PKR is a sentinel kinase constitutively expressed in all cells as an inactive protein that is subsequently activated by virus RNA produced during an infection. The active kinase perturbs virus replication by phosphorylating protein substrates in the cell. RNA helicase A (RHA) is a novel substrate for PKR. Viruses usurp this helicase to replicate their own genome. Phosphorylation of RHA by PKR perturbs the ability of the helicase to bind virus RNA. Correspondingly, PKR prevents the capacity of RHA to enhance expression of genetic elements encoded by the human immunodeficiency virus (HIV). In addition, HIV virions packaged within cells that also express protein fragments of RHA have enhanced infectivity. These fragments of RHA occur within a protein domain previously established to bind RNA but increasingly recognized to mediate protein–protein interactions. This supports an emerging role for these protein domains to coordinate the cell’s response to pathogen-associated RNA. The findings identify a new cell-signaling pathway important in the response to virus infection.
An Antiviral Response Directed by PKR Phosphorylation of the RNA Helicase A. 2009 PLoS Pathog 5(2): e1000311
The double-stranded RNA-activated protein kinase R (PKR) is a key regulator of the innate immune response. Activation of PKR during viral infection culminates in phosphorylation of the α subunit of the eukaryotic translation initiation factor 2 (eIF2α) to inhibit protein translation. A broad range of regulatory functions has also been attributed to PKR. However, as few additional PKR substrates have been identified, the mechanisms remain unclear. Here, PKR is shown to interact with an essential RNA helicase, RHA. Moreover, RHA is identified as a substrate for PKR, with phosphorylation perturbing the association of the helicase with double-stranded RNA (dsRNA). Through this mechanism, PKR can modulate transcription, as revealed by its ability to prevent the capacity of RHA to catalyze transactivating response (TAR)–mediated type 1 human immunodeficiency virus (HIV-1) gene regulation. Consequently, HIV-1 virions packaged in cells also expressing the decoy RHA peptides subsequently had enhanced infectivity. The data demonstrate interplay between key components of dsRNA metabolism, both connecting RHA to an important component of innate immunity and delineating an unanticipated role for PKR in RNA metabolism.
Kaposi’s sarcoma (KS) is a multifocal tumour only found in a few groups of people, including elderly Mediterranean men, individuals in Africa and patients with immune disorders. The tumours arise from the formation of new blood or lymphatic vessels (angiogenesis or lymphangiogenesis) due to the proliferation of endothelial cells. In 1994 Chang and Moore identified a new virus, Kaposi’s sarcoma-associated herpesvirus (KSHV) as the cause of these tumours.
Unlike other herpesviruses, the seroprevalence of KSHV is not ubiquitous, perhaps only 5% in those countries with low KSHV rates such as the USA and Northern Europe). Like all herpesviruses, KSHV infection persists for the life of the host and can enter either of two states: latency or lytic reactivation. In latency, the minimum number of viral genes is expressed to maintain the virus genome in dividing cells, evading immune detection. Lytic reactivation occurs when the virus re-enters productive replication to generate new progeny, lysing the host cell in the process. And like all herpesviruses KSHV likes to mess with the immune system of its host. The KSHV genome contains 86 genes, almost a quarter of which encode proteins with immunoregulatory activities such as T- and B-cell function, complement activation, the innate antiviral interferon response and natural killer cell activity. Many of these gene are homologues of cellular proteins.
The KSHV proteins MIR1 and MIR2 ubiquitinate the cytoplasmic tail of MHC-I which triggers endocytosis and proteasomal degradation. This protects KSHV-infected cells from NK-mediated lysis. MIR2 can also down-regulate other components of the immune synapse, ICAM (CD54) and PECAM (CD31) by the same mechanism.
The KSHV vOX2 protein causes the cellular CD200 receptor to deliver an inhibitory signal to granulocytes, although the mechanism by which this acts is not yet well defined.
The KCP protein is present on the surface of KSHV virions and infected cells and protects them from complement attack by accelerating the decay of the classical pathway C3 convertase enzyme complex.
The K15 protein activates MAP kinases and this affects immune function.
KSHV encodes a family of 12 miRNAs. These regulate both B- and T-cell function.
The K1 protein reduces the presence of B cell receptors on the surface of B cells and interferes with the production of cytokines, and inhibits apoptosis.
The MIR2 protein down-regulates tetherin, which is involved in normal B-cell differentiation.
Three KSHV chemokine homologues (vCCL1–3) have affinity for chemokine receptors (CCRs) and this affects T-cell responses.
KSHV proteins inhibit interferon pathways.
Since KSHV infection results in lifelong persistence of the virus, these immunomodulation activities are clearly successful in preventing its elimination by the immune system. Many questions about KSHV infection remain unanswered, but we have learned valuable lessons about the normal function of the immune system through studying this virus.
HIV is evolving rapidly to escape the human immune system. Researchers have shown HIV is able to adapt rapidly to counter human genes controlling immune system molecules that can target it for destruction. Progression to AIDS is tied to genes which control production of key immune system molecules called human leucocyte antigens (HLAs). Humans differ in the HLA genes they have, and even small differences can have a big impact on how quickly AIDS develops. Researchers found mutations that enabled HIV effectively to neutralise the effect of a particular HLA gene were more frequent in populations with a high prevalence of that specific gene.
Adaptation of HIV-1 to human leukocyte antigen class I. Nature, 25 February 2009
The rapid and extensive spread of the human immunodeficiency virus (HIV) epidemic provides a rare opportunity to witness host–pathogen co-evolution involving humans. A focal point is the interaction between genes encoding human leukocyte antigen (HLA) and those encoding HIV proteins. HLA molecules present fragments (epitopes) of HIV proteins on the surface of infected cells to enable immune recognition and killing by CD8+ T cells; particular HLA molecules, such as HLA-B*57, HLA-B*27 and HLA-B*51, are more likely to mediate successful control of HIV infection1. Mutation within these epitopes can allow viral escape from CD8+ T-cell recognition. Here we analysed viral sequences and HLA alleles from >2,800 subjects, drawn from 9 distinct study cohorts spanning 5 continents. Initial analysis of the HLA-B*51-restricted epitope, TAFTIPSI (reverse transcriptase residues 128–135), showed a strong correlation between the frequency of the escape mutation I135X and HLA-B*51 prevalence in the 9 study cohorts (P = 0.0001). Extending these analyses to incorporate other well-defined CD8+ T-cell epitopes, including those restricted by HLA-B*57 and HLA-B*27, showed that the frequency of these epitope variants (n = 14) was consistently correlated with the prevalence of the restricting HLA allele in the different cohorts (together, P < 0.0001), demonstrating strong evidence of HIV adaptation to HLA at a population level. This process of viral adaptation may dismantle the well-established HLA associations with control of HIV infection that are linked to the availability of key epitopes, and highlights the challenge for a vaccine to keep pace with the changing immunological landscape presented by HIV.
The discovery of a previously unknown mechanism of immunity may lead to a better way to protect vulnerable children and adults against Streptococcus pneumoniae (pneumococcus) infection. The findings may aid the development of novel pneumococcal vaccines that would be less expensive and cover a greater number of known pneumococcal strains than that currently available. Pneumococcus causes serious infections in children and the elderly, including pneumonia and meningitis (inflammation of the brain). Since 2000, U.S. infants have been routinely immunized against pneumococcus, but most developing countries (where nearly one million children die from pneumococcal infections annually) cannot afford the existing vaccine. Researchers have been studying how natural immunity against pneumococcus develops, and have shown that in addition to antibodies, T-cells can provide broad protection against this pathogen. This new study identifies the specific protective T-cells – so-called TH17 cells – and show that they protect against infection by releasing IL-17, a protein that enables human blood cells to kill pneumococcus in the nose more efficiently. This is significant, since colonizing a person’s nose is the first necessary step of infection. Researchers knew that as children get older, they carry pneumococcus in the nose for shorter periods of time and have less risk of disease, but it hadn’t been known how this resistance develops. This works shows that adults and older children, but not newborn babies, have TH17 cells that target pneumococci, suggesting that exposure to pneumococcus normally leads to production of these cells. In mice, they show directly that exposure to pneumococcus triggers the development of these T cells and shortens the duration of nasal carriage of the pathogen. The investigators also describe an efficient way of measuring TH17 cells, which could help determine whether a new vaccine is rallying an effective response. A vaccine that induces both protective antibodies and T-cell immunity to pneumococcus may be a very effective way to protect against this potentially devastating disease.
Interleukin-17A Mediates Acquired Immunity to Pneumococcal Colonization. 2008 PLoS Pathog 4(9): e1000159
Although anticapsular antibodies confer serotype-specific immunity to pneumococci, children increase their ability to clear colonization before these antibodies appear, suggesting involvement of other mechanisms. We previously reported that intranasal immunization of mice with pneumococci confers CD4+ T cell–dependent, antibody- and serotype-independent protection against colonization. Here we show that this immunity, rather than preventing initiation of carriage, accelerates clearance over several days, accompanied by neutrophilic infiltration of the nasopharyngeal mucosa. Adoptive transfer of immune CD4+ T cells was sufficient to confer immunity to naïve RAG1-/- mice. A critical role of interleukin (IL)-17A was demonstrated: mice lacking interferon-gamma or IL-4 were protected, but not mice lacking IL-17A receptor or mice with neutrophil depletion. In vitro expression of IL-17A in response to pneumococci was assayed: lymphoid tissue from vaccinated mice expressed significantly more IL-17A than controls, and IL-17A expression from peripheral blood samples from immunized mice predicted protection in vivo. IL-17A was elicited by pneumococcal stimulation of tonsillar cells of children or adult blood but not cord blood. IL-17A increased pneumococcal killing by human neutrophils both in the absence and in the presence of antibodies and complement. We conclude that IL-17A mediates pneumococcal immunity in mice and probably in humans; its elicitation in vitro could help in the development of candidate pneumococcal vaccines.
Successful replication of a virus within a host cell requires a remarkable and complex series of interactions between the virus and host, starting with recognition of the cell receptor and ending with the release of progeny virions. Viruses utilize many cellular components in order to replicate, everything from cellular enzymes and transcription factors to membranes and organelles such as ribosomes. However, cells do not sit idly and allow themselves to be hijacked by viruses. Apoptosis is a genetically and biochemically controlled process of cell death that functions in development and homeostasis of multicellular organisms through selective removal of unwanted or damaged cells. Apoptosis also provides a potent antiviral response, and limits both the time and cellular machinery available for virus replication.
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To prolong cell viability and facilitate their own replication, viruses have evolved multiple mechanisms to inhibit the host apoptotic response. Cellular proteases such as caspases and serine proteases are instrumental in promoting apoptosis. Thus, these enzymes are logical targets for virus-mediated modulation to suppress cell death. There are at least four major classes of virus caspase inhibitors:
Serpins
p35 family members
Inhibitor of apoptosis proteins
Virus FLICE-inhibitory proteins
Viruses also subvert activity of the serine proteases, granzyme B and a range of other targets to avoid cell death before replication can occur. However, although viruses go to some lengths to suppress apoptosis, some viruses actually utilize caspases during replication to aid virus maturation, particle release, or both. Little is known regarding how caspase activity is positively regulated while still limited to allow replication, the temporal relationships between pro- and antiapoptotic signals are poorly understood.
A multifaceted relationship clearly exists between viruses and the apoptotic response they induce. Examination of these interactions contributes to our understanding of both virus pathogenesis and the regulation of apoptotic enzymes in normal cellular functions.
Viral Subversion of Apoptotic Enzymes: Escape from Death Row. Annual Review of Microbiology, 62: October 2008doi:10.1146/annurev.micro.62.081307.163009
To prolong cell viability and facilitate replication, viruses have evolved multiple mechanisms to inhibit the host apoptotic response. Cellular proteases such as caspases and serine proteases are instrumental in promoting apoptosis. Thus, these enzymes are logical targets for virus-mediated modulation to suppress cell death. Four major classes of viral inhibitors antagonize caspase function: serpins, p35 family members, inhibitor of apoptosis proteins, and viral FLICE-inhibitory proteins. Viruses also subvert activity of the serine proteases, granzyme B and HtrA2/Omi, to avoid cell death. The combined efforts of viruses to suppress apoptosis suggest that this response should be avoided at all costs. However, some viruses utilize caspases during replication to aid virus protein maturation, progeny release, or both. Hence, a multifaceted relationship exists between viruses and the apoptotic response they induce. Examination of these interactions contributes to our understanding of both virus pathogenesis and the regulation of apoptotic enzymes in normal cellular functions.
The cytotoxin nitric oxide (NO) can be converted by bacteria to the relatively harmless nitrous oxide (N2O). In this article in Microbiology Today, David Richardson, Andrew Thomson and Nicholas Watmough describe how this envirotoxin causes different problems.
NO is one of the most versatile and important molecules in living organisms. In higher animals and plants it is an important signalling molecule, for example it is the effector responsible for stimulating the dilation of blood vessels. However, it is also a potent cytotoxin and specialized cells called macrophages produce NO as part of a generalized response to invasion by pathogenic bacteria. Such bacteria have evolved a number of enzymic systems to defend themselves against this “gas attack”. Soil bacteria which can denitrify also need to protect themselves from the autotoxic effects of NO produced through their own metabolism.
Gastroenteritis is a common disease in both developed and developing countries. The two main causes of this affliction are bacteria and viruses. One of the primary viruses implicated in gastroenteritis has been shown to be noroviruses, which include Norwalk virus, notorious for numerous recent outbreaks where people live close together, such as on cruise ships, nursing homes, military bases and schools and hospital wards. Antibiotics are ineffective, because they fight bacteria, not viruses. Only recently have scientists been able to grow noroviruses in the laboratory and study them.
Scientists have just identified the primary immune sensor that detects the presence of noroviruses in the body. They found that the sensor – a protein called melanoma differentiation associated protein-5 (MDA-5) – triggers an immune response that increases up the body’s defenses to fight off the infection. This knowledge may help develop a treatment that prevents or reduces infection. MDA-5 is the primary sensor for norovirus infection, but the body’s ability to detect the virus is so important that it doesn’t just rely on one sensor. Another protein sensor – Toll-like receptor 3 (TLR3) – serves as a back-up and there may be others that have not yet been discovered.
The team demonstrated their work in mice but says the same proteins are likely to be responsible for detecting norovirus infection in humans. MDA-5, and to a lesser extent, TLR3, respond by causing other cells to release interferon, which shuts down production of the virus and initiates a full-scale immune attack. MDA-5 and TLR3 are both intracellular proteins. The researchers suspected that these two proteins may be important in detecting noroviruses because they are known to be important in recognizing similar types of viral infections. Cells that lack the MDA-5 protein do not mount an appropriate immune response against norovirus infection. The team then investigated two groups of mice – one group was bred without the ability to produce MDA-5 and the other was bred to lack TLR3. Again, both groups of mice had a defective immune response against noroviruses. In particular, mice without MDA-5 had higher levels of norovirus in their bodies and a defect in the ability to signal other immune cells to respond. Mice that lacked TLR3 also had a decreased response to norvirus infection. Interestingly, some people have common variations of the MDA-5 gene that could make them more susceptible to norovirus infection. Future norovirus treatments could be especially helpful to these individuals. These findings allow us to better understand the pathogenesis of norovirus infection and may provide clues for controlling the disease.
MDA-5 Recognition of a Murine Norovirus. 2008 PLoS Pathog 4(7): e1000108
Noroviruses are important human pathogens responsible for most cases of viral epidemic gastroenteritis worldwide. Murine norovirus-1 (MNV-1) is one of several murine noroviruses isolated from research mouse facilities and has been used as a model of human norovirus infection. MNV-1 infection has been shown to require components of innate and adaptive immunity for clearance; however, the initial host protein that recognizes MNV-1 infection is unknown. Because noroviruses are RNA viruses, we investigated whether MDA5 and TLR3, cellular sensors that recognize dsRNA, are important for the host response to MNV-1. We demonstrate that MDA52/2 dendritic cells(DC) have a defect in cytokine response to MNV-1. In addition, MNV-1 replicates to higher levels in MDA52/2 DCs as well as in MDA52/2 mice in vivo. Interestingly, TLR32/2 DCs do not have a defect in vitro, but TLR32/2 mice have a slight increase in viral titers. This is the first demonstration of an innate immune sensor for norovirus and shows that MDA5 is required for the control of MNV-1 infection. Knowledge of the host response to MNV-1 may provide keys for prevention and treatment of the human disease.
Antigenic variation is one of the most elegant systems that have evolved to evade host immune defenses. The surface of Trypanosoma brucei, a unicellular parasite that lives in the bloodstream of its mammalian host, is coated with glycoprotein (VSGs) molecules. To evade elimination by the immune system, this parasite replaces its coat with one tailored from another glycoprotein variant. Even though there are hundreds of VSG genes in the genome, this process, called antigenic variation, works because all are silenced except for the one that encodes the current coat. In this week’s issue of PLoS Biology, new research shows show that the chromatin modifying enzyme DOT1B helps to epigenetically regulate the number of VSGs each parasite can have at a time and how fast each parasite can switch from one coat to another. Post-transcriptional histone modifications play important roles in the regulation of chromatin structure and gene expression. Unlike acetylation, which is in general associated with transcription activation, histone methylation can activate or repress transcription depending upon the genomic location and the position of the modified amino acid in the histone chain. In cells lacking DOT1B, silent VSG genes become partially active and the switch from one VSG to another slows down, allowing two different VSGs to appear on the surface of an individual parasite at the same time. This study reveals the importance of epigenetics in regulating VSG genes and provide new insights toward the understanding of this unique survival device.
A histone methyltransferase modulates antigenic variation in African trypanosomes. 2008 PLoS Biol 6(7): e161
To evade the host immune system, several pathogens periodically change their cell-surface epitopes. In the African trypanosomes, antigenic variation is achieved by tightly regulating the expression of a multigene family encoding a large repertoire of variant surface glycoproteins (VSGs). Immune evasion relies on two important features: exposing a single type of VSG at the cell surface and periodically and very rapidly switching the expressed VSG. Transcriptional switching between resident telomeric VSG genes does not involve DNA rearrangements, and regulation is probably epigenetic. The histone methyltransferase DOT1B is a nonessential protein that trimethylates lysine 76 of histone H3 in Trypanosoma brucei. Here we report that transcriptionally silent telomeric VSGs become partially derepressed when DOT1B is deleted, whereas nontelomeric loci are unaffected. DOT1B also is involved in the kinetics of VSG switching: in DDOT1B cells, the transcriptional switch is so slow that cells expressing two VSGs persist for several weeks, indicating that monoallelic transcription is compromised. We conclude that DOT1B is required to maintain strict VSG silencing and to ensure rapid transcriptional VSG switching, demonstrating that epigenetics plays an important role in regulating antigenic variation in T. brucei.
A University of Leicester researcher has discovered how a protein in the blood linked to defence against meningitis plays a more vital role than previously understood in the body’s immune defence system. The published research has helped to advance medical understanding of how the body defends against disease and heals itself. The study also reveals that the same protein, properdin – discovered half a century ago – can also harm internal organs under certain circumstances. Lack of the protein in the human body has previously been linked to susceptibility to meningitis. But the new findings by Cordula Stover of the Department of Infection, Immunity and Inflammation at the University of Leicester assign hitherto unappreciated importance to this protein of the immune defence. Dr Stover, a Lecturer in Immunology, said:
I have a broad interest in immune mechanisms of health and disease, though recently, I have focused on a particular component of the first line immune defence, a protein called properdin. Properdin deficiency in families, though rare, predisposes people to develop meningococcal meningitis, usually with poor outcome of the infection. I hypothesised that the importance of properdin extends beyond this particular infectious disease, and that indeed it is an important player in health generally, and that its importance becomes apparent in conditions involving both acute and chronic states of inflammation.
Now Dr Stover’s paper published in the Journal of Immunology demonstrates that properdin plays a significant role in the survival of conditions relating to surgical perforation of the gut and activation of the immune system by wall components of bacteria. In conditions relating to multi-organ dysfunction, a complication which can occur in response to severe sepsis, properdin however aggravates organ damage.
So far, the system properdin is a part of – the so-called complement system – is classified as a first line, innate, acutely effective immune activation mechanism. This work shows that the activity of properdin extends beyond the acute phase and, importantly, that properdin is stepping onto the stage as an important player in different inflammatory conditions. As the worldwide burden of chronic inflammatory disease increases, it is of practical relevance to understand the contribution of this immune protein.
PKR is a sentinel kinase constitutively expressed in all cells as an inactive protein that is subsequently activated by virus RNA produced during an infection. The active kinase perturbs virus replication by phosphorylating protein substrates in the cell. RNA helicase A (RHA) is a novel substrate for PKR. Viruses usurp this helicase to replicate their own genome. Phosphorylation of RHA by PKR perturbs the ability of the helicase to bind virus RNA. Correspondingly, PKR prevents the capacity of RHA to enhance expression of genetic elements encoded by the human immunodeficiency virus (HIV). In addition, HIV virions packaged within cells that also express protein fragments of RHA have enhanced infectivity. These fragments of RHA occur within a protein domain previously established to bind RNA but increasingly recognized to mediate protein–protein interactions. This supports an emerging role for these protein domains to coordinate the cell’s response to pathogen-associated RNA. The findings identify a new cell-signaling pathway important in the response to virus infection.
Kaposi’s sarcoma (KS) is a multifocal tumour only found in a few groups of people, including elderly Mediterranean men, individuals in Africa and patients with immune disorders. The tumours arise from the formation of new blood or lymphatic vessels (angiogenesis or lymphangiogenesis) due to the proliferation of endothelial cells. In 1994 Chang and Moore identified a new virus, Kaposi’s sarcoma-associated herpesvirus (KSHV) as the cause of these tumours.
