Human papillomaviruses (HPVs) are DNA viruses associated with major human cancers. As such there is a strong interest in developing new means, such as vaccines and microbicides, to prevent HPV infections. Developing the latter requires a better understanding of the infectious life cycle of HPVs. The HPV infectious cycle is closely linked to the differentiation state of the stratified epithelium it infects, with progeny virus only made in the terminally differentiating suprabasal compartment. It has long been recognized that HPV must first establish its infection within the basal layer of stratified epithelium, but why this is the case has not been understood. In part this restriction might reflect specificity of expression of entry receptors. However, this hypothesis could not fully explain the differentiation restriction of HPV infection, since many cell types can be infected with HPVs in monolayer cell culture.
Chemical biology approaches have been used to reveal that cell cycle progression through mitosis is critical for HPV infection. Using infectious HPV16 particles containing the intact viral genome, G1-synchronized human keratinocytes as hosts, and early viral gene expression as a readout for infection, researchers learned that the recipient cell must enter M phase (mitosis) for HPV infection to take place. Late M phase inhibitors had no effect on infection, whereas G1, S, G2, and early M phase cell cycle inhibitors efficiently prevented infection. They concluded that host cells need to pass through early prophase for successful onset of transcription of the HPV encapsidated genes. These findings provide one reason why HPVs initially establish infections in the basal compartment of stratified epithelia. Only this compartment of the epithelium contains cells progressing through the cell cycle, and therefore it is only in these cells that HPVs can establish their infection. By defining a major condition for cell susceptibility to HPV infection, these results also have potentially important implications for HPV control.
Establishment of Human Papillomavirus Infection Requires Cell Cycle Progression. 2009 PLoS Pathog 5(2): e1000318
Human papillomaviruses (HPV), which comprise more than 100 genotypes, are the most prevalent sexually transmitted infection and are associated with multiple human cancers including all cervical cancers, many other anogenital cancers, and 25% of head and neck cancers. The HPV life cycle is closely linked to epithelial differentiation of skin keratinocytes, with initial infection occurring only in the undifferentiated proliferating basal compartment of the epithelium and progeny virus production only in the terminally differentiated suprabasal compartment. So far, little is known about how host cells restrict the HPV life cycle to specific stages of skin cell development. Here, by identifying small molecule inhibitors of HPV infection, we discovered that cell cycle progression through mitosis is critical for the establishment of HPV infection. In addition, our further chemical genetic dissection of this process showed that early steps of mitosis are required for HPV infection and early gene expression. Our findings provide one reason why HPV only infects undifferentiated proliferating cells and provide new leads for the development of preventive and therapeutic strategies against HPV infection.
One of my sons asked me this question a few days ago. I said no, but I’m happy that others agree with me:
Ten reasons to exclude viruses from the tree of life. 2009 Nature Reviews Microbiology 7: 306-311
When viruses were discovered, they were accepted as missing links between the inert world and living organisms. However, this idea was soon abandoned as information about their molecular parasitic nature accumulated. Recently, the notion that viruses are living organisms that have had a role in the evolution of some essential features of cells has experienced a renaissance owing to the discovery of unusually large and complex viruses that possess typical cellular genes. Here, we contend that there is strong evidence against the notion that viruses are alive and represent ancient lineages of the tree of life.
Viruses do not reproduce by division, but are replicated by the self-assembly of preformed components. This, not size, differentiates them from cellular living organisms such as bacteria. A virus-infected cell is more like a car factory than a womb.
Also, unlike living organisms, no virus has the means of generating its own energy – they are all energy pirates.
Lentiviruses are mammalian retroviruses known to infect cattle, cats, horses, sheep, and primates. They are the focus of intense study due to their causative association with AIDS in human. Although our knowledge on the origin and early evolution of HIV has grown exponentially over the past few years, much remains unresolved about the deeper relationships between primate and non-primate lentiviruses, the origin of lentiviruses, and their mode of structural evolution over long periods of evolutionary time. This is because these viruses evolve extremely rapidly, in a conflicting relationship with their hosts, and while their high mutation rate provides a wealth of information documenting their recent history, it also quickly erases evidence of their deeper ancestry. The lifecycle of retroviruses is atypical compared to other viruses in that after appropriate receptor recognition and entry in a specific cell type, their RNA genome is reverse transcribed into double-stranded DNA and integrated into the host genome as a provirus. Occasionally this process can take place in the host germline, and the integrated copy, also called endogenous retrovirus (ERV), may be transmitted vertically from parent to offspring and reach fixation in the host population. As such, ERVs constitute a fossil record of past viral infections that potentially provide an alternative way of gaining insights into the deep evolutionary history of present day exogenous retroviruses.
Although many ERVs have been characterized in mammals (e.g. 8% of the human genome), apparently very few derive from lentiviruses. Two reasons have traditionally been put forward to explain their absence in mammalian genomes: (i) they are of relatively recent evolutionary origin and endogenization has not yet commonly occurred, and/or (ii) they were not able to enter germ cells because of a very specific cell tropism. Recently however, an endogenous lentivirus called RELIK has been identified in the genome of rabbits and hares, whose germline integration was dated at least 12 millions years old. This discovery not only showed that lentiviruses were able to infiltrate mammalian germlines, but also demonstrated that this group of viruses is probably much older than what could previously be inferred based on sequence comparison of extant exogenous lentiviruses. New research now shows that a retrovirus related to HIV became stably integrated into the genomes of lemurs around 4.2 million years ago. The discovery of prosimian immunodeficiency virus (pSIV) offers new insights into the evolution of lentiviruses.
Based on “fossil” sequences collected from different lemur species, the researchers computationally reconstructed an apparently intact and complete DNA sequence for the ancestral prosimian lentivirus. The discovery that two different species of lemurs endemic to Madagascar suffered, independently and quasi-simultaneously, multiple germline infections of pSIV provides evidence that lentiviruses have repeatedly infiltrated the germline of prosimian species. These findings should allow future functional analysis of the extinct virus and advance our understanding of the biology of lentiviruses, including HIV. In addition, the characterization of this ancient lentivirus in lemurs raises the possibility that HIV-like retroviruses are still circulating today in the mammalian fauna of Madagascar.
Parallel Germline Infiltration of a Lentivirus in Two Malagasy Lemurs. 2009 PLoS Genet 5(3): e1000425
Retroviruses normally infect the somatic cells of their host and are transmitted horizontally, i.e., in an exogenous way. Occasionally, however, some retroviruses can also infect and integrate into the genome of germ cells, which may allow for their vertical inheritance and fixation in a given species; a process known as endogenization. Lentiviruses, a group of mammalian retroviruses that includes HIV, are known to infect primates, ruminants, horses, and cats. Unlike many other retroviruses, these viruses have not been demonstrably successful at germline infiltration. Here, we report on the discovery of endogenous lentiviral insertions in seven species of Malagasy lemurs from two different genera – Cheirogaleus and Microcebus. Combining molecular clock analyses and cross-species screening of orthologous insertions, we show that the presence of this endogenous lentivirus in six species of Microcebus is the result of one endogenization event that occurred about 4.2 million years ago. In addition, we demonstrate that this lentivirus independently infiltrated the germline of Cheirogaleus and that the two endogenization events occurred quasi-simultaneously. Using multiple proviral copies, we derive and characterize an apparently full length and intact consensus for this lentivirus. These results provide evidence that lentiviruses have repeatedly infiltrated the germline of prosimian species and that primates have been exposed to lentiviruses for a much longer time than what can be inferred based on sequence comparison of circulating lentiviruses. The study sets the stage for an unprecedented opportunity to reconstruct an ancestral primate lentivirus and thereby advance our knowledge of host–virus interactions.
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.
The press was buzzing with speculation about the chances of a vaccine to prevent diabates. Very good news for diabetics? Well not so fast. Before we look at the science, let me tell you two things about myself. First, I have two close relatives who are affected by diabetes, so this is a disease I care a lot about. Second, I’ve been in the virology business a long time – and we’ve been here before.
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The new paper claimed to have detected “enterovirus capsid protein vp1″ in 44 out of 72 pancreases from children who had died of type-1 diabetes shortly after becoming ill, but in only three out of 50 neonatal and paediatric normal control specimens. Statistically there is a strong correlation in this study between diabetes and the presence of the virus protein, but a correlation does not indicate a cause. Are diabetics more susceptible to enterovirus infection? We don’t know. While it’s not ethically possible to satisfy Koch’s postulates in humans, we need to be very careful in inferring from small scale studies such as this one:
The microorganism must be found in abundance in all organisms suffering from the disease.
The microorganism must be isolated from a diseased organism and grown in pure culture.
The cultured microorganism should cause disease when introduced into a healthy organism.
The microorganism must be reisolated from the inoculated, diseased experimental host and identified as being identical to the original specific causative agent.
There are over a hundred different enteroviruses and the antibody used for detection of virus protein in this study (yes, that’s right, just one non-specific antibody) does not identify the virus involved. Vaccine against diabetes? I don’t think so.
But as I said, we’ve been here before. There are reports of viruses associated with diabetes dating from the 1960s, and a very well known model of Coxsackie virus B4 causing diabetes in mice dating from the 1970s (Coxsackie Viruses and Diabetes Mellitus. BMJ 1973 November 3; 4(5887): 260–262). So does the latest work add anything new, and is a vaccine against diabetes just around the corner? No. I wish it was.
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.
Burkitt’s lymphoma (BL) was first described 50 years ago, and the first human tumour virus Epstein–Barr virus (EBV) was discovered in BL tumours soon after. Since then, the role of EBV in the development of BL has become more and more enigmatic. Only recently have we finally begun to understand, at the cellular and molecular levels, the complex and interesting interaction of EBV with B cells that creates a predisposition for the development of BL. This review discusses the intertwined histories of EBV and BL and their relationship to the cofactors in BL pathogenesis: malaria and the MYC translocation.
A recent paper in Trends in Microbiology examined the possibilities for using bacteriophages in controlling biofilms which might result in healthcare-associated infections (Preventing biofilms of clinically relevant organisms using bacteriophage. 2009 Trends in Microbiology 17: 66-72). Biofilms are firmly attached microbial communities in which the organisms produce an extracellular polymeric matrix. Biofilm organisms might cause disease by detachment of individual cells or clumps of cells, by production of endotoxin, or by providing a niche for the development of antibiotic-resistant organisms. Biofilm organisms are usually tolerant to antimicrobial agents and the treatment of indwelling medical device-associated infections with systemic antimicrobial agents is usually ineffective.
Bacteriophages have been used for the treatment of infectious diseases in plants and animals, although few clinical trials with stringent negative controls have been carried out. There is a renewed interest in phage therapy in light of growing concerns with antimicrobial resistance in healthcare institutions worldwide. The use of phages for the treatment of device-associated infections could reduce the use of antibiotics and might limit the spread of resistant organisms.
There is some evidence for the potential of phages in biofilm control. Bacteriophage T4 phage was effective against E. coli biofilms in a glucose-limited chemostat, although the rate of phage synthesis and assembly were directly proportional to the amount of protein synthesis in the host cell. Some phages produce polysaccharide depolymerases that have the potential to degrade the biofilm matrix.
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However, there are several important characteristics of phage that should be considered when evaluating the potential of phages to control clinically relevant biofilms. The specificity of receptors for a single phage strain will determine its host range; some phage have specificity at the strain level, such as strain typing phages, whereas some are more broad-spectrum and can infect multiple strains or related species. This high degree of specificity could be a drawback, especially in the case of polymicrobial biofilms.
Few studies have explored the role of biofilms in the development of phage-resistance. Phage cocktails developed via the isolation of host range mutants and broad spectrum phage could be advantageous. Another important question is how a patient’s immune system will respond to the therapeutic introduction of phage. Phages are antigenic and elicit a response by serum antibodies and the cellular immune system. Repeated exposure to the phage results in increasing antibody titers and studies in animal models have shown that phage is cleared from the bloodstream by the cellular immune system. In a short-lived treatment, the antibody response to phage is weak except in cases were serum antibody titers are present before phage treatment. It is possible, although unproven, that phage might associate with biofilms and thereby be protected from inactivation. It might also be possible to design mutant phages with enhanced ability to resist clearance by the cellular immune system.
Human papillomaviruses (HPVs) are DNA viruses associated with major human cancers. As such there is a strong interest in developing new means, such as vaccines and microbicides, to prevent HPV infections. Developing the latter requires a better understanding of the infectious life cycle of HPVs. The HPV infectious cycle is closely linked to the differentiation state of the stratified epithelium it infects, with progeny virus only made in the terminally differentiating suprabasal compartment. It has long been recognized that HPV must first establish its infection within the basal layer of stratified epithelium, but why this is the case has not been understood. In part this restriction might reflect specificity of expression of entry receptors. However, this hypothesis could not fully explain the differentiation restriction of HPV infection, since many cell types can be infected with HPVs in monolayer cell culture.
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.
