Mystery Rays from Outer Space

CTL (green) and HSV-infected cells (red) (from Akiko Iwasaki)

Last time I talked about herpesvirus immune evasion of cytotoxic T lymphocytes, I cautiously wondered if there might be a theme emerging: Do these genes mainly help the virus with latent infection?

Immune evasion of CTL seems to be pretty much universal among the millions of different herpesvirus species — at least, as far as I know, in every case where people have looked for it, the virus has some way of blocking antigen presentation. Although other virus families also block antigen presentation (HIV, some poxviruses, and human adenoviruses are probably the best known instances), immune evasion isn’t as universal among those other families.

For example, although human adenoviruses mostly have immune evasion function, adenoviruses of other species do not (as far as we can tell); and for that matter not even all human adenoviruses have the ability to block antigen presentation. What’s more, there is a trend for those non-herpesvirus viruses that do evade CTL, to also establish long-term latent or persistent infection.

A recent paper from Frank Carbone’s lab¹ offers a little more, indirect, support for this theme. They asked what CTL actually do to herpes simplex virus in the initial infection.

We usually blithely call CTL “antiviral lymphocytes”, but what exactly does that mean for specific virus infections? For example, I’ve previously pointed out experiments that show that CTL have more than one way of clearing away virus infections — they can use cytokine secretion as well as, or instead of, cell lysis, as their weapon, which opens up the opportunity for activity over a broad range rather than one cell at a time. In another example, Luis Sigal’s lab has shown that CTL can protect against extromelia (mousepox) infection at a very early stage, by blocking the virus’s spread from the skin to the liver, cutting them off at the bottleneck of the lymph through which the virus intially spreads.

On the other hand, herpes simplex virus often seems to do just fine even when CTL are present. The virus sets up an initial infection in the skin, and rapidly tracks up through neurons to ganglia, where it sets up a latent infection. By the time CTL are up and running, the virus is comfortably snuggled down in the neuron, shutting down all its protein expression to the point where CTL don’t see it very well. Even if you have an active CTL response already, the virus seems to be able to get in to the neurons and set up latency anyway.

So what do CTL do to herpes simplex? Carbone’s lab set up mice with and without specific anti-HSV CTL, and infected them with the virus. As you’d expect (and as has been shown lots of times in the past) the CTL markedly reduced the amount of virus replication and shedding, but did not prevent the virus from setting up a latent infection.

Though the presence of herpes-specific effector CD8+ T cells early during viral challenge attenuated the primary infection and prevented the development of disease, these cells failed to block the skin to nervous system transmission of the virus, and hence substantial latent infections were established in the face of this CTL immunity.

(My emphasis.) How come? Part of the answer seems to be that the CTL didn’t respond quite fast enough. ² Virus infects the skin, replicates, and moves up into neurons in about 24 hours. (If they surgically removed the infected skin prior to 24 hours after infection, neurons weren’t infected; if they removed the infected region more than 24 hours after infection, neurons were infected.) CTL, on the other hand, move into the infected region of skin within about 15 hours of infection. At this point the CTL start to shut down virus replication; but the window of opportunity, as you can see, is very narrow. The CTL need to shut down the virus in the skin very rapidly, and to very low levels, within just a few hours of entering the site of infection.

In fact, if you start off with a relatively small amount of virus, then CTL can shut the replication down enough to make a difference.  It’s mainly when there’s a lot of virus to start with that the CTL can’t get the virus down under some threshold level that allows efficient latent infection:

Thus, virus-specific CTL can reduce the average viral copy number of the residual latent infection, but this is only achievable when a substantial attenuation of the skin infection is observed.

There are a bunch of fascinating points that arise from this work. First, it helps account for the fact that superinfection with herpes simplex is actually quite rare — that is, if you’re infected with HSV already, then you’re unlikely to get re-infected with a second virus. Normal exposure to HSV probably is at a very low level, with only a handful of virions entering the skin; it’s more like the low-range infection in Carbone’s experiments than the high-range, where they put in some 10 million virions, and at the low range, CTL can move in and check the initial infection fast enough to make a difference.

Second, a critical point about these experiments is that they were done in the absence of CTL evasion. That’s because there experiments were done in mice, and the HSV immune evasion molecule ICP47 doesn’t work in mice, as opposed to in humans.

One of the puzzling things about immune evasion genes, to me, has been how ineffectual they seem to be in authentic infections. But these experiments suggest if your interest is in establishing latency, then immune evasion doesn’t need to be hugely effective: It just needs to keep the window open a crack for a few more hours, letting the virus replicate through the first wave of CTL invasion. If ICP47 can hold off the CTL for an extra 8 hours, then it’s probably done its job, allowing HSV to set up a latent infection and thus reactivate and infect new hosts on and off over the next 60 or 70 years.

So, even though this paper really didn’t look at immune evasion per se, I think it does offer some support for the concept that (for herpesviruses, anyway) immune evasion really isn’t for the acute infection at all.  Instead, it’s a mechanism to help the virus establish latent infection.

1. Wakim, L.M., Jones, C.M., Gebhardt, T., Preston, C.M., Carbone, F.R. (2008). CD8+ T-cell attenuation of cutaneous herpes simplex virus infection reduces the average viral copy number of the ensuing latent infection. Immunology and Cell Biology DOI: 10.1038/icb.2008.47[↩]
2. I wonder whether they miigh have seen a faster response if they had started with skin-specific T cells, though.[↩]

Cytotoxic T lymphocytes (CTL) are important destroyers of virus, recognizing MHC class I on infected cells and killing the cell before virus replication is complete. Unsurprisingly, many viruses (especially, but not only, the large and complex viruses like herpesviruses and adenoviruses) have evolved mechanisms to block MHC class I antigen processing and, therefore, T cell recognition.

What has been surprising is how ineffective these viral immune evasion mechanisms seem to be. (I’ve talked about this before in a bit more detail.) In cells in the incubator (which is where most of the experiments with the antigen presentation immune evasion molecules have been done), the effects often look pretty dramatic; but when you look at the immune response in humans infected with human cytomegalovirus¹ or herpes simplex virus² (both of which have formidable immune evasion functions in tissue culture³) there are pretty hearty and effective CTL responses, suggesting that the immune evasion doesn’t work all that well in vivo.

Most of the viruses that have had immune evasion genes identified are human viruses; that’s where the clinical interest and the money are. One problem with this is that the large DNA viruses that are likely to have these immune evasion functions tend to be pretty species-specific. Human herpesviruses and adenoviruses don’t infect mice well, if at all, and even if they do infect lab animals it’s not really clear how accurately this infection mimics the natural course in humans. There are a couple of lab animal herpesviruses that may be useful models (though even here the effects of immune evasion molecules are quite small.⁴) Still, it would be nice to understand what’s going on with immune evasion in, say, herpes simplex infection.

Herpes simplex virus actually will infect mice (the alphaherpesviruses tend to be somewhat less host-restricted than the beta or gammaherpesviruses), and while mouse infection isn’t exactly like that in humans it does seem to be similar enough to offer useful insights. Not into immune evasion, though, because the immune evasion gene of herpes simplex (called “ICP47″) works very poorly in mice.⁵ Recently, though, Chris Wilson’s group took an ingenious approach to looking at the question, and a theme may be starting to emerge.

Wilson reasoned that if ICP47 doesn’t work in mice,⁶ why not replace it with a functionally-similar gene that does? They made recombinant herpes simplex viruses that have immune evasion genes from mouse or human cytomegalovirus that block mouse MHC class I fairly well. These viruses are more neurovirulent in mice than the control viruses⁷ and this was because the CTL didn’t work as well in the recombinants. ^8,9

Their latest paper¹⁰ shows something new and exciting with the recombinant virus: It reactivates better from neurons.

Trigeminal ganglion

Remember that HSV establishes a latent infection in neurons and, in humans, periodically reactivates and may shed and infect new hosts this way. HSV latency is still a mysterious and little-understood field in spite of huge amounts of work over the years, but a host of recent findings have started to explain at least bits and pieces. It used to be felt that latent HSV didn’t express any proteins, and so should be invisible to the immune system. As I observed here, it’s now known that the immune system can detect HSV in neurons. That offers a mechanism for reactivation to be coupled with immune suppression. The Orr et al paper here offers more support for this concept, and suggests that one of the (most important?) functions for HSV immune evasion is to tip the scales a little further toward the virus being able to escape recognition in the neuron, and therefore being more easily able to reactivate and infect new hosts — something this ubiquitous virus does with extraordinary efficiency. Orr et al call this an “actively testing the waters” model, in which HSV constantly sticks its toes out into the stream of the immune response to see if it’s safe to reactivate.

What especially interesting about this is that we now have (that I know of) three models of MHC class I immune evasion in more or less authentic infections, and each case the immune evasion seems to be designed to support latency, reactivation, and transmission. Here we have HSV using immune evasion in latency and persistence. Ann Hill’s group has recently shown that mouse cytomegalovirus MHC class I immune evasion proteins help the virus persist in the salivary glands,¹¹ so that (as with the HSV story) it is more able to infect new hosts. And in the third model I know of, mouse herpesvirus 68, the MHC class I immune evasion genes are important for, you guessed it, latency and persistence.¹²

Three data points aren’t very many, but it does suggest that this may be a common feature of the herpesviruses. Perhaps in acute infection the virus doesn’t really “want” to block immune responses all the effectively, because their lifestyle involves only a brief burst of acute infection followed by a long-term latency; and it’s that latency that allows infection of new hosts. The viruses “let” themselves be driven into apparent submission, but then use their immune evasion functions to mount intermittent stealth campaigns to recruit new hosts.

If this is true for herpesviruses (and of course it’s just a hypothesis) is it also true for other viruses that have MHC class I immune evasion functions? Maybe for some (I wonder about the adenoviruses in particular, since they’re also very capable of long-term persistence in spite of immune responses) but I doubt it’s universal — HIV, for example, doesn’t obviously fit into the same lifestyle pattern as the herpesviruses, and yet if has a protein (nef) that targets MHC class I. Still, if offers a framework for thinking about the problem, which is very valuable.

1. Manley, T. J., Luy, L., Jones, T., Boeckh, M., Mutimer, H., and Riddell, S. R. (2004). Immune evasion proteins of human cytomegalovirus do not prevent a diverse CD8+ cytotoxic T-cell response in natural infection. Blood 104, 1075-1082.[↩]
2. Posavad, C. M., Koelle, D. M., and Corey, L. (1996). High frequency of CD8+ cytotoxic T-lymphocyte precursors specific for herpes simplex viruses in persons with genital herpes. J. Virol. 70, 8165-8168.[↩]
3. York, I. A., Roop, C., Andrews, D. W., Riddell, S. R., Graham, F. L., and Johnson, D. C. (1994). A cytosolic herpes simplex virus protein inhibits antigen presentation to CD8+ T lymphocytes. Cell 77, 525-535.
   and
   Jones, T. R., Hanson, L. K., Sun, L., Slater, J. S., Stenberg, R. M., and Campbell, A. E. (1995). Multiple independent loci within the human cytomegalovirus unique short region down-regulate expression of major histocompatibility complex class I heavy chains. J. Virol. 69, 4830-4841.[↩]
4. For example, Munks, M. W., Pinto, A. K., Doom, C. M., and Hill, A. B. (2007). Viral interference with antigen presentation does not alter acute or chronic CD8 T cell immunodominance in murine cytomegalovirus infection. J Immunol 178, 7235-7241.[↩]
5. Jugovic, P., Hill, A. M., Tomazin, R., Ploegh, H., and Johnson, D. C. (1998). Inhibition of major histocompatibility complex class I antigen presentation in pig and primate cells by herpes simplex virus type 1 and 2 ICP47. Journal of Virology 72, 5076-5084.[↩]
6. To be strictly accurate, ICP47 does work in mice, just not very well — 100 times less than in humans, maybe, though what the unit of comparison is or should be isn’t clear[↩]
7. I first wrote “than wild-type virus”, but I don’t think we know that — the process of recombination made the virus less virulent and the comparisons were done to this reduced-virulence control virus; I don’t see a direct comparison to wild-type virus.[↩]
8. Orr, M. T., Edelmann, K. H., Vieira, J., Corey, L., Raulet, D. H., and Wilson, C. B. (2005). Inhibition of MHC class I is a virulence factor in herpes simplex virus infection of mice. PLoS Pathog 1, e7.[↩]
9. This actually parallels and supports previous work showing that ICP47 does in fact have a small effect on neurovirulence in mice.[↩]
10. ORR, M., MATHIS, M., LAGUNOFF, M., SACKS, J., WILSON, C. (2007). CD8 T Cell Control of HSV Reactivation from Latency Is Abrogated by Viral Inhibition of MHC Class I. Cell Host & Microbe, 2(3), 172-180. DOI: 10.1016/j.chom.2007.06.013 [↩]
11. Lu, X., Pinto, A. K., Kelly, A. M., Cho, K. S., and Hill, A. B. (2006). Murine cytomegalovirus interference with antigen presentation contributes to the inability of CD8 T cells to control virus in the salivary gland. J Virol 80, 4200-4202.[↩]
12. Bennett, N. J., May, J. S., and Stevenson, P. G. (2005). Gamma-herpesvirus latency requires T cell evasion during episome maintenance. PLoS Biol 3, e120.[↩]

TcR interacting with artificial membrane1

Earlier this week I talked about the phenomenon of viruses that downregulate MHC class II. The “purpose” of this blockade is kind of unclear to me, because the immunity driven by MHC class II is not focused on the cell it’s attached to, but rather spills out broadly over a wide area; it seems that a virus would have to very rapidly infect a very large number of MHC class II-expresing cells to have much effect on the anti-viral immune system.

One possible explanation is that the downregulation may be only peripherally related to MHC class II-based immunity. Instead, the virus could be simply going about its cell-biological business and either targeting MHC class II as an accidental side-effect, or because of some function of MHC class II that isn’t directly related to immunity. Here’s a parallel case that may help think about the problem.

The paper is
Sullivan, B.M., Coscoy, L. (2007). Downregulation of the T-Cell Receptor Complex and Impairment of T-Cell Activation by Human Herpesvirus 6 U24 Protein. Journal of Virology, 82(2), 602-608. DOI: 10.1128/JVI.01571-07

The T-cell receptor (TcR) is what recognizes MHC class I or II. Human herpesvirus 6 (HHV6) infects T helper (CD4) cells and (depending on viral strain) also does a number of things to modulate the immune system.² Sullivan and Coscoy show here that the virus also down-regulates the TcR on infected T cells. (It’s altogether a more solid paper than the last one I mentioned, with nice experiments that directly show what’s happening to the TcR: “HHV-6 U24 protein inhibits CD3 recycling to the cell surface and, as a consequence, downregulates CD3 cell surface expression and prevents T-cell activation.“)

U24 blocks CD3ε access to Rab11 recycling endosomes.3

At first glance this raises exactly the same question as does Vpu’s effect on MHC class II. How does reducing TcR on infected cells benefit the virus? The infected T cell will be less able to recognize its target, but what are the odds that its target is HHV6? Pretty minimal; there are (at least) tens of billions of different TcRs and only a handful of them recognize any particular antigen. The virus might be causing generalized immune suppression if it infects a large fraction of the T cells, but that’s not a particular benefit for the virus. If HHV6 specifically infected cells with TcRs that are specific for the virus then this would be a targeted immune evasion technique, but as far as I know there’s no evidence for this, nor is there an obvious mechanism by which HHV6 could target antigen-specific CD4 T cells.

There is, however, a nice explanation for TcR downregulation that doesn’t involve direct effects on T cell recognition. HHV6 (like all herpesviruses) has two choices when it infects a cell. It can either enter lytic replication — replicating the genome, producing more viruses, and eventually destroying the infected cell — or enter latency — a long-term, perhaps life-long, infection with minimal protein expression and little if any effect on the infected cell. As with any virus, the more prepared a cell is to replicate, the easier it is for a virus to replicate it’s own genome. T cells that receive a signal through their TcR become activated⁴ and divide very rapidly. In this environment, it’s very easy for HHV6 to replicate — that is, to enter lytic replication and kill the cell, releasing more viruses into the system.

Human herpesvirus 6

Probably HHV6 downregulates the TcR “because” it prevents its host from becoming activated by whatever its random antigen is. That prevents the virus from entering lytic replication and allows it to enter a persistent state, where it can hang about and await the best opportunity to infect a new person.

I know of one other viral protein that downregulates the TcR — the herpesvirus saimiri Tip protein ⁵ — and there seems to be controversy⁶ over whether this protein activates T cell signalling (potentially driving the virus into lytic replication) or blocks it (preventing lytic replication and facilitating persistence). ⁷ The original paper describing the TcR downregulation found that Tip blocked signaling, and proposed the same explanation as Sullivan and Coscoy:

… these associations ultimately block lymphocyte receptor signal transduction. … these interactions likely play an important role in the establishment and maintenance of HVS persistent infection by protecting infected cells from surveillance by the immune system. In fact, animals infected with recombinant HVSΔTip have been shown to have higher levels of cell-associated infectious virus titer compared to other recombinant HVS.

So in this case the downregulation of the TcR (a quintessentially immune molecule) apparently isn’t directly related to immune evasion, but is a way of switching between the lytic and the persistent lifestyles. (It’s also a reminder of the fairly obvious point that we shouldn’t think of viruses as blind replicators, desiring nothing more than maximal replication. At least some viruses have a range of lifestyle options, and can switch between them quite comfortably.)

I still don’t see a direct analogy to the MHC class II downregulation imposed by the HIV Vpu protein. but it’s an example of why we shouldn’t get too focused on single causes and single functions. Life is more complicated than that.

1. By Raghuveer Parthasarath, then in the Groves lab[↩]
2. Lusso, P., 2006. HHV-6 and the immune system: mechanisms of immunomodulation and viral escape. Journal of Clinical Virology, 37(Supplement 1), p.S4-S10. doi:10.1016/S1386-6532(06)70004-X [↩]
3. Sullivan & Coscoy, Fig 4[↩]
4. I am simplifying immensely![↩]
5. Park, J. et al., 2002. Herpesviral Protein Targets a Cellular WD Repeat Endosomal Protein to Downregulate T Lymphocyte Receptor Expression. Immunity, 17(2), p.221-233. doi:10.1016/S1074-7613(02)00368-0 [↩]
6. Brinkmann, M.M. & Schulz, T.F., 2006. Regulation of intracellular signalling by the terminal membrane proteins of members of the Gammaherpesvirinae. J Gen Virol, 87(5), p.1047-1074. DOI 10.1099/vir.0.81598-0[↩]
7. I’m more convinced by the argument for blocking signaling, but only because of the very bad reason that I know the people involved. I haven’t looked at the papers pro and con very carefully.[↩]