Mystery Rays from Outer Space

Meddling with things mankind is not meant to understand. Also, pictures of my kids

August 24th, 2010

Adenoviruses and the occupied sign

“Adenovirus” (by Mapposity)

There are two aspects about virology that constantly amaze me: How much we know about viruses, and how little we know about viruses.

Adenovirus research offers examples of both. Adenoviruses are probably among the best-studied virus groups.1 We really do know an amazing amount about them. But it was only last year that Linda Gooding’s group offered the most convincing demonstration yet that adenoviruses actually establish a truly latent infection — a really basic aspect of their lifestyle, 2 and a new paper from her group3 is looking at some equally-basic implications of that finding. (I talked about Gooding’s earlier latency finding here.)

It’s been known pretty much since day 1 that adenoviruses persistently infect tonsils;4 that was why they were first isolated, when the virus grew out of apparently-normal tonsil tissue in culture. The critical distinction is between mere “persistence” and true “latency”. In a latent infection, the virus shuts down production of new viruses, and is maintained basically as DNA within the host cell. Persistence is cruder — the virus continues to replicate, but at a low level that balances its destruction. Simplistically, latency is a destruction-free process, while persistence can include viral and cellular destruction.

Adenoviruses establish their latency in tonsils, which of course have lots of lymphocytes, but we usually think of adenoviruses as infecting epithelial-type cells, or hepatocytes, or whatever. Clinically, these guys typically cause cold-type symptoms, which you tend to get from fairly superficial infections of the respiratory tract lining. We don’t tend to think of adenoviruses as effective infectors of lymphocytes, but it turned out that their latent infection was, in fact, in T lymphocytes.  It looks like adenoviruses have one cell type (epithelial-type cells) for a lytic infection that leads to shedding of infectious virus, and another cell type for latent infection, allowing the virus to remain in the host and potentially re-infect an epithelial type later on.

Accordingly, Gooding and her team set up infections of cultured T lymphocytes in vitro, to see what would happen. In particular, they wanted to know whether, and how, the viral replication cycle would be controlled; and whether and how the host cell would be affected by the infection. I will skip over most of their findings and and highlight a couple that surprised me:

Occupied!(1) The “Occupied!” sign. To get into a cell, adenoviruses usually need to bind to their cellular receptor, the CAR receptor.5 But latently-infected cells almost permanently shut off this receptor. For hundreds of days after the initial infection, cells express little or no CAR. The latent virus doesn’t want any competition; it has found a congenial long-term environment, and it doesn’t want some interloper infecting its cozy cell and perhaps destroying it.

There seem to be several mechanisms for the shutdown, but at least part of it is that the virus apparently permanently modifies the host DNA:

CAR synthesis and expression remained repressed even after the viral genome was lost (Fig. 8 and data not shown), suggesting a virus-induced epigenetic change to the cells that does not require the continued presence of the virus.3

And in fact the CAR isn’t the only thing to be modified for this purpose:

Even when CAR levels were restored by transduction with a CAR-containing retrovirus, the previously infected cells could not be reinfected3

We don’t know how the latent viruses were blocking superinfection, but it’s clear that the latent viruses really don’t want company.

(2) Rearranging the furniture.  The latent virus doesn’t stop at hanging an “occupied” sign; it modifies its host cell in other ways as well, apparently again by long-term or even permanent epigenetic modification of the DNA. That means that even after the virus itself is altogether gone, not even latently present, there are modified cells hanging about:

Remembering that adenoviruses infect just about everyone, that may mean that we’re all walking around carrying cells that are tagged and functionally altered by these viruses.

There’s been speculation for many years that adenovirus infection may underlie some forms of human tumors. One argument against this has been that there’s no evidence of adenovirus DNA in tumors, for the most part.6 (One rule of thumb in determining if a virus is actually causing a tumor is if it’s actually present in the tumor.) But of course, if adenoviruses leave a permanent scar on cellular DNA that lasts longer than the virus itself, this may not be relevant:

One compelling reason to gain an understanding of this nonlytic infection is the likelihood that adenovirus gene products cause damage to the host cell genome. … While these functions are irrelevant to the lytic infection of epithelial cells where all infected cells die, they are of serious concern when infected lymphocytes have carried the viral genome and survived. … Despite this normal appearance, the cells display altered gene expression long after the virus is lost.3

  1. There are over 40,000 papers on adenoviruses, or at least mentioning them, in PubMed.[]
  2. To be fair, it’s been suspected for decades that they do go latent, but that was the first time it was actually proven.[]
  3. Zhang, Y., Huang, W., Ornelles, D., & Gooding, L. (2010). Modeling Adenovirus Latency in Human Lymphocyte Cell Lines Journal of Virology, 84 (17), 8799-8810 DOI: 10.1128/JVI.00562-10[][][][]
  4. I’m going to limit this discussion to the Group C adenoviruses — the latency concept may be true for other groups of adenoviruses but that hasn’t been directly shown.[]
  5. “CAR” stands for the “Coxsackie B virus and Adenovirus Receptor”. Can anyone guess what other virus uses this receptor? Bueller? Anyone?[]
  6. Also, the epidemiological links between tumors and adenoviruses are not very strong, at least in humans.[]
May 11th, 2009

On emerging pathogens

The recent much-publicized report World at Risk predicts that we are soon to experience a biological or nuclear weapons attack.  … we are at least equally likely, if not more likely, to soon experience large-scale morbidity through epidemics of emergent pathogens. As was illustrated by the severe acute respiratory syndrome–associated coronavirus, when a ubiquitous nuisance pathogen suddenly becomes more virulent, its reign of destruction needs little help from rogue nations or terrorist cells. Humankind is quite efficient in spreading such pathogens around.

Adenovirus (Mapposity)
“Adenovirus” (by Mappposity)

That paragraph was written last December and published in early April, a couple of weeks before the new H1N1 influenza cases were detected in San Diego, making the author look prescient.  Except, of course, the article has nothing to do with influenza.

This is from an editorial1 in the Journal of Infectious Disease, and refers to a series of papers2 showing that adenovirus strain 14 has suddenly exploded throughout Oregon and Texas in the past year or two, hospitalizing dozens and causing at least 17 deaths.

Adenovirus infections are very common in humans, and usually don’t cause much in the way of disease; they’re one of the “common cold” cluster of agents (see an earlier post on adenoviruses here, and other posts here).  But there are over 50 different human adenoviruses, and some are both more severe, and less common, than others.  Adenovirus Type 14 (Ad14) was actually one of the sporadic, fairly moderate, strains, until 2005 when a new variant of the virus (poetically called “Ad14a”) abruptly started causing epidemics.  It’s this new variant that’s continued to cause these outbreaks.

Most Ad14a infections are probably minor.  Probably; we don’t really know, because “Ad surveillance is generally passive. Additionally, relatively few laboratories look for Ad, and even fewer can distinguish Ad14 from other Ad types.” 1  So we don’t know how many Ad14a infections there actually are.  This is the same “missing denominator” problem I alluded to with the Mexican cases of the new H1N1 — when we find that 18% of patients with Ad14a infection die, does that mean it’s a very serious disease, or does it mean doctors only look for Ad14a in seriously ill patients?  In all likelihood, there are a vast number of Ad14a infections that are missed (“With its propensity for rapid transmission, it seems likely that Ad14a is now circulating throughout the United States and may have been introduced from another country“).

Still, the virus clearly can cause serious problems.  It’s just another warning that we’re constantly under siege by pathogens.  We probably should understand better what’s attacking us.

  1. Gray, G., & Chorazy, M. (2009). Human Adenovirus 14a: A New Epidemic Threat The Journal of Infectious Diseases, 199 (10), 1413-1415 DOI: 10.1086/598522[][]
  2. Lewis PF, Schmidt MA, Lu X, et al. A community-based outbreak of severe respiratory illness caused by human adenovirus serotype 14. J Infect Dis 2009;199:1427–34

    Tate JE, Bunning ML, Lott L, et al. Outbreak of severe respiratory disease associated with emergent human adenovirus serotype 14 at a US Air Force training facility in 2007. J Infect Dis 2009;199:1419–26

    Centers for Disease Control and Prevention. Acute respiratory disease associated with adenovirus serotype 14—four states, 2006–2007. MMWR Morb Mortal Wkly Rep 2007;56:1181–4.[]

January 26th, 2009

55 years, 40,000 papers, and still surprises

Adenovirus infecting HeLa cells
Adenovirus infecting HeLa cells

I’ve quoted before that “the stupidest virus is smarter than the smartest virologist”. Adenoviruses are far from the stupidest viruses, and even after 55 years of study, and nearly 40,000 papers in PubMed, adenoviruses still throw surprises at us on a regular basis. Last week, while talking about herpesviruses, I added that “the other virus families that are known to evade MHC class I are human adenoviruses, which now turn out to establish true latency”. True latency has been hinted at for a while, but Linda Gooding’s group has added much more support for it in a paper in press at J Virol.1

Adenoviruses are very, very common viruses, both in humans and in many other species. In humans there are over 40 different adenovirus “species”, mostly causing cold-like symptoms and mild gastrointestinal disease. Occasionally, and perhaps with increasing frequency, there’s a more or less widespread outbreak of moderate disease, but in general these guys are not huge problems as far as mortality in immune-competent people.

As well as being common, many adenovirus species are pretty easy to grow in cultured cells, and so it’s not surprising that they were isolated and described a long time ago. In fact, the first identification of adenoviruses was as an accidental isolation in 1953, when Rowe et al noticed that the tonsil cell cultures they were growing were showing signs of viral infection.2 Since then, adenoviruses have been used as models for a huge number of cell biology functions (the Nobel on splicing came from work on adenoviruses, just as one example), as well as cancer biology and lots of other things; adenoviruses have also been used as workhorses for the viral vector field, moving into gene therapy as well.

Tonsillectomy (Greenfield Sluder: 1923)

If you look in lots of tonsils, you’ll find lots of adenovirus; something like 80% of samples turn up positive. 3 That’s much higher than the disease rate, of course, and it’s also much higher than any plausible incidence rate — that is, those can’t all be new infections, in the past week or so, that simply haven’t been cleared by the immune system. That means that adenoviruses must be able to persist for some fairly significant period — months to years — after their initial infection, without causing symptoms.

This long-term persistence by adenovirus is one of its most characteristic, um, characteristics, but it’s been completely mysterious. (If you don’t believe me, here’s what Bill Wold and Marshall Horwitz say in the latest version of the authoritative Fields Virology: “We really do not understand whether and how adenovirus persists at very low levels in humans … “)

For adenoviruses, usually the term “persistence” is used, rather than latency, because “latency” has a specific meaning: Basically, a latent virus is still present at the genome level, but isn’t capable of forming a new virus particle. Herpesviruses are the main viruses that are known to do this. Some retroviruses set up latent infection by intergrating into the host genome, but that’s a little different story. Herpesviruses can maintain their own genomes in the latently-infected cell, but in a form that’s independent of the host genome. As I’ve noted before, this latency may be tightly linked to immune evasion functions. The other option is mere “persistence”, where presumably the virus would remain in a replicating form, and would be capable of forming a new virus, perhaps at a very slow turnover rate of replication.

A handful of papers have suggested that adenoviruses might establish truly latent state4, but this new paper from Gooding’s lab1 is the most convincing I’ve seen yet. They compared infectious virus to the amount of viral DNA — that is, to viral genomes — and concluded that “only a small amount of viral DNA is present as infectious virus, even in samples with large amounts of viral DNA. … time in culture also appears to activate latent virus in the tissues, which was detected by transferring “activated” lymphocyte-derived virus onto permissive A549 cells.” (This is exactly how you detect latent infection by herpesviruses, in general — you transfer latently-infected tissue onto other cells, and over time the virus reactivates from latency to become infectious virus, and shows up on the permissive cells.) They showed that on initial exam the virus wasn’t making any transcript, again a requirement for true latency, and that over time transcripts began to appear as the virus reactivated.

As a side note, Gooding notes dryly that sloppy technique may be part of the reason adenoviruses were so often found in tonsil explants, a suggestion I hadn’t heard before.

Reports of infection of laboratory workers in the early adenovirus groups suggest that some exogenous contamination might have elevated the frequency with which live virus was found in these studies.

Finally, Gooding re-emphasizes the same point I’ve made here:

Furthermore, like all DNA viruses that form latent or persistent infections, human species C adenoviruses encode a variety of gene products, primarily within the E3 transcription unit, that function to counteract host anti-viral defense mechanisms. We have previously reported that the E3 promoter is up-regulated when cells are exposed to signals that activate T lymphocytes. Hence, it appears likely that the immune evasion strategies of these viruses are directed toward protecting the T lymphocyte from destruction during the period of viral activation from latency.

(My emphasis) As far as that goes, it’s particularly interesting to me that only human adenoviruses, and not even all of them, have MHC class I immune evasion functions. Does that mean that that particular function is less critical for latency, or does it mean that non-human adenoviruses don’t establish true latency (even in these humans, they really only found group C adenoviruses to be latent, though that may just reflect tissue preferences), or what?

  1. C. T. Garnett, G. Talekar, J. A. Mahr, W. Huang, Y. Zhang, D. A. Ornelles, L. R. Gooding (2008). Latent species C adenoviruses in human tonsil tissues Journal of Virology DOI: 10.1128/JVI.02392-08[][]
  2. Rowe WP, Huebner RJ, Gillmore LK, et al. Isolation of a cytopathic agent from human adenoids undergoing spontaneous degeneration in tissue culture. Proceedings of the Society for Experimental Biology and Medicine. 1953;84:570-573. []
  3. Garnett CT, Erdman D, Xu W, et al. Prevalence and quantitation of species C adenovirus DNA in human mucosal lymphocytes. J Virol 2002;76:10608-10616. []
  4. Neumann R, Genersch E, Eggers HJ. Detection of adenovirus nucleic acid sequences in human tonsils in the absence of infectious virus. Virus Res 1987;7:93-97. []
March 10th, 2008

Viral T cell evasion in vivo: The vanishing evidence

Cotton rat, Sigmodon hispidusI’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).

Hela cells infected with adenovirusThere 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):

Cotton rat + adenovirus lungs (Ginsberg et al 1989)

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.

AdenovirusThe problem is that it’s likely not true.

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.

  1. And apparently no one else has realized it yet[]
  2. Pacini, D. L., Dubovi, E. J., and Clyde, W. A. J. (1984). A new animal model for human respiratory tract disease due to adenovirus. J Infect Dis 150, 92-97.[]
  3. Ginsberg, H. S., Horswood, R. L., Chanock, R. M., and Prince, G. A. (1990). Role of early genes in pathogenesis of adenovirus pneumonia. Proc Natl Acad Sci U S A 87, 6191-6195.
    Ginsberg, H. S., Moldawer, L. L., Sehgal, P. B., Redington, M., Kilian, P. L., Chanock, R. M., and Prince, G. A. (1991). A mouse model for investigating the molecular pathogenesis of adenovirus pneumonia. Proc Natl Acad Sci U S A 88, 1651-1655.[]
  4. Babiss, L. E. (2003). In memoriam: Harold S. Ginsberg (1917-2003). Arch Virol 148, 1655-1657.[]
  5. Patterson, L. J., Prince, G. A., Richardson, E., Alvord, W. G., Kalyan, N., and Robert-Guroff, M. (2002). Insertion of HIV-1 genes into Ad4DeltaE3 vector abrogates increased pathogenesis in cotton rats due to E3 deletion. Virology 292, 107-113.[]
February 25th, 2008

Viral evasion of NK cells

NK cells ganging up on a tumor cell
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.)

NK and tumor cellRecently, 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.)

Blood cells including NK cellsK5 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.

  1. Burgert, H.-G., and S. Kvist. 1985. An adenovirus type 2 glycoprotein blocks cell surface expression of human histocompatibility class I antigens. Cell 41:987-97. []
  2. Bennett, E. M., Bennink, J. R., Yewdell, J. W., and Brodsky, F. M. (1999). Cutting edge: adenovirus E19 has two mechanisms for affecting class I MHC expression. J Immunol 162, 5049-5052.[]
  3. There’s a review in Immune evasion of natural killer cells by viruses
    Stipan Jonjića,Marina Babića, Bojan Polića and Astrid Krmpotića
    Current Opinion in Immunology 20:30-38 (February 2008) doi:10.1016/j.coi.2007.11.002 []
  4. Thomas, M., Boname, J.M., Field, S., Nejentsev, S., Salio, M., Cerundolo, V., Wills, M., Lehner, P.J. (2008). Down-regulation of NKG2D and NKp80 ligands by Kaposi’s sarcoma-associated herpesvirus K5 protects against NK cell cytotoxicity. Proceedings of the National Academy of Sciences, 105(5), 1656-1661. DOI: 10.1073/pnas.0707883105[][]
  5. McSharry, B.P., Burgert, H., Owen, D.P., Stanton, R.J., Prod’homme, V., Sester, M., Koebernick, K., Groh, V., Spies, T., Cox, S., Little, A., Wang, E.C., Tomasec, P., Wilkinson, G.W. (2008). Adenovirus E3/19K Promotes Evasion of NK Cell Recognition by Intracellular Sequestration of the NKG2D Ligands MICA and MICB. Journal of Virology DOI: 10.1128/JVI.02251-07[]
  6. Ishido, S., Wang, C., Lee, B. S., Cohen, G. B., and Jung, J. U. (2000). Downregulation of major histocompatibility complex class I molecules by Kaposi’s sarcoma-associated herpesvirus K3 and K5 proteins. J Virol 74, 5300-5309.[]
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January 10th, 2008

Oncolytic viruses and immune clearance

Oncolytic VSV
Oncolytic VSV (gold) infecting lung tumors1

Oncolytic viruses are a concept I’d like to be more excited by than I am.2 It’s an idea that seemed really exciting when I first came across it, but the more I thought about it the more dubious I was. But a recent paper helps me feel better about at least two of my worries.

The concept is a straightforward one. Viruses are good at killing cells.3 Why not have them infect cells that we want to die? That would be, for example, cancer cells. So all you need to do is find or make a virus that only grows in cancer cells, and you’re cured. Simple! Tomorrow we’ll fix global warming!

There’s the obvious problem with this: How can you find (or make) a cancer-specific virus? In principle the answer is the same as with chemotherapy; you use the ways cancer cells are different from normal as targets. This isn’t as hard as you might think. Lots of the things that make cancer cells cancerous are similar to the things viruses like. Viruses often drive infected cells into a cancer-like state that is more hospitable to the virus — friendly to nucleic acid replication, replication, unresponsive to death signals, independent of the signals that normally regulate growth. So lots of viruses are already kind of pre-adapted to replicate well in cells with a cancerous phenotype, and it doesn’t take all that much tweaking to make them adapted to only replicate well in cancer cells.

(After writing this post, it occurred to me that this is actually topical! I don’t usually do the topical blog post thing, but the background in “I Am Legend” has an anti-cancer virus, isn’t it? I haven’t seen it myself, with the grant-writing and the teaching and the two little kids,4 but have I actually tied a current entertainment topic into “Mystery Rays”? Fame and fortune is certain to come my way!)

Oncolysis through the ages

Jennerex Onolytic virusThe first runs at this technique that I knew of5 used mutant herpesviruses,6 but I think that much of the buzz came from work with defective adenoviruxes, especially the ONYX-015 virus.7 The approach here was based on the observation that adenoviruses (like many other viruses) normally inactivate p53 during infection. p53 is a multifunctional growth regulator that is very often also inactivated in cancers, for the same reason as viruses like to inactivate it:it oten triggers death in cells with unchecked growth. Adenoviruses lacking the gene that inactivates p53 (their E1B gene) can only efficiently infect cells lacking p53 — which would usually be, of course, cancer cells.

Blogging on Peer-Reviewed ResearchAs well as herpesviruses and adenoviruses, though, all sorts of other viruses have been used.8 One interesting approach is vesicular stomatitis virus. This is a very, very innocuous virus in normal people, partly because VSV is extremely sensitive to interferon. (VSV is used in bioassays for interferon release, because even tiny amounts of interferon completely block the virus’s replication.) So which kind of cells often aren’t responsive to interferon? Right; cancer cells, as part of their own immune evasion pathway, frequently disable their interferon responses. VSV doesn’t infect normal cells, but does infect, and kill, tumor cells.9

Questions and (maybe) answers

Anyway, the first question, of specificity, is more or less under control.10 Three questions that had made me rather dubious about the concept, though, still remained:

1. Getting the virus to the tumor …
2. Especially in the face of an immune response.
3. Killing all of the cancer cells, not a mere 99% of them (from which the cancer will rapidly recover).

Malignant melanoma cells in lymph node
Malignant melanoma cells in lymph node

A paper in Nature Medicine11 offers encouragement on all of those.

They used VSV as their cancer killer, and their twist here was to deliver it by loading it onto T cells. T cells naturally traffic to lymph nodes, and quite a few tumors metastasize through lymph nodes; the T cell therefore acts as a ferry to deliver its deadly viral cargo to the metastasizing tumor. (The goal here was not to clear the primary tumor, but to prevent metastases, which are often the major problem.  However, they did see some effect on the primary tumor, too, in some cases.) When it reaches the lymphoid tissue, it delivers the passenger virus to the cancer cells, the only ones that the VSV can productively infect (since the cancer cells are the only ones that have mutated their interferon pathway). This is an interesting idea, though limited in this form — I wonder about using antigen-specific T cells instead, to target the virus to a specific site — and it seemed to work quite well.

The two more interesting points to me were kind of peripheral to their main point. First, they find that once the virus killed some cancer cells, there was anti-tumor protection even after the virus was all cleared, and this was probably because of the immune response,12 which was triggered by the cell death initially caused by the virus:

In vivo tumor cell purging resulted both from direct viral oncolysis by virus released from the T cell carriers and from the priming of protective antitumor immunity, which prevents repopulation by further waves of cells metastasizing from the primary tumor.

— just as described in the paper by Apetoh et al13 that I talked about here. The authors suggest that because the cancer metastases are being killed in the lymph nodes, rather than in the bulk of the tumor (which is generally a highly immunosuppressive environment) the immune response was more efficient. That starts to get past my concern #3 above, because it offers multiple attacks on the tumor, not just the virus.

The other point is that the virus could reach the cancer reasonably well even in the face of an anti-viral immune response; the trick was to use just enough virus to kill the tumor cells, without getting enough on the T cells to trigger an immune response:

In virus-immune mice, T cells loaded with large amounts of VSV (MOI 1 or 10) could not keep DLNs or spleens free of tumors. However, T cells loaded with fewer viruses (MOI 0.1) still protected even virus-immune animals from tumor colonization of the DLN and spleen

The data are still very preliminary and inconclusive, but certainly it’s a step in the right direction, and I feel better about this whole approach than I did before reading the paper.

  1. Carrier Cell-based Delivery of an Oncolytic Virus Circumvents Antiviral Immunity. Anthony T Power, Jiahu Wang, Theresa J Falls, Jennifer M Paterson, Kelley A Parato, Brian D Lichty, David F Stojdl, Peter A J Forsyth, Harry Atkins and John C Bell. Molecular Therapy (2007) 15, 123-130. []
  2. That sentence needs a road map, but you got here eventually, didn’t you.[]
  3. At least, lytic viruses are.[]
  4. And the running and the screaming and the monkeys in the hair[]
  5. I realize now, though, that the concept arose long before that, apparently in the 1950s. For example: Love R, Sharpless GR. Studies on a transplantable chicken tumor, RPL-12 lymphoma. II. Mechanism of regression following infection with an oncolytic virus. Cancer Res. 1954 Oct;14(9):640-7. though I don’t know much about those studies other than the titles []
  6. Experimental therapy of human glioma by means of a genetically engineered virus mutant. Martuza RL, Malick A, Markert JM, Ruffner KL, Coen DM. Science. 1991 May 10;252(5007):854-6. []
  7. ONYX-015, an E1B gene-attenuated adenovirus, causes tumor-specific cytolysis and antitumoral efficacy that can be augmented by standard chemotherapeutic agents. Heise C, Sampson-Johannes A, Williams A, McCormick F, Von Hoff DD, Kirn DH. Nat Med. 1997 Jun;3(6):639-45.
    An adenovirus mutant that replicates selectively in p53-deficient human tumor cells. Bischoff JR, Kirn DH, Williams A, Heise C, Horn S, Muna M, Ng L, Nye JA, Sampson-Johannes A, Fattaey A, McCormick F. Science. 1996 Oct 18;274(5286):373-6. []
  8. And I have no idea which, if any, is the most promising.[]
  9. Exploiting tumor-specific defects in the interferon pathway with a previously unknown oncolytic virus. David F. Stojdl, Brian Lichty, Shane Knowles, Ricardo Marius, Harold Atkins, Nahum Sonenberg & John C. Bell. Nature Medicine 6, 821 – 825 (2000) doi:10.1038/77558[]
  10. One other point is that you can probably get away with a virus that isn’t completely restricted to tumor cells, because these are usually viruses that cause very mild disease anyway, so even if they can spread to normal cells it’s no more worry than exposure to a standard subway car. Maybe more concern for immunosuppressed cancer patients, of course, but likely not an insurmountable worry.[]
  11. Qiao, J., Kottke, T., Willmon, C., Galivo, F., Wongthida, P., Diaz, R.M., Thompson, J., Ryno, P., Barber, G.N., Chester, J., Selby, P., Harrington, K., Melcher, A., Vile, R.G. (2007). Purging metastases in lymphoid organs using a combination of antigen-nonspecific adoptive T cell therapy, oncolytic virotherapy and immunotherapy. Nature Medicine DOI: 10.1038/nm1681[]
  12. The short-term clearance worked in immune-deficient mice, but the long-term did not[]
  13. Toll-like receptor 4-dependent contribution of the immune system to anticancer chemotherapy and radiotherapy. Apetoh, L., Ghiringhelli, F., Tesniere, A., Obeid, M., Ortiz, C., Criollo, A., Mignot, G., Maiuri, M. C., Ullrich, E., Saulnier, P., Yang, H., Amigorena, S., Ryffel, B., Barrat, F. J., Saftig, P., Levi, F., Lidereau, R., Nogues, C., Mira, J. P., Chompret, A., Joulin, V., Clavel-Chapelon, F., Bourhis, J., Andre, F., Delaloge, S., Tursz, T., Kroemer, G., and Zitvogel, L. (2007). Nat Med 13, 1050 – 1059. []