passaged in tissue culture. Ginsberg infected cotton rats with some of these mutants. Overall, the mutants spread and replicated in the rats just as well as wild-type virus did. But there was one big difference. Here’s the money shot (Ginsberg et al, Fig. 2; click for a larger version):
These are cotton rat lungs. On the left, infected with wild-type virus; and on the right, infected with a deletion mutant virus lacking E3gp19k. There’s much more infiltrate (inflammation) in the lungs on the right. The obvious explanation, and the one I’ve used for nearly 20 years, is that E3gp19k actually protects the host as much as the virus. In many cases (especially in pneumonia), it’s inflammation that causes the clinical signs of disease. By reducing CTL recognition of infected cells, E3gp19k reduces inflammation and should reduce the amount of disease.
There are some puzzling parts of this story (why E3gp19k, instead of, say, 14.7k, which reduces cytokines and should have a larger effect on inflammation?) and some weaknesses in the paper (they never actually showed that E3gp19k works in cotton rat cells, for example) but overall it was a clean, clear story that made sense. When Lee Babiss wrote Ginsberg’s obituary in 20034 he included this among Ginsberg’s particularly important acheivements:
They also began to investigate the role in pathology of the adenoviral early gene 3 region, and determined that the proteins encoded by the E3 transcripts influenced the host inflammatory response. This observation led the way to the creation of adenoviral gene delivery vectors that could persist in the host cell for long periods of time, thus promoting prolonged transgene expression.
The problem is that it’s likely not true.
Counterevidence
The E3gp19k mutants were naturally-occurring mutants with actual deletions in the E3 region. There’s no obvious reason why this should present a problem, but an obscure paper in 1994 showed that in fact, it is a problem:
Berencsi, K., Uri, A., Valyi-Nagy , T., Valyi-Nagy, I., Meignier, B., Peretz, F.V., Rando, R.F., Plotkin, S.A., Gönczöl, E. (1994). Early region 3-replacement adenovirus recombinants are less pathogenic in cotton rats and mice than early region 3-deleted viruses. Laboratory Investigations, 71(3), 350-358.
I admit that I haven’t read the whole paper yet. It’s only available on paper (how quaint!) and my request to the library for a copy hasn’t been answered yet. Still, the abstract is very clear. The authors compared the deletion mutant with a replacement mutant — still eliminating E3gp19k, but replacing it with an unrelated gene that restores the genome size to normal (actually greater than normal). The replacement mutant doesn’t show the pathology that the deletion mutant does.
An Ad5 recombinant, Ad-human cytomegalovirus glycoprotein B (Ad-HCMV.gB), in which the E3 region is replaced by the full-length gB gene of HCMV and with a genome size exceeding that of Wt-Ad, induced mild histopathologic responses in cotton rat and mouse lungs, comparable with those of Wt-Ad, but less severe than those of Ad5-delta E3. Analysis indicated that neither class I major histocompatibility complex expression on the cell surface nor differential expression of the protective E3-14.7 kilodalton protein underlies the pathologic differences observed in cells infected with Ad5-delta E3 or the Ad-HCMV.gB recombinant. … Pathogenicity and replication of the recombinant viruses inversely correlate with the genomic size.
(My emphasis.) The lung inflammation is a genome size effect, not an E3gp19k effect.
What’s more, this has been reproduced. In 2001, a second group found exactly the same thing using adenovirus type 4 instead of type 5. 5 They didn’t even know about the Berencsi paper, and didn’t make a connection to genome size (they floundered about trying to explain the effect as a function of the inserted genes), but the actual observation was essentially exactly the same: Pathogenicity is related to reduced genome size, but not to loss of E3gp19k.
As found previously for Ad5, deletion of Ad4 E3 genes resulted in increased lung pathology. Surprisingly, insertion of HIV genes into this region significantly restored protection attributed to E3 gene products, diminishing overall pathologic effects to Ad4WT levels (P<= 0.0001).
Ginsberg’s original paper has been cited over 200 times. Berencsi’s has been cited just 13 times, mainly for technical aspects; as far as I can tell none of the citations actually note the critical observation. Patterson’s paper? Only 4 citations (and as I say, they themselves didn’t cite Berencsi either). Fields Virology, the authoritative source, mentions Berencsi et al in passing but doesn’t describe or comment on the actual finding, let alone its significance, and doesn’t mention Patterson et al at all. Fields offers the party line, Ginsberg’s interpretation, on pulmonary inflammation.
It’s possible that people in the field are aware of the observation and are discounting it for some good reason, but if so, it’s apparently an unpublished good reason. (Maybe when I see the paper I’ll decide it’s a load of dingo’s kidneys, but it’s hard to see how it could go that wrong; what’s more, the replication by Patterson et al make it much more likely that the phenomenon is real.) If it’s not E3gp19k deletion that’s causing the causing the inflammation, what is it? I have no idea. Perhaps the deletion alters regulation or expression of another gene (perhaps by altering splicing); perhaps it alters the rate of genome replication; perhaps (because this is 2008) there’s some microRNA effect. Who knows? The important thing is now I have one less piece of evidence that CTL evasion is important in vivo.
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| NK cells ganging up on a tumor cell |
A couple of recent papers describe immune evasion of natural killer cells by viruses. One of the interesting things is that both of the viral genes responsible are multifunctional, apparently blocking both T cell and NK cell recognition simultaneously.
Immune evasion of cytotoxic T lymphocytes (CTL) by blocking the class I major histocompatibility complex (MHC class I) pathway was first described over 20 years ago. The first viral gene shown to block MHC class I was in adenoviruses, the E3gp19K gene of adenovirus types 2 and 5. That was way back in 1985,1 but though E3gp19K has been studied pretty extensively in the interim it still throws out occasional surprises. For example, the original description of E3gp19K showed that it binds physically to MHC class I molecules, preventing them from reaching the surface, and it wasn’t until 15 years later that Frances Brodsky’s group showed that E3gp19K can also bind to the TAP peptide transporter,2 blocking MHC class I antigen presentation in a completely different way.
So viral evasion of CTL has been described for a long time, but our understanding of natural killer (NK) evasion lagged for a while, mostly because our understanding of NK target recognition lagged that of CTL recognition and MHC class I antigen presentation. (See my previous article here for more detail, including a rather attractive graph of the number of references for each field.)
Recently, as tools and understanding improved there’s been quite a bit more attention paid to the subject, and a number of well-defined viral NK evasion mechanisms have been described.3 The two I’m talking about today are the Kaposi’s sarcoma herpesvirus (HSHV) gene K5,4 and none other than our old friend E3gp19K. 5
K5, like E3gp19K, was first identified as a CTL evasion molecule;6 it grabs MHC class I on the cell surface and forces it to be degraded. It’s a remarkably versatile molecule, in that it can also cause degradation of at least seven other cell-surface receptors,7 and one of the very early observations was that K5 also renders cells resistant to NK cells.8 The latest paper4 adds a ninth, tenth, and eleventh notches to K5’s gun: MICA, MICB, and AICL, all of which are NK ligands. (In fact, they’re NK ligands in two separate pathways, so the famous redundancy of NK cell recognition is being attacked here.)
K5 acts on these ligands in the same general way it acts on its other targets: It ubiquitinates them and causes them to be internalized and (in some cases) degraded. Similarly, E3gp19K’s new activity is in line with its previously-described talents: It binds to MICA and MICB and prevents them from leaving the ER, so they’re not available for NK cell to bind to. MICA and MICB are in the same general family as MHC class I (see my Guide to the MHC Family) and E3gp19K seems to bind to them in the same way as it does to other MHC class I molecules.
In the big picture, I think it’s not at all surprising that these viruses apparently block NK cell recognition — I’m sure that most, if not all, of the large DNA viruses do so — but it’s nice to have some molecular targets and interactions identified. It’s pretty impressive that these viruses are able to perform such a complex set of actions with single (small!) molecules, and at the molecular level it’s going to be a fascinating story to find out how K5 handles such a diverse range of targets.
One other thing I wonder about — it’s been assumed that E3gp19K is an anti-CTL molecule, but (as I observed here) the actual evidence for this is pretty feeble. What’s more, I’ve been looking at adenovirus effects on MHC class I lately myself, and the most striking thing about it is just how pathetic it is — the effect of E3gp19K on MHC class I expression is pretty unimpressive (as was noted by Routes and Cook many years ago9 ). I wonder if the effect on classical MHC class I is a mere side effect, with the major function of E3gp19K in pathogenesis being NK cell evasion. And given that thought, I wonder if some other viral immune evasion molecules that have been described as CTL resistance factors are in fact mainly NK resistance factors, with CTL being minor or accidental targets.
To the extent that I’m a virologist at all, I’m mostly a DNA virus kind of guy, so I can’t give a lot of deep background about noroviruses. I know what everyone knows — noroviruses are a major cause of gastoinstestinal symptoms, especially where people congregate in groups — cruise ships are notorious sites for norovirus epidemics — but also pretty much anywhere; hundreds of thousands of people are infected weekly in Britain at the moment, for example. The virus is a smallish RNA jobbie (a member of the caliciviruses: single-stranded positive-strand RNA, a bit over 7500 bases long). And it turns out to be extraordinarily interesting in its evolution.
This is from
Lindesmith, L.C., Donaldson, E.F., LoBue, A.D., Cannon, J.L., Zheng, D., Vinje, J., Baric, R.S. (2008). Mechanisms of GII.4 Norovirus Persistence in Human Populations . PLoS Medicine, 5(2), e31. DOI: 10.1371/journal.pmed.0050031
They were able to track the sequences of noroviruses involved in epidemics over the past 20 years, and analyzed them functionally. They found two functional changes over time: First, the viruses shift their targets (so that people who are resistant to infection today, may not be in five years time); and second, the viruses drift antigenically, so they avoid the previous year’s immune response.
Both of these evolutionary directions surprise me, at any rate. First, I’m not used to viruses being able to blithely switch their receptor over time; and second, my impression has been that immunity to noroviruses is so weak and transient that the virus wouldn’t need to worry about last year’s immunity to any significant effect.
The receptor thing is apparently because noroviruses use a family of carbohydrates as their receptor; the carbohydrates are variable among the human population, so that:
Variation in the capsid carbohydrate-binding domain is tolerated because of the large repertoire of similar, yet distinct HBGA carbohydrate receptors available on mucosal surfaces that could interface with the remodeled architecture of the capsid ligand-binding pocket.
As for the transient immunity, it seems that I’m a little out of date, though I have company — the accompanying review article in the same issue of PLoS Medicine says:1
Acquired immunity is not thought to last until a subsequent norovirus season, though a few individuals may acquire longer-lasting immunity. With these factors combined, one might think that immune selection pressure would be rather transient-only heavy at the end of a season-and that an evolutionarily stable strategy for norovirus might be to wait out the summer low season and attack again when population immunity has waned. This is not what Baric and colleagues have found.
It’s true that early studies on noroviruses did show only transient immunity, but apparently a number of recent studies have shown that long-term immunity is possible. 2 Critically, in the years following outbreaks of a new norovirus strain, infection rates dropped, suggesting that at least some herd immunity exists.3 That being the case, it’s not surprising that noroviruses evolve to escape from this pressure:
not only does antigenic drift occur in the capsid region of GII.4 norovirus strains over time, but that the variation greatly influences the ability of preexisting herd immunity to neutralize extant strains, based on carbohydrate blockade assays.
Finally, just to make Larry Moran happy, the authors point out that most of the changes in noroviruses over time are due to random drift:
In our analyses, the shell domain appears to be evolving by random drift, as only 5% of changes are informative (i.e., became fixed in the population).