Mystery Rays from Outer Space

Vesicular stomatitis virus (VSV), like the related rabies virus, is a bullet-shaped virus.  Hong Zhou has just added VSV to his collection of cryo-electron microscopy virion structures,¹ and as always with viruses, it’s just gorgeous.

“Architecture of the VSV virion. … A montage model of the tip and the cryo-EM map of the trunk”1	“A plausible process by which the nucleocapsid ribbon generates the virion head, starting with its bullet tip.”1

1. Peng Ge, Jun Tsao, Stan Schein, Todd J. Green, Ming Luo, & Hong Zhou (2010). Cryo-EM Model of the Bullet-Shaped Vesicular Stomatitis Virus. Science, 327 (5966), 689-693 DOI:10.1126/science.1181766[↩][↩][↩]

Clonal evolution during in situ to invasive breast carcinoma progression1

What’s a tumor?

In some ways, that’s a bad question (never mind the answer) because it implies that a tumor is a single thing. But we know that’s not true. A tumor, by the time we can detect it, is a collection of many cells, at least billions of them, and those cells are not all the same. I’m not even talking about the normal cell types that are incorporated into a tumor (things like blood vessels and support cells). Even cells that are unambiguously cancerous are very different within a tumor. And of course, that’s important for the things we’re most interested in, prognosis and treatment, because it’s not the average tumor cell that we’re most concerned about, it’s the subset of tumor cells that are most resistant to treatment, or that are most aggressive.

The development of this variation is really fundamental to how we understand tumor formation and tumor growth. Cancerous cells don’t just appear, fully ready to metastasize and grow. What happens is that a normal cell mutates slightly and gains a little advantage. Most of its progeny stay like that, but one of them mutates again and changes a little more, and then one of that cell’s progeny mutates again, and so on. It probably takes at least a half-dozen mutations, over many cell generations, before a normal cell has progressed through to a detectably cancerous cell.  (I’ve talked about this before, here.)

Also, since truly normal cells simply don’t mutate that many times — there are too many checks and repair systems to allow a half-dozen mutations to accumulate in a single human’s² lifetime — one of the mutations is probably in the check/repair system, turning the cancerous pathway into a mutator pathway as well.

So we expect tumors to be made up of many different cell types, and this is indeed what we see:

With rare exceptions, human malignancies are thought to originate from a single cell, yet by the time of diagnosis, most tumors display startling heterogeneity in cell morphology, proliferation rates, angiogenic and metastatic potential, and expression of cell surface molecules. ¹

So how diverse are tumors?

That’s been a hard question to answer, because you’d need tools to look at individual cells, and you’d also need some way of expressing that diversity. A recent paper¹ looked at diversity in breast cancer using some individual-cell tools, which I’m not going to discuss, and took an interesting approach to describing the variability:

… we applied diversity measures from the ecology and evolution sciences to our copy number data. These diversity measures estimate the number and distribution of species in a certain geographical area or environmental niche. In our context, a species is a cancer cell population … Hence, a region of a tumor containing cancer cells with 3 different copy number ratios is interpreted to contain 3 distinct “species.” ¹

They suggest that this way of describing tumors could be a useful aid to prognosis and to predicting response to therapy, offering a quantitative description of tumor variability (which might correlate with the tumor’s potential for spread and escaping treatment).

I hadn’t thought of tumors as ecosystems before, but I wonder if the analogy could be taken further by considering, say,cytotoxic T lymphocytes as predators …

1. Park, S., Gönen, M., Kim, H., Michor, F., & Polyak, K. (2010). Cellular and genetic diversity in the progression of in situ human breast carcinomas to an invasive phenotype Journal of Clinical Investigation DOI: 10.1172/JCI40724[↩][↩][↩][↩]
2. let alone a mouse’s lifetime[↩]

Every so often — not often enough — I run across a paper that’s so ridiculously ingenious that it just makes me laugh with pleasure.

Ladies and gentlemen, a round of applause, please, to Shou-Wei Ding, of the Center for Plant Cell Biology at UC Riverside, for his Rube Goldberg-esque brilliant technique for identifying new viruses. ¹

Background: Small interfering RNA (siRNA, ² or RNAi) is pretty well known nowadays, especially since three of its discoverers were given Nobel Prizes a few years ago. These small RNAs are found in most eukaryotes — plants, insects, worms, as well as birds and mammals. In fact, siRNA was first discovered in plants and then was widely used in insects and worm research well before it was shown in mammals. In mammals, we tend to think of small RNAs as having regulatory functions. In plants, insects, and worms, there are certainly regulatory siRNAs, but siRNAs are also used as an important part of their antiviral immune response. (Unsurprisingly, plant and insect viruses themselves have defenses against these siRNAs, by producing anti-siRNA genes.)

siRNAs are small (duh), maybe 20-30 bases long. Without going into mechanisms more than necessary, they recognize specific sequences of their target RNA (by being complementary to the target). When they bind to their target, they cause that target RNA to be chopped up into small pieces. Some of those small pieces can then act as new siRNA and the cycle continues.

Ding’s group reasoned that (1) if insect viruses are being attacked by siRNA, and (2) the viruses are then chopped up into new siRNA, then (3) all you have to do is piece together the siRNA, to recover the virus sequence.

Seems sort of obvious now that I put it together that way, but I would have said that there’s no way it would work — not enough coverage, I would have said, you’d only see a tiny fraction of the genome. Even if there was enough coverage, how would you find the virus pieces in the huge pool of other siRNAs? And even if you could do that, how would you piece them together? It would be like a jigsaw puzzle where every piece was just a tiny snippet of blue sky.

But, wonderfully, it actually worked for Ding’s group. Not only did they identify viruses that they knew should be there, they also pulled out five brand-new viruses out of their insect cells:

In this study, we found that viral small silencing RNAs produced by invertebrate animals are overlapping in sequence and can assemble into long contiguous fragments of the invading viral genome from small RNA libraries sequenced by next-generation platforms. Based on this finding, we developed an approach of virus discovery in invertebrates by deep sequencing and assembly of total small RNAs (vdSAR) isolated from a host organism of interest. Use of this approach revealed mix infection of Drosophila cell lines and adult mosquitoes by multiple RNA viruses, five of which were previously undescribed.

“Virus discovery in OSS cells by viral genome assembly from sequenced viral piRNAs of 25–30 nucleotides in length after viral siRNAs were removed.”

The ability to piece these tiny fragments together is a spin-off of the new genome sequencing platforms, which by their nature make very short reads that have to be computationally stitched back together. Most of these new platforms make slightly longer fragments than siRNA size, but they’re in the same ballpark and I guess the same approaches work.

As far as the coverage, they didn’t find 100% of the genomes, but they got a really surprisingly high fraction back — 80% to 95% or more of the various viruses.

Not only that, they got really new viruses:

As a result, none of the four viruses could be assigned into an existing virus genus. This suggests that vdSAR is capable of discovering viruses that are only distantly related to known viruses.

Giving credit where it’s due, another group recently, and independently, used a similar approach to identify viruses in sweet potatot plants³ but I didn’t notice that article until Ding pointed it out.

Whether or not this technique proves useful in the long run, it’s just so ingenious that I want it to succeed.

1. Wu, Q., Luo, Y., Lu, R., Lau, N., Lai, E., Li, W., & Ding, S. (2010). Virus discovery by deep sequencing and assembly of virus-derived small silencing RNAs Proceedings of the National Academy of Sciences, 107 (4), 1606-1611 DOI: 10.1073/pnas.0911353107[↩]
2. I’m going to call all of the various types of small RNAs “siRNA” here, but that’s just shorthand, there are different subclasses that I won’t go into[↩]
3. Kreuze, J., Perez, A., Untiveros, M., Quispe, D., Fuentes, S., Barker, I., & Simon, R. (2009). Complete viral genome sequence and discovery of novel viruses by deep sequencing of small RNAs: A generic method for diagnosis, discovery and sequencing of viruses Virology, 388 (1), 1-7 DOI: 10.1016/j.virol.2009.03.024[↩]

I didn’t post anything about the recent study¹ showing that handwashing + face masks reduces influenza spread, because other blogs covered it fairly extensively (for example, here’s Avian Flu Diary’s commentary). Here’s another study giving a common-sense check:

… in a household setting, simple, readily available products such as 1% bleach, 10% vinegar and 0.01% washing up liquid all make convenient, easy to handle killing agents for influenza virus A/H1N1. These findings can be readily translated into simple public health advice, even in low resource settings. The public do not need to source more sophisticated cleaning products than these.

–Greatorex, J., Page, R., Curran, M., Digard, P., Enstone, J., Wreghitt, T., Powell, P., Sexton, D., Vivancos, R., & Nguyen-Van-Tam, J. (2010). Effectiveness of Common Household Cleaning Agents in Reducing the Viability of Human Influenza A/H1N1 PLoS ONE, 5 (2) DOI: 10.1371/journal.pone.0008987

(My emphasis) Their figures show that these common solutions almost immediately reduced the numbers of virus from between 1 and 100 million at the start, to undetectable levels (less than 200). Hot water, not surprisingly, didn’t work.

They also added that “branded anti-bacterial wipes and anti-viral tissues were encouragingly effective at inactivating the virus“, so if you’d rather buy something expensive, go ahead.

1. Aiello, A., Murray, G., Perez, V., Coulborn, R., Davis, B., Uddin, M., Shay, D., Waterman, S., & Monto, A. (2010). Mask Use, Hand Hygiene, and Seasonal Influenza?Like Illness among Young Adults: A Randomized Intervention Trial The Journal of Infectious Diseases, 201 (4), 491-498 DOI: 10.1086/650396[↩]

Smallpox pustules (R. Carswell, 1831)

But despite these advances, there is far more that we simply do not understand about smallpox disease or its causative virus. The smallpox vaccine, vaccinia virus, remains the poster-child for human vaccines, but we have only begun to understand how vaccinia-induced immune responses protect vaccinees from orthopoxvirus infections.  …  In contrast, we still do not understand why smallpox disease was so lethal in humans, or if host responses such as the oft-quoted and still poorly-understood “cytokine storm” is really a key instrument of the disease pathophysiology. In fact, we do not comprehend the basis for the strict host tropism of variola virus for humans, nor why there are no animal reservoirs. So, there is really no scientific debate about whether variola virus still has much to teach us about human immunology and viral pathogenesis in general. Instead, the main flashpoint for debate remains the issue of risk versus benefit at acquiring any more scientific information with live variola virus.

– McFadden, G. (2010). Killing a Killer: What Next for Smallpox? PLoS Pathogens, 6 (1) DOI: 10.1371/journal.ppat.1000727

(My emphasis)

The arguments pro and con for smallpox destruction have been made over and over, and I’m assuming that anyone interested in such things¹ has already seen them; but McFadden here does a nice job of outlining the situation.  The article is open-access, so you should read it.  Giants in the field, who know far more about the virus than I ever will, have historically been split on the subject. ²

Not that anyone asks me, but even if they did, I don’t know which side of the destroy smallpox stocks/keep smallpox stocks debate I come down on.³ I lean slightly toward destroying the stocks, but I also think there’s not a lot of point to it; there are almost certainly unregistered stocks out there, whether in a terrorist’s hands (I doubt that), or some nation’s official-unofficial stocks (I do believe this, and suspect there may be quite a few such stocks, but that’s just my cynicism speaking), or in the bottom of some old-time virologist’s freezer marked “AFR45UNK0450″ (I did my Master’s degree with one such old-time virologist, and I’ll tell you, the bottom of his freezer was a marvel indeed, though I don’t think there was any smallpox per se).

Still, history makes it very clear that viruses can escape from research labs.  I’ve pointed out a number of such cases: Foot and mouth disease escape from the Pirbright research station, probable Dengue escape from a lab, probable escape of H1N1 influenza leading to the 1976 pandemic. ⁴ And, of course, the last case of smallpox in the world was itself a lab escape.  Reducing the number of labs containing smallpox stocks should reduce the risk of an escape.

Further reading:

• Lab escapees
• Life & Death, pre-vaccination
• Immune evasion as a vaccine target
• Malaria eradication: The smallpox precedent
• Dogma supported: Antiviral immunity

1. And if you’re not, why are you reading this blog?[↩]
2. Destroy the stocks – Frank Fenner and many more:

   Science. 1993 Nov 19;262(5137):1223-4. The remaining stocks of smallpox virus should be destroyed. Mahy BW, Almond JW, Berns KI, Chanock RM, Lvov DK, Pettersson RF, Schatzmayr HG, Fenner F.

   Keep the stocks: Joklik, Moss, Fields, and many more:

   Science. 1993 Nov 19;262(5137):1225-6. Why the smallpox virus stocks should not be destroyed. Joklik WK, Moss B, Fields BN, Bishop DH, Sandakhchiev LS.[↩]

3. Just to forestall a common argument that I’ve seen (not in the scientific circles): Should we keep the stocks so if there’s a new outbreak we have vaccines?  This is not a good argument, of course, because smallpox is not its own vaccine; no one sane is arguing that we should get rid of vaccinia virus, the vaccine against smallpox.[↩]
4. Recent human influenza A (H1N1) viruses are closely related genetically to strains isolated in 1950. Nakajima K, Desselberger U, Palese P.  Nature. 1978 Jul 27;274(5669):334-9[↩]

OK, last time I thought H1N1 influenza was coming back (just after Christmas) it turned out to be just a blip.  But I notice that according to Google Flu Trends, 30 states are showing increases in flu activity this week, compared to last week. Mostly very small increases,¹ but 10 of the states have shown a sustained increase (at least two weeks of increasing numbers). Over the past two weeks, Alabama, Kansas, Louisiana, Oregon, and Utah have each shown a 10-20% increase in flu activity.

This is pretty much the time that flu season normally begins, but there’s been very little evidence of the normal seasonal flu strains circulating this year, so odds are these cases are almost all H1N1 swine-origin influenza virus.

Just saying.

1. None are more than about 10% higher than last week[↩]

Last week, the Effect Measure blog¹ talked about a paper that offered a new way of treating influenza.² Briefly, the approach is to attack the virus by treating the host cell: Eliminating host functions that the virus requires, but that the host cell does not.

The authors of the paper commented that “targeting host cell determinants temporarily dispensable for the host but crucial for virus replication could prevent viral escape,” and Effect Measure observed that “It’s not obvious to me why the virus can’t as easily mutate in ways to adapt to a missing “office tool” as to a drug that affects an important viral function.” In the comments, I said:

I think the fundamental difference is that in the latter case, the virus needs just modify an already-present function; but if the tool is missing altogether, the virus would have to develop a whole new function from scratch, an altogether more difficult task.

That’s not to say that a virus could not do it — we see examples of this all the time, with viruses that have co-opted host functions and in some cases even host genes. But the process is usually much slower; we tend to recognize those events in hindsight, whereas we can often watch viral genes adapting to drugs in real time.

I still think my explanation is generally true, but here’s a counterexample, of what appears to be a virus evolving a brand-new function in just 5 weeks.

Background: Mammals have what seems to be a general defense against retroviruses like HIV. Several members of the APOBEC family of proteins are anti-retroviral;³ they force widespread mutations into the HIV genome, so many mutations that the virus can’t replicate or produce normal proteins.³ The reason HIV is able to replicate in spite of the APOBECs, is that HIV has in turn an anti-APOBEC protein, vif, that causes rapid destruction of several APOBECs. (I’ve mentioned this before, here, here, and here.)

Predicted vif structure (Zhang et al.)

Variants of HIV that don’t have vif (either natural or artificial) can replicate pretty much normally in cells that don’t produce APOBECs; but they’re dead in cells that do have APOBECs, and their natural targets for infection do have APOBECs. So HIV is pretty much absolutely dependent on vif for its life-cycle.

There’s a lot of interest in trying to use this fact as an anti-HIV treatment. If there was a safe, effective anti-vif drug, then the APOBECs that are normally present could go ahead and destroy the virus. Now, we know from experience with other anti-HIV drugs that the virus could probably mutate vif to avoid this hypothetical drug, but let’s say it couldn’t. Let’s say the drug was completely effective in blocking vif, and there was no way for HIV to build a drug-resistant vif. Would the virus be completely helpless, or would it be able to develop a whole new anti-APOBEC function from scratch?

At least in a specific and somewhat limited set of conditions, that’s just what happened. Hache et al⁴ took a vif-deleted virus and tossed it into cells that have APOBEC3G. For several weeks, as you’d expect, there was almost no virus recovered (because without vif, the virus was destroyed by the APOBEC3G). But (see the figure to the left here; click for a larger version) after about 45 days, in 3 of the 48 cultures, virus abruptly started to grow again.

“Highlights of the long-term spreading-infection experiments for Vif-deficient viruses on vector-control- or APOBEC3G-expressing CEM-SS cell lines. Of the cultures, 45/48 showed no virus replication on APOBEC3G-expressing cells (flat lines not graphed).” 4

These new viruses still didn’t have any vif, but they were pretty much resistant to APOBEC3G — they had developed a brand-new function that conferred resistance to APOBEC3G. This new function behaves quite differently from vif. For one thing, vif protects against several different members of the APOBEC family, while the new variants were only resistant to APOBEC3G (they were still susceptible to APOBEC3F). And it took two simultaneous changes in the viruses for this to work: “Virus replication was only detectable after two mutations appeared: a noncoding A200T(C) transversion and a Vpr null mutation.“

The mechanisms underlying this aren’t quite clear, and it’s really mysterious why getting rid of vpr would help make HIV resistant to APOBEC3G (vpr is a fairly mysterious protein in its own right, so it doesn’t offer a lot of handles to work it out). Anyway, although the authors did offer a number of possible explanations, that’s not really what I wanted to talk about. The point I wanted to make is that viruses can acquire new functions out of nothing, as well as modifying already-present functions.

Co-expression of Vif and APOBEC3G in HeLa cells

Having said that, I think this actually does support my answer to some extent. Would this sort of resistance actually arise in vivo? Remember the question the authors were looking at originally: If we have an anti-vif drug, will resistance to it quickly arise? And if we look at the characteristics of the resistance in this artificial system, it’s actually somewhat encouraging:

• The resistance took quite a while to pop up — several weeks, anyway ⁵
• The resistance was rare even over that timescale. Only three of the 48 cultures threw out resistance variants. Each culture was infected with thousands of viruses originally,⁶ so you could say that the rate was lower than 3 in 400,000⁷

(Both of these are sort of what you’d expect from variants that require multiple mutations for resistance. Single mutations occur very frequently, but multiple mutations are exponentially⁸ less frequent.)

• The resistance was partial. These viruses were only resistant to APOBEC3G. In a natural infection, these resistant viruses would probably still be killed, because they’re still susceptible to APOBEC3F

So: Yes, viruses can develop new functions, but it’s probably still fair to say they’re not as adept at this as at modifying existing functions.

1. Which you should be reading, if you’re not already[↩]
2. Karlas, A., Machuy, N., Shin, Y., Pleissner, K., Artarini, A., Heuer, D., Becker, D., Khalil, H., Ogilvie, L., Hess, S., Mäurer, A., Müller, E., Wolff, T., Rudel, T., & Meyer, T. (2010). Genome-wide RNAi screen identifies human host factors crucial for influenza virus replication Nature DOI: 10.1038/nature08760[↩]
3. [↩][↩]
4. HACHE, G., SHINDO, K., ALBIN, J., & HARRIS, R. (2008). Evolution of HIV-1 Isolates that Use a Novel Vif-Independent Mechanism to Resist Restriction by Human APOBEC3G Current Biology, 18 (11), 819-824 DOI: 10.1016/j.cub.2008.04.073[↩][↩]
5. How does this compare to the time it would take HIV to pop up resistant variants to one of the drugs in HAART therapy?  Anyone know? Bueller? Anyone?[↩]
6. It’s not clear from their methods exactly how many, but 10,000 seems like a reasonable guess[↩]
7. That’s not really a valid interpretation but it offers an upper limit, anyway[↩]
8. Exponentially? Geometrically? Anyway, “much less”[↩]

“Untitled (Green Silver)” – Jackson Pollock

In the past few weeks not only did I post a short update on the DRiPs hypothesis here, but coincidentally a bunch of papers on DRiPs have also been published. I’ll probably cover some of these in more detail at some point, but here are some of the recent papers and my brief comments.

Just as a reminder: the DRiPs (“Defective ribosomal products”) hypothesis proposes that most of the peptides presented to cytotoxic T lymphocytes don’t come from the actual proteins that we normally measure — rather, the immunologically relevant peptides come from deformed and defective proteins that are mis-read and misfolded during their translation. (More explanation of DRiPs here and here; more explanation of how T cells recognize cells and where peptides come in, here.)

Jon Yewdell’s insight,¹ which is still somewhat controversial, was that defective proteins may actually be very common. Instead of being rare and abnormal events, he argued, protein production is a highly error-prone business, and a large fraction of newly synthesized proteins are broken. These defective products are very rapidly recycled into peptides and amino acids, and because of this rapid recycling they are the major source of peptides for T cell recognition.

On his original publication I had no problem with the underlying concept, but wasn’t overwhelmed by the data, and felt that there were too many counterexamples; since then he, and others, have put forward more and more examples, and I think it’s also fair to say that Jon has softened a little on the original hypothesis.² I’m more or less convinced that DRiPs are one important source of peptides, though I remain dubious that they are the only, or (and here I get very uncertain) even the major source.

Anyway, in the past few weeks, we’ve seen these papers:

• The Synthesis of Truncated Polypeptides for Immune Surveillance and Viral Evasion³

This is from Nilabh Shastri, and it’s not a big conceptual departure from some of his previous work. He’s argued for quite a while that aberrant proteins are major sources of T cell targets (see my posts here and here, for examples). Here he extends the argument to the EBNA1 protein from Epstein-Barr virus. This is a remarkably interesting protein for many reasons, one of which is that there’s reason to believe that DRiPs must be the only real source of T cell targets from EBNA1. Here, Shastri shows that in fact DRiPs (in the forms of truncated synthesis products) are in fact targets for T cells (“Thus, translation of viral mRNAs as truncated polypeptides is important for determining the antigenicity of virus proteins“). (I don’t know if it’s fair to generalize to all viral mRNAs from this very unusual protein, though.)  Very intriguingly, he also shows that DRiPs seem to be specifically blocked by EBNA1 mRNA!

Regulating production of DRiPs at the level of mRNA translation may serve as an immune evasion strategy for latent viruses. …  It is tempting to speculate that episome maintenance proteins, found in herpesviruses of various species, might have evolved to inhibit pMHC I presentation by interfering with production of DRiPs.

Is this a new viral immune evasion mechanism? And if so, how widespread is it? I know Nilabh (or someone from his lab) reads this blog occasionally, and I’d be interested in hearing their ideas on this — is it pure speculation, or do they have reason to extend the observation?

• Viral adaptation to immune selection pressure by HLA class I–restricted CTL responses targeting epitopes in HIV frameshift sequences⁴

HIV-1 frameshift inducing element

These authors looked at proteins produced by reading frame shifts from HIV.  Although HIV does a lot of frame-shifting “deliberately”, here we’re looking at frame-shifts that are (probably) not “real”.  That is, while it’s possible that some of these proteins have a biological function, for the most part they’re probably nonsense proteins, the product of incorrect selection of reading frames by the ribosome, and therefore you’d expect them to be recognized as improper proteins by the quality-control system and rapidly destroyed. In that sense they fit into the “DRiPs” concept. This fits neatly with Shastri’s previous work on frame-shifting, as well as providing modest support of the DRiPs concept.

The interesting thing here is that this paper offers evidence for large-scale immunological importance of peptides from frame-shifted proteins.  Shastri has previously shown convincing evidence that peptides derived from frame-shifted proteins can be recognized by T cells, but I always wondered if that was just a test-tube novelty. In this paper, though, Berger et al. argue that these frame-shifted potential targets show evidence of evolutionary selection, suggesting that they are recognized often enough to be a significant factor in the viral life-cycle.

• CD8 T cell response and evolutionary pressure to HIV-1 cryptic epitopes derived from antisense transcription. ⁵

And this is a very similar paper, showing the same thing for antisense-derived peptides. Like the frame-shifted proteins discussed above, these antisense proteins would probably be nonsense and rapidly degraded — defective ribosomal products, in other words — and again, there’s some evidence that these are under immunological selection, suggesting that this recognition is a real-world phenomenon.

These findings indicate that the HIV-1 genome might encode and deploy a large potential repertoire of unconventional epitopes to enhance vaccine-induced antiviral immunity.⁵

• The antiviral factor APOBEC3G improves CTL recognition of cultured HIV-infected T cells. ⁶

This is a particularly cool paper.⁷ We know that APOBEC3G — a host protein that evolved, apparently, to provide protection against infection with retroviruses such as HIV — acts by driving hypermutation of infecting retroviral genomes. HIV resists this effect through its protein vif, which in turn drives rapid degradation of several APOBECs.

But in spite of this vif-mediated protection, it’s probably true that APOBECs still have some effect on HIV, especially very early in an infection before vif can take them out; so there’s a background of mutation in HIV driven by APOBECs. This paper shows that APOBEC-driven mutation improves T cell recognition of HIV-infected cells, and the effect is probably because the mutations force HIV to make even more defective proteins, so that there are more T cell targets. This was done in rather an artificial system (mainly by either eliminating vif altogether, or by cranking up the levels of APOBEC3G artificially), so it’s not clear how important it would be in a natural infection.

I also wonder if this argues against the notion that DRiPs are normally a big factor, because if so the background of DRiP-derived peptides should be quite high and increasing it might not be a big factor; but that’s a quantitative issue that’s hard to deal with. Still, an interesting take on antiviral effects.

• Defective Ribosomal Products Are the Major Source of Antigenic Peptides Endogenously Generated from Influenza A Virus Neuraminidase ⁸

“Drips” (Inger Taylor)

This is the paper that most explicitly tests DRiPs, which is not surprising, since it comes from Jon Yewdell himself.⁹ The paper starts with quite a fair summary of the hypothesis’s status, including some of the problems with previous experiments:

In all of these studies, we used recombinant vaccinia viruses (VVs) to express SIINFEKL-containing source Ags. It is possible that we grossly overestimated the contribution of DRiPs to Ag processing in these studies due to the use of VV to express non-VV genes. We recently showed that differences in viral translation mechanisms can greatly increase the fraction of DRiPs; expression of influenza A virus (IAV) nuclear protein by an Alphavirus vector resulted in the defective translation of >50% of nuclear protein recovered from cells. VV expression is known to modify the Ag processing pathway of some inserted viral gene products compared with their natural infection context. Further, the fusion of multiple genes to create chimeric proteins can greatly decrease the fidelity of protein synthesis or protein folding …⁸

In an attempt to get around some of these problems, they tried to come up with a more natural system.  What they built is more natural, but still is fairly artificial (as they acknowledge); still, their findings did add more support to the basic idea. (As a sign that Jon has softened his position some in the past decade, their comment “Although DRiPs are clearly a major source of antigenic peptides, it is important to recognize that peptides are also generated from natural protein turnover” is one that I think all but the most hardened anti-DRiPers would agree with; it’s coming down to a question of quantitation, of what “major” actually means, rather than absolutes.)

I still suspect that there are cases where DRiPs are critical, and cases where they’re not particularly important, and I don’t have a good sense for how many instances of each there are. My gut feeling is about half and half, but it’s not something I’d defend with my life.

1. Yewdell, J. W., Aton, L. C., and Benink, J. R. (1996). Defective ribosomal products (DRiPs): A major source of antigenic peptides for MHC class I molecules? J. Immunol. 157, 1823-1826[↩]
2. Which has made it a bit of a moving target when it comes to disproving it, unfortunately[↩]
3. Cardinaud, S., Starck, S., Chandra, P., & Shastri, N. (2010). The Synthesis of Truncated Polypeptides for Immune Surveillance and Viral Evasion PLoS ONE, 5 (1) DOI: 10.1371/journal.pone.0008692[↩]
4. Berger, C., Carlson, J., Brumme, C., Hartman, K., Brumme, Z., Henry, L., Rosato, P., Piechocka-Trocha, A., Brockman, M., Harrigan, P., Heckerman, D., Kaufmann, D., & Brander, C. (2010). Viral adaptation to immune selection pressure by HLA class I-restricted CTL responses targeting epitopes in HIV frameshift sequences Journal of Experimental Medicine, 207 (1), 61-75 DOI: 10.1084/jem.20091808[↩]
5. Bansal, A., Carlson, J., Yan, J., Akinsiku, O., Schaefer, M., Sabbaj, S., Bet, A., Levy, D., Heath, S., Tang, J., Kaslow, R., Walker, B., Ndung’u, T., Goulder, P., Heckerman, D., Hunter, E., & Goepfert, P. (2010). CD8 T cell response and evolutionary pressure to HIV-1 cryptic epitopes derived from antisense transcription Journal of Experimental Medicine, 207 (1), 51-59 DOI: 10.1084/jem.20092060[↩][↩]
6. Casartelli, N., Guivel-Benhassine, F., Bouziat, R., Brandler, S., Schwartz, O., & Moris, A. (2009). The antiviral factor APOBEC3G improves CTL recognition of cultured HIV-infected T cells Journal of Experimental Medicine, 207 (1), 39-49 DOI: 10.1084/jem.20091933[↩]
7. I’m presenting this one on Friday in the Immunology Journal Club I run here.[↩]
8. Dolan, B., Li, L., Takeda, K., Bennink, J., & Yewdell, J. (2009). Defective Ribosomal Products Are the Major Source of Antigenic Peptides Endogenously Generated from Influenza A Virus Neuraminidase The Journal of Immunology, 184 (3), 1419-1424 DOI: 10.4049/jimmunol.0901907[↩][↩]
9. Interestingly, it looks as if Jon has turned his attention back to influenza viruses in the past year — he cut his teeth on influenza, quite a number of years back, but it hasn’t been his main focus for a while. I guess H1N1 gave him the excuse he needed to move back that way.[↩]

Ladies & Gentlemen, I give you The Fever Districts of the United States, as of 1856 (click for a larger version):

Note the outlining of the Intermittent Fever districts, including Lansing, MI, where I live. Note the intense yellow rim of Yellow Fever.  Note the Small Pox Measles Scarlatina Consumption Endemic region along the Eastern seaboard, the large-case TYPHUS, the DYSENTERY, the casual “And many epidemics” tacked on to the main Yellow Fever, the serpentine red band tracing cholera. ¹ There’s goitre in the Midwest and Mexico, elephantiasis down in South America, “Dia. & Dys. (severe)” in tiny writing down in the Bahamas, and the Bermudas are “generally healthy: Influenza, Rheumatism, Dysentery, Yellow Fever”.  And so much more.  (Compare to the map of Malaria in the USA, 1870.)

This amazing map is a mere afterthought, an inset of a map whose awesomeness goes up to 11.  The US map to the right² (again, click for a larger version) is still just a small fraction of the whole, and that’s not even mentioning the jaw-dropping charts and graphs, also inset, showing “Consumption: Proportion of Deaths in the different quarters of the Globe”, “Comparative Value of Life in Different Countries”, “Proportionate Mortality of European Residents in Foreign Countries” and still more and more.

This map is “The geographical distribution of health & disease, in connection chiefly with natural phenomena. (with) Fever districts of United States & W. Indies, on an enlarged scale,” and it’s from:

The physical atlas of natural phenomena
by Alexander Keith Johnston, F.R.S.E., F.R.G.S., F.G.S.
William Blackwood and Sons, Edinburgh and London, MDCCCLVI ³

I’d run across reverent mentions of this map — especially the Fever Districts inset — here and there in old books, and I just stumbled across it in downloadable form.  You must go at once to The David Rumsey Collection and pore over it for several hours, at the highest resolution.

1. Lansing seems to have been just barely cholera-free, at least in 1856.[↩]
2. The colors refer to “zones of disease” – Torrid (brown), Sub-torrid & temperate (green), sub-temperate & arctic (blue) [↩]
3. That’s 1856, for those of you who, like me, need to pause a while in thought when confronted with years in Roman numerals[↩]

“Drips” (Susan S. Roberts. 2007)

A while ago, I posted a brief extract from a 2008 paper from Drummond & Wilke¹ that determined that 18% of proteins have at least one misincorporated amino acid, and linked that to my commentary on Yewdell’s “DRiPs” hypothesis here (also see here for more commentary).  At the time, I took the 18% figure as surprisingly good support for Yewdell’s notion that a large fraction of proteins are misfolded (and therefore rapidly degraded) on translation.

Claus Wilke just gave a really interesting seminar here and I had a chance to chat with him about this paper and much more, so I want to clarify that he does not take this finding as support for (or disproof of) the DRiPs hypothesis.  Claus’s point, supported more strongly in subsequent papers,² is that proteins incorporate this level of aberrant amino acids without experiencing misfolding as a consequence.  He argues that genes have evolved enough robustness in their folding parameters that they can and do tolerate this level of mistranslation.  Since Yewdell’s hypothesis is based on mis-folding, I think Claus’s data are more or less neutral with regard to DRiPs.

1. Mistranslation-Induced Protein Misfolding as a Dominant Constraint on Coding-Sequence Evolution. D. Allan Drummond and Claus O. Wilke. Cell 134:341-352 (25 July 2008) doi:10.1016/j.cell.2008.05.042[↩]
2. For example:  The evolutionary consequences of erroneous protein synthesis. D. Allan Drummond & Claus O. Wilke.  Nature Reviews Genetics 10, 715-724 (October 2009) doi:10.1038/nrg2662[↩]