Mystery Rays from Outer Space

Last week I talked about the evolution of noroviruses, which have repeatedly thrown out new strains and caused new epidemics over the past 20-odd years. In contrast — and rather unexpectedly, at least for me — it seems that HIV is not changing its virulence over time; at least, not in North America.

You can think of reasons why HIV “should have” either increased or decreased its virulence since entering North America. For example, HIV entered the human population relatively recently; perhaps it’s still adapting to its new host, and is learning how to more efficiently infect us. On the other hand, it’s widely (though incorrectly) believed that pathogens inevitably evolve toward reduced virulence; perhaps HIV follows this path as well. (Similarly, at least in theory, the human population could be adapting to the virus, something that certainly happens with other species and viruses. However, 30 years or so seems pretty short for this kind of evolution in humans.) The HIV epidemic in North America started in the early 1980s, which has given the virus lots of time to evolve one way or another (compare to noroviruses, which have thrown out six epidemic-causing variants¹ since 1995).

Experimental evidence has pointed in all directions — some suggests that HIV is becoming less virulent, some that it is becoming more virulent, and some says it’s staying the same. There are all sorts of complications in measuring this. What exactly is “virulence“, anyway — time to death? Death rate? Rate of replication? Transmission? In the absence of a suitable animal model the most direct answers can’t really be tested directly. What’s more, because of the vast improvement in anti-retroviral treatment over the years, it’s hard to compare clinical course. And the virus is intrinsically so variable that you’d need large numbers just to be sure you’re analyzing a real trend.

A recent paper in PLoS ONE² tried to resolve the question by looking at clinical correlates of virulence, and they reached the conclusion that the virus hasn’t changed significantly (in terms of virulence, by this definition) over time:

We tested for associations between calendar year of seroconversion and three prognostic markers of disease progression in the MACS cohort between 1984 and 2005. Our results showed no significant trends in set point plasma viral RNA load, CD4 cell count after seroconversion, or the rate of CD4 cell decline in the first three years after seroconversion. Moreover, estimates of change in these markers over time were very close to zero. Thus, the results of this study do not support the hypothesis that there has been any important change in the virulence of HIV-1 over this time period in this cohort.

Is this study likely to end the controversy? I really doubt it; there are just too many limitations in this kind of study. (I do think, though, that it helps establish bounds: it’s unlikely that HIV virulence has changed massively either way since it arrived in North America.) Nor does it help us going forward: HIV strains with different virulence may be arising even now. Still, it’s an interesting finding. My own prejudice would have been that HIV virulence would be increasing, but I’m happy to set that aside for now.

1. Simplified![↩]
2. Herbeck, J.T., Gottlieb, G.S., Li, X., Hu, Z., Detels, R., Phair, J., Rinaldo, C., Jacobson, L.P., Margolick, J.B., Mullins, J.I., Tripathy, S. (2008). Lack of Evidence for Changing Virulence of HIV-1 in North America. PLoS ONE, 3(2), e1525. DOI: 10.1371/journal.pone.0001525[↩]

My papers from my PhD research (not counting previous and subsequent work) have now been cited exactly 1000 times.

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.

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.[↩]
7. Mansouri M, Douglas J, Rose PP, Gouveia K, Thomas G, Means RE, Moses AV, Fruh K (2006) Blood 108:1932-1940;
   Sanchez DJ, Gumperz JE, Ganem D (2005) J Clin Invest 115:1369-1378;
   Coscoy L, Ganem D (2001) J Clin Invest 107:1599-1606;
   Bartee E, McCormack A, Fruh K (2006) PLoS Pathogens 2:e107;
   Lehner PJ, Hoer S, Dodd R, Duncan LM (2005) Immunol Rev 207:112-125;
   Li Q, Means R, Lang S, Jung JU (2007) J Virol 81:2117-2127[↩]
8. Ishido, S., Choi, J. K., Lee, B. S., Wang, C., DeMaria, M., Johnson, R. P., Cohen, G. B., and Jung, J. U. (2000). Inhibition of natural killer cell-mediated cytotoxicity by Kaposi’s sarcoma-associated herpesvirus K5 protein. Immunity 13, 365-374.[↩]
9. Routes, J. M., and Cook, J. L. (1990). Resistance of human cells to the adenovirus E3 effect on class I MHC antigen expression. Implications for antiviral immunity. J. Immunol. 144, 2763-2770. [↩]

In case anyone cares, I’m still slowly¹ working on making a non-beta version of XPlasMap. (The present release is 0.96 [see comments here], so it’s getting incrementally closer to 1.0.) I’ve squashed a few bugs, and added a few features, but much of what I’ve been doing has been kind of shuffling things behind to scenes to make it a little easier to maintain, and also to prepare for adding a couple of features that would have stepped on the toes of the original setup.

I spent the morning trying to figure out why printing was suddenly low-resolution, a bug I thought I had fixed several iterations ago. Turns out the fix only works with the most recent versions of wxPython, whereas I was still working with the builtin 2.8.4.0 in the hope of making the file more portable on Leopard. There’s nothing in the release notes that I can see, but when I switched back to 2.8.7.1 print resolution was back to the proper level again.

There are a couple of things in the ToDos that probably won’t make it into v. 1.0, but most of them should, I think.

XPlasMap ToDos for v 1.0

• BUG - Clicking on anything often selects the plasmid rather than the feature itself. (Take the plasmid out of the pathlist)
• BUG – Changing gene text parameters overrides text position
• BUG – Fix highlights in Cut Dialog
• BUG – Split MCS doesn’t work with no downstream sequence/site
• BUG – Hide/show text in List View is really slow for a batch of genes!
• BUG – writeXMLFile intermittently throws error at self.recentFilesMenu.Delete(deleteId) – “invalid item in wxMenu.Delete”
• BUG – Show/hide comment contextual menu doesn’t work
• BUG – When importing from FastA make sure first save prompts for a name
• “Info” icon in toolbar
• Highlight features when selected
• Flip text orientation manually
• Insert fragments by restriction site
• More fine-grained control of fonts
• Determine PNG and JPG resolution at export time
• When importing and identifying sequences, put enzyme sequence into “Note”
• When opening a file, put MCS center (etc) in if missing. Also put in during creation in dialog
• Convert restriction sites to MCS
• Magnifying-glass-type zoom
• Error message if “Import” tries to open an incorrect file type
• Checkbox – Identify ORFs in Genbank imports (Default on or off in preferences)
• Annotation menu
• Add special symbols for Annotations (Arrows, brace-brackets, etc)
• Status bar – show mouse position in nucleotides
• Speed up changes in List View
• Improve batch handling of genes
• Clean up the code for the copy and cut fragment sections
• Turn plasmid name and description into a free-standing comment
• (With special double-click characteristics?)
• Import from ApE gb files (APE gb files are broken, repair their LOCUS line spacing and then import; or write a less restrictive GenBank interpreter)
• Import from GB as circular when noted in LOCUS line
• Report error when ImportFromGB module fails
• Print from ListView
• BUG – short gene goes all the way around when crossing the 90^o point
• BUG – Printing more than one page doesn’t work (Use GetVirtualSize instead of GetSizeTuple; But this causes problems for the next page, if it’s smaller)
• BUG – Printing is low-resolution (again!)
• BUG – Highlight color is too opaque
• BUG – “No features are selected” in List View
• BUG – Text height of new import is off the window
• BUG – Short genes only show clockwise arrow
• Grab “Product” from GenBank imports for names
• Batch entry for restriction sites
• Batch entry for genes
• Change “Insert fragment” shortcut from “⌘I” to “⌘V”; change “Edit plasmid info” shortcut to “⌘I”

1. XPlasMap is lower priority than writing grants, teaching, doing lab work, and taking kids to birthday parties and tobogganing, so that leaves about an hour twice a week, late at night when the kids are asleep, for XPlasMap.[↩]

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).

1. Lopman B, Zambon M, Brown DW (2008) The Evolution of Norovirus, the “Gastric Flu”. PLoS Med 5(2): e42 doi:10.1371/journal.pmed.0050042[↩]
2. Lindesmith L, Moe C, Lependu J, Frelinger JA, Treanor J, et al. (2005) Cellular and humoral immunity following Snow Mountain virus challenge. J Virol 79: 2900-2909.
   Siebenga JJ, Vennema H, Duizer E, Koopmans MP (2007) Gastroenteritis caused by norovirus GGII.4, The Netherlands, 1994-2005. Emerg Infect Dis 13: 144-146.
   Lindesmith L, Moe C, Marionneau S, Ruvoen N, Jiang X, et al. (2003) Human susceptibility and resistance to Norwalk virus infection. Nat Med 9: 548-553.[↩]
3. Siebenga JJ, Vennema H, Renckens B, de Bruin E, van der Veer B, et al. (2007) Epochal evolution of GGII.4 norovirus capsid proteins from 1995 to 2006. J Virol 81: 9932-9941[↩]

I have a very sporadic and idiosyncratic series in which I talk about “classic papers”, and in my idiosyncratic series Vic Engelhard’s paper on tyrosinase processing counts as a classic paper. It was one of the early indications that proteins in the ER must be degraded in the cytosol, and as such it’s one of a number of ways that antigen presentation has helped fundamental understanding of cell biology; but I think it hasn’t received as much recognition as it could have.

But perhaps I should begin at the beginning.

Proteolysis is a normal part of cell function. Proteins that are damaged, misfolded, or mis-translated, as well as proteins that have simply reached the end of their useful life, are degraded and converted to amino acids that can be recycled into new proteins. In the early- to mid-1990s, there were three general systems that were known to degrade proteins, depending on which subcellular compartment they were in:

• In the cytosol and nucleus, proteins are predominately degraded by proteasomes.
• Proteins taken up from the exterior of the cell can be degraded in acidic lysosomes
• Mitochondrial proteins are degraded by a number of proteases within the mitochondrion¹

That leaves an obvious gap. What happens to proteins that are in the endoplasmic reticulum (ER)? This is particularly relevant because the ER is a ferociously active site of protein synthesis, folding, and assembly; when any of those steps goes awry, the protein is supposed to be degraded, a process known as “quality control”. It was clear in the 1980s that proteins that failed quality control in the ER were degraded; in human cells, a well-known example was the cystic fibrosis transmembrane conductance regulator (CFTR), which folds inefficiently and is rapidly degraded². But it was not clear where the degradation happened (in the ER? The cytosol? Somewhere else?), and what proteases were responsible.

At first it was believed that “what happens in the ER stays in the ER” — ER proteins were degraded in the ER, by ill-defined proteases in that compartment. But I don’t think there was much enthusiasm for that belief, and basically, the field was a mess, as you can see from this introductory paragraph from the time:³

Other membrane proteins are also known to be degraded at the ER, but the process is poorly understood, and the responsible enzymes have not been identified. For example, some of these proteolytic events are ATP dependent, but some are not; some occur within the lumen while others take place on the cytoplasmic side; some exhibit inhibitor sensitivities characteristic of serine proteases, whereas others do not.

This was relevant to me as an immunologist, because viral glycoproteins (which, of course, are synthesized in the ER) are popular targets for cytotoxic T lymphocyte (CTL) recognition (a quick review of MHC class I antigen presentation is here). We knew in the early 1990s that most if not all CTL targets — even those derived from ER proteins — were generated in the cytosol. As I wrote in a 1996 review:⁴

Proteins targeted into the ER by signal sequences can also be presented on MHC I molecules. Since these molecules are cotranslationally transported into the endoplasmic reticulum, they might be expected to bypass hydrolysis in the cytosol. However, where analyzed, the presentation of most of these antigens is dependent on the TAP-transporter and on proteasome activity, and therefore the presented peptides are probably being generated in the cytosol.

The three obvious possible explanations were that either the putative glycoprotein never made it into the ER and was degraded as a mistargeted protein (Jon Yewdell would call that a “DRiP”; I can’t remember exactly when I heard him propose that first, but it was around that time); that the glycoprotein went in to the ER, got degraded there, and the peptides were first transfered to the cytosol; or that the glycoprotein was transfered from the ER to the cytosol and degraded there.

The simplest explanation, at the time, seemed to be the first one –  proteins never made it in to the ER, and were degraded in the cytosol. However, it wasn’t an explanation that we liked very much, for various reasons. Vic Engelhard’s paper (Remember Engelhard’s paper? This here’s a post about Engelhard’s paper) cleared that question up, at least for one epitope.

Skipper, J. C., Hendrickson, R. C., Gulden, P. H., Brichard, V., Van Pel, A., Chen, Y., Shabanowitz, J., Wolfel, T., Slingluff, C. L., Jr., Boon, T., Hunt, D. F., and Engelhard, V. H. (1996). An HLA-A2-restricted tyrosinase antigen on melanoma cells results from posttranslational modification and suggests a novel pathway for processing of membrane proteins. J. Exp. Med. 183, 527-534.

His finding is simple enough to describe, although it relied on a technically very difficult mass spec analysis:

1. A peptide presented on MHC class I was derived from an ER protein (which they knew from its sequence);
2. the protein had actually gone into the ER, because it had been N-glycosylated, which only happens in the ER;
3. yet the peptide itself was probably generated in the cytosol, because enzymes that modified it are mainly found in the cytosol.

The most surprising and exciting part was the second point: Clear evidence that the protein had actually gone into the ER before the peptide was generated. ⁵ This wasn’t by any means definitive proof that ER proteins are degraded in the cytosol (a follow-up paper in 1998⁶ took it a bit further) but it certainly was suggestive.

If Vic’s paper had come out a year or two earlier, it would probably have made much more of a splash than it did, but in February of 1996 it was only a nose ahead of several more focused papers. The field had started to clear up in the mid-1990s, with some observations in yeast in 1993 and 1994 ⁷ and around 1995 moving into mammalian cells with (among others) the Jensen et al. paper I quoted above. ⁸  And later in 1996, the iceberg tipped over altogether, with a whole bunch of almost simultaneous papers that showed quite clearly that ER degradation wasn’t done by ER proteases at all, but was in fact performed by proteasomes. ⁹ All the papers demonstrated that there’s an export step before degradation: ER proteins that fail quality control are shunted out into the cytosol, where the proteasomes can grab onto them and chop them up. (The export step is still not all that well understood in molecular detail, though in 2007 it started to open up some, I think.)

Though Engelhard’s 1996 paper is reasonably widely cited (256 citations as I write this) it clearly didn’t have the impact on cell biology in general that it did on me, probably because it came out around the same time as a bunch of more specific papers. This blogpost is an attempt to give a bit more retroactive credit to a very nice example of logical reasoning from indirect evidence.

1. I believe that some, though not all, of the mitochondrial proteases were identified in the late 1980s/early 1990s, and that the broad outline of mitochondrial proteolysis was understood in the early 1980s (Desautels, M. and Goldberg, A. L. (1982) Liver mitochondria contain an ATP-dependent, vanadate-sensitive pathway for the degradation of proteins. Proc Natl Acad Sci USA 79 , pp. 1869-1873.). I don’t know all that much about mitochondrial proteolysis, though, so if someone wants to correct me, please do so.[↩]
2. For example, Ward C, Kopito R (October 14, 1994) Intracellular turnover of cystic fibrosis transmembrane conductance regulator. Inefficient processing and rapid degradation of wild-type and mutant proteins. J. Biol. Chem. 269.:25710-25718[↩]
3. Taken from Jensen TJ, Loo MA, Pind S, Williams DB, Goldberg AL, et al. (October 6, 1995) Multiple proteolytic systems, including the proteasome, contribute to CFTR processing. Cell 83:129-35. with references removed[↩]
4. York, I. A., and Rock, K. L. (1996). Antigen processing and presentation by the class I major histocompatibility complex. Annual review of immunology Annu Rev Immunol 14, 369-396.[↩]
5. Technical explanation: The mass spec analysis showed that the asparagine that is encoded in the DNA was actually an aspartic acid in the presented peptide; deglycosylating enzymes that were believed to only be present in the cytosol remove carbohydrates from Asn to generate Asp.[↩]
6. Mosse, C. A., Meadows, L., Luckey, C. J., Kittlesen, D. J., Huczko, E. L., Slingluff, C. L., Shabanowitz, J., Hunt, D. F., and Engelhard, V. H. (1998). The class I antigen-processing pathway for the membrane protein tyrosinase involves translation in the endoplasmic reticulum and processing in the cytosol. J Exp Med 187, 37-48.[↩]
7. (Sommer T, Jentsch S (September 9, 1993) A protein translocation defect linked to ubiquitin conjugation at the endoplasmic reticulum. Nature 365.:176-9.
   and
   Kölling R, Hollenberg CP (July 15, 1994) The ABC-transporter Ste6 accumulates in the plasma membrane in a ubiquitinated form in endocytosis mutants. EMBO J 13.:3261-71.[↩]
8. Jensen TJ, Loo MA, Pind S, Williams DB, Goldberg AL, et al. (October 6, 1995) Multiple proteolytic systems, including the proteasome, contribute to CFTR processing. Cell 83:129-35. [↩]
9. I may be missing some:
   Hampton RY, Gardner RG, Rine J (December 1996) Role of 26S proteasome and HRD genes in the degradation of 3-hydroxy-3-methylglutaryl-CoA reductase, an integral endoplasmic reticulum membrane protein. Mol Biol Cell 7.:2029-44.
   Werner ED, Brodsky JL, McCracken AA (November 26, 1996) Proteasome-dependent endoplasmic reticulum-associated protein degradation: an unconventional route to a familiar fate. Proc Natl Acad Sci U S A 93.:13797-801.
   Hiller MM, Finger A, Schweiger M, Wolf DH (September 20, 1996) ER degradation of a misfolded luminal protein by the cytosolic ubiquitin-proteasome pathway. Science 273.:1725-8.
   Qu D, Teckman JH, Omura S, Perlmutter DH (September 13, 1996) Degradation of a mutant secretory protein, alpha1-antitrypsin Z, in the endoplasmic reticulum requires proteasome activity. J Biol Chem 271.:22791-5.
   Wiertz EJ, Jones TR, Sun L, Bogyo M, Geuze HJ, et al. (March 8, 1996) The human cytomegalovirus US11 gene product dislocates MHC class I heavy chains from the endoplasmic reticulum to the cytosol. Cell 84.:769-79.[↩]

When Michael Specter’s article, ”Darwin’s Surprise”, ran in the New Yorker, I had a peevish post complaining about its comments on “junk DNA”.

Now T Ryan Gregory, at Genomicron,¹ is saying what I wanted to say (but that I mangled to the point of incoherence). “Junk DNA” — in spite of lazy journalists’ claims — was not ignored or “dismissed” (the journalists’ favourite word), but was studied with an open mind:

The important message being offered is that there was plenty of research into possible functions or lack thereof in noncoding sequences of all types, and that whichever way authors concluded was based on the evidence available at the time, not ideology.

As well as the whole coherence thing, Genomicron is providing definitive proof with quotes from the literature. Read all about it:
Quotes of interest — 1980s edition (part one).
Quotes of interest — long neglected, some noncoding DNA is actually functional.
Quotes of interest — Nobel Prize special edition.
Quotes of interest — 1980s edition (part two).
and especially
Quotes of interest — pseudogene.

1. And from my old alma mater, though I think I had already left before he started there[↩]

What with visiting speakers and new faculty recruitment, I’ve been out late every night this week; what with committee meetings¹ and trying to squeeze in experiments, I’ve been up early every morning; and what with teaching starting up again, seminars from visiting speakers and recruitees, and faculty meetings, I’ve had little time for other stuff. So this is going to be a short post. ²

A while ago I talked about lamprey immune systems. The key points are that lampreys …

• have an immune system
• that works pretty well
• and in concept looks a lot like our immune system, with lymphocytes and specific receptors
• but the receptors are utterly unlike our T cell receptors and antibodies
• using instead of the immunoglobulin domain structure, a leucine-rich repeat (LRR) structure;
• and, instead of using RAG-based recombination, uses a gene conversion system to generate diversity.

There’s a temptation, even for those who intellectually know better, to assume that “primitive” animals have “worse” systems; so because lampreys are more like the common ancestor of vertebrates, their immune system must be “worse”. (Hagfish and lampreys, which may have diverged some some 500 million years ago — see the figure to the right;³ click for a larger version — have very similar immune systems, so this system must be at least that old.) In some ways the mammalian immune response does seem to have some advantages — faster memory response, for example. Still, lamprey immune systems have served them well for 500 million years, which is more than we can say about ours; and in some other ways lampreys do better than we do. They have if anything a greater diversity to their receptors, for example, potentially generating more than 10¹⁴ different receptors — compare to our roughly 10⁸ T cell receptors.

Max Cooper, who has done much of the work on lamprey immunity,⁴ has just published a paper showing off some other unusual properties of lamprey immune receptors. ⁵ Since there’s no system for making lamprey monoclonal antibodies that’s analogous to the mouse monoclonal antibody systems, he used a molecular cloning approach to express monoclonal variable lymphocyte receptor (VLR) -B cDNAs from immunized lampreys.

What did they get?

They got soluble “antibodies”, capable of the highly specific recognition that’s seen in conventional monoclonal antibodies. The VLR-B antibodies are extraordinarily stable, maintaining binding at pH 1.5 and maintaining structure at pH 11, as well as after incubation at 56 ^oC for a couple days or at room temperature for weeks. ⁶ Although the individual LRR subunits have relatively low binding affinity, they are secreted as multimers of eight to ten subunits (see the diagram to the left), and as a result the VLR-B binding ability can be at least as good as mouse monoclonals: “Equal concentrations of VLR4 and EA2-1, starting at 0.5 mg/ml, were serially diluted in 10-fold increments and scored for the degree of spore agglutination. Spore agglutination by VLR4 was detected at a concentration 1,000-fold more dilute (5 pg/ml) than the mouse monoclonal antibody (5 ng/ml).”

Finally, as opposed to mouse monoclonals, these are single proteins; conventional mouse monoclonals have two components, a heavy and a light chain. That makes VLR-B easier to work with in some ways: “The single peptide composition of VLR-B antibodies makes them more amenable to molecular engineering, including manipulation of the antigen binding site by mutagenesis and fusions to the coding sequences of other peptides, such as enzymes, toxins, and epitope tags to extend their functional capabilities.” ⁷

These things clearly have potential to be useful in all kinds of things — a nice example of basic research giving rise to clinically and commercially useful tools.

1. Proof, if proof were needed, that deans are evil: 7:30 AM meeting with the dean[↩]
2. Also, I just realized I really, really need to get some flowers for my wife today, don’t I.[↩]
3. From: Modern look for ancient lamprey. Philippe Janvier. Nature 443, 921-924(26 October 2006 [↩]
4. It was his talk at the Autumn Immunology Conference in Chicago a couple of years ago that made me realize how fascinating the subject is[↩]
5. Herrin, B.R., Alder, M.N., Roux, K.H., Sina, C., Ehrhardt, G.R., Boydston, J.A., Turnbough, C.L., Cooper, M.D. (2008). Structure and specificity of lamprey monoclonal antibodies. Proceedings of the National Academy of Sciences, 105(6), 2040-2045. DOI: 10.1073/pnas.0711619105[↩]
6. Conventional antibodies are pretty stable, but not up to this level.[↩]
7. I think it’s camels that make single-chain antibodies, and there’s been interest in developing monoclonal systems based on camel antibodies for the same reason.[↩]

A while ago I talked about a paper that demonstrated that cancers establish a long-term equilibrium with the immune system. That paper provided a formal demonstration of a concept that was already widely accepted — that cancers can arise and persist for a long time in balance with the immune system, so that the immune system isn’t able to completely eliminate the cancerous cells, but the tumor is not able to grow or spread, either. Evidence in humans shows that this equilibrium can persist for many years. In some cases, the tumor may eventually mutate to a form that can more completely avoid the immune system, and those tumors become clinically detectable. In other cases, perhaps, the immune system may finally destroy all vestiges of the tumor, and the person won’t ever know that he was carrying the potentially-cancerous cells; or the balance may persist for a lifetime, until the person dies of something altogether different.

There was an interesting implication to that concept that I had overlooked, because it doesn’t fit with the conventional understanding of cancer development. A paper from last month¹ proposes a new and rather troubling model.

People rarely die of a primary tumor; the direct cause of death is usually metastatic cancer. The prevailing models of cancer development suggest that metastasis is a late event in cancer development. (I believe this concept was first articulated by Fidler in 1978,² although in retrospect this paper only argues that metastasis is the product of a subset of selected cells from the tumor — the concept that a tumor is not a homogenous entity — not that the selection process that leads to the metastatic subset must necessarily occur late in the cancer’s progression.) Hanahan and Weinberg, in their immensely influential 2000 paper “The Hallmarks of Cancer”,³ discussed “Tissue Invasion and Metastasis” as the sixth and final “acquired capabilities” of a tumor, and show metastasis as the final event in four of the five pathways they illustrated, and as the penultimate event in the other one (see the figure to the right here; click for a larger version).

However, there’s also some evidence that metastasis isn’t necessarily a very late event in cancer development. For example, Engel et al. worked out the point at which metastases from breast cancers must have occurred, based on their growth rates, and determined that metastases must often happen quite early in the course of the tumor: ⁴

Our hypothesis of an early MET [metastasis. IY] initiation questions the morphological and genetic correlation between the primary tumour and MET, because the primary tumour at the time of dissemination would have been much smaller and probably had prognostically favourable characteristics.

The paper I’m talking about here,¹ from Hüsemann et al, takes this argument a couple of steps further. Not only can metastases arise very early in the course of cancer development, they say, metastases can even arise before the cells are fully malignant. Using a mouse mammary carcinoma model in which mammary tumors arise in a predictable fashion, they were able to detect the premalignant mammary cells in the bone marrow as early as 4 weeks – before outright tumors had arisen in the mammary tissue itself — and these early-spreading cells were able to cause cancer in irradiated (immune-deficient) mice after bone marrow transplant.

One provocative finding of our study is that, in mouse models of breast cancer, large tumors seed neither more nor genetically further-advanced cancer cells than do small lesions… Thus, the ability of metastatic dissemination does not appear to be the result of selection of tumor cells within the tumor. Rather, the data suggest that tumor cells disseminate early and will be selected for outgrowth at distant sites.

(My emphasis.)

If metastases spread very early, then why do we even see “primary tumors”? Why don’t the metastases arise at the same time as the “primaries”, or even earlier — so that all we see is a metastatic cancer with no primary tumor? (In fact, this is exactly what is seen in a lot of tumors. Some 5 – 10% of tumors present as metastases with an unknown primary tumor. ⁵ However, the question still applies to the remaining 90-95% of tumors.)

The most likely explanation is that there is some growth restriction on metastases. For example, perhaps the primary tumor secretes growth factors systemically, which permits the metastases to expand. Or — and this is a possibility the authors didn’t really discuss, so I’m not sure if there’s some obvious flaw that I’m missing — the immune system may hold the small tumors in check for a while until the metastases manage to evade equilibrium with the immune system. I wonder if this could be a regulatory T cell (TReg) effect, in which the primary tumor is able to expand and then conditions infiltrating lymphocytes to become TRegs; we know that TRegs circulate, so perhaps these circulating TRegs then shut down the ongoing response to the metastases as well, and allow them to grow out.

In any case, this observation (assuming it holds up) changes some of the concepts of cancer therapy, I think. If the metastases are already out there, then some of the rationale for removing the primary tumor is gone. On the other hand, there’s a potential opportunity there, too: If something prevents metastases from growing out and causing a problem for a long time, perhaps that something can be harnessed and used to prevent outgrowth for a lifetime.

1. Yves Hüsemann, Jochen B. Geigl, Falk Schubert, Piero Musiani, Manfred Meyer, Elke Burghart, Guido Forni, Roland Eils, Tanja Fehm, Gert Riethmüller and Christoph A. Klein. (2008). Systemic Spread Is an Early Step in Breast Cancer. Cancer Cell, 13(1), 58-68. DOI: 10.1016/j.ccr.2007.12.003[↩][↩]
2. Fidler, I. J. (1978). Tumor heterogeneity and the biology of cancer invasion and metastasis. Cancer Res 38, 2651-2660.[↩]
3. Hanahan, D., and Weinberg, R. A. (2000). The hallmarks of cancer. Cell 100, 57-70.[↩]
4. Engel, J., Eckel, R., Kerr, J., Schmidt, M., Furstenberger, G., Richter, R., Sauer, H., Senn, H. J., and Holzel, D. (2003). The process of metastasisation for breast cancer. Eur J Cancer 39, 1794-1806.[↩]
5. van de Wouw AJ, Janssen-Heijnen MLG, Coebergh JWW, Hillen HFP (2002) Epidemiology of unknown primary tumours; incidence and population-based survival of 1285 patients in Southeast Netherlands, 1984-1992. European Journal of Cancer 38.:409-413 [↩]

A while ago I talked about evolution of the herpesviruses, and I said:

We know of 200-odd herpesviruses so far, and more are being identified practically daily. It’s likely that virtually every animal species has its own set of unique herpesviruses. This is probably because herpesviruses are very host-restricted (rarely infecting more than a single species) and set up latent, life-long infection. When an animal population speciates, its complement of herpesviruses speciates along with it.

Word of my blog post spread like wildfire (as is inevitable for a blog that is read by upward of five people, including my mother) and Duncan McGeoch hastened to correct me with a fascinating paper now in pre-print form at the Journal of Virology:
Ehlers, B., Dural, G., Yasmum, N., Lembo, T., de Thoisy, B., Ryser-Degiorgis, M., Ulrich, R.G., McGeoch, D.J. (2008). Novel mammalian herpesviruses and lineages within the Gammaherpesvirinae: Cospeciation and interspecies transfer. Journal of Virology DOI: 10.1128/JVI.02646-07

McGeoch’s group went herpesvirus hunting (“Be vewwy, vewwy quiet!”) and — supporting at least the first part of my quote here — found no less than 14 brand-new gammaherpesviruses. (They would have found 16 new viruses, but their elephant gammaherpesvirus was described earlier this year when Wellehan et al earlier this year found 6 new gammaherpesviruses of elephants, rock hyraxes, and manatees,¹ and their gorilla virus was described a few years ago by Gessain’s group² )

The new gammaherpesviruses come from shrews, tapirs, rhinocerous, zebras, babirussas, pygmy hippos, and so forth (see the table to the right, and see the paper itself for accession numbers, I’m not going to copy them all down). They used a fiddly, if not conceptually difficult, technique to get a reasonable sequence length (3.4 kb) for phylogenetic analysis, rather than the couple hundred base pairs that this sort of virus fishing expedition usually yields, and then lined up the newly expanded complement of gammaherpesviruses, which now includes no less than 45 viruses.

I won’t reproduce their phylogenetic tree here (the actual figure from the preprint has the “ACCEPTED” watermark stamped all over it, and I don’t have time to get 45 virus sequences and re-build the alignment according to their description). The interesting thing about this tree is that it tells a somewhat different story than an earlier and more limited analysis.

In earlier analyses of Herpesviridae phylogeny, it was possible to discern within each of the subfamilies a substantial degree of congruence in tree branching pattern with the corresponding pattern for lineages of the mammalian hosts, and this was taken as indicating extensive cospeciation of herpesviruses and hosts.

Even in these previous analyses, the gammaherpesviruses were the worst match for the co-speciation hypothesis — that is, closely-related gammaherpesviruses were found in mammalian hosts that were not very closely related, suggesting that the viruses’ ancestors might have jumped hosts at some point. That observation was even more true in this larger set, especially in the more ancient divisions:

… applicability of cospeciational interpretation declines further with the extensive detail now available for the GHV tree. … the deeper branching details of the tree prove rather unproductive for constructing any unified coevolutionary correspondence across host lineages. In particular, the two deepest distinct lineages, i.e. LCV and EmaxGHV-1, are not simultaneously compatible with a single cospeciational scheme, and in the MF2 clade the unresolved nodes for major lineages do not enable any compelling interpretation. On the other hand, clear dispersed examples of cospeciation can be seen in the terminal branchings within major lineages. … In summary, there are substantial indications in the GHV tree of evolution both by cospeciation with host lineages and by transfer between widely distinct hosts.

(My emphasis.)

There are at least two really interesting implications from this work. First, McGeoch’s group supports the previously-proposed notion that there may be more human herpesviruses yet to be found, because there are major divisions of the gammaherpesvirus families that don’t yet have human representatives. Second, although I’m not aware of any instances of non-human herpesviruses successfully³ infecting humans, this work suggests that at least in the past, and therefore likely today, gammaherpesviruses could make interspecies leaps.

Just something else to watch for.

1. Wellehan JFX, Johnson AJ, Childress AL, Harr KE, Isaza R (August 19, 2007) Six novel gammaherpesviruses of Afrotheria provide insight into the early divergence of the Gammaherpesvirinae. Vet Microbiol. doi:10.1016/j.vetmic.2007.08.024 [↩]
2. Either Lacoste V, Mauclre P, Dubreuil G, Lewis J, Georges-Courbot M, et al. (2000) Virology: KSHV-like herpesviruses in chimps and gorillas . Nature 407.:151-152
   or
   Lacoste V, Mauclère P, Dubreuil G, Lewis J, Georges-Courbot MC, et al. (September 2001) A novel gamma 2-herpesvirus of the Rhadinovirus 2 lineage in chimpanzees. Genome Res 11.:1511-9.
   I didn’t check the sequences to see which reference is McGeoch’s gorilla virus.[↩]
3. Where “successfully” means not merely infecting and killing occasional individuals, but actually spreading from person to person — I know about herpesvirus B and so on[↩]