Two fundamental events in virus replication cycles are the delivery of virus genomes into host cells and the packaging of these genomes into virus protein capsids. In bacteriophages and herpesviruses these processes occur linearly along the genome, base pair after base pair, through a single portal located at a unique site in the viral capsid. New research has addressed the question of whether such a linear translocation through a single portal also takes place for viruses with very large genomes by studying genome delivery and packaging in the amoeba-infecting virus Acanthamoeba polyphaga Mimivirus. With 1.2 million base pairs, this double-stranded DNA genome is the largest documented viral genome. Using electron tomography and cryo-scanning electron microscopy researchers identified a large tunnel in the Mimivirus capsid that is formed shortly after infection, following a large-scale opening of the capsid. The tunnel allows the whole virus genome to exit in a rapid, one-step process. DNA encapsidation is mediated by a transient aperture in the capsid that, they suggest, may promote concomitant entry of multiple segments of the viral DNA molecule. These unprecedented modes of viral genome translocation imply that Mimivirus - and potentially other large viruses - evolved mechanisms that allow them to cope effectively with the exit and entry of particularly large genomes.
Distinct DNA exit and packaging portals in the virus Acanthamoeba polyphaga mimivirus. 2008 PLoS Biol 6(5): e114
Icosahedral double-stranded DNA viruses use a single portal for genome delivery and packaging. The extensive structural similarity revealed by such portals in diverse viruses, as well as their invariable positioning at a unique icosahedral vertex, led to the consensus that a particular, highly conserved vertex-portal architecture is essential for viral DNA translocations. Here we present an exception to this paradigm by demonstrating that genome delivery and packaging in the virus Acanthamoeba polyphaga mimivirus occur through two distinct portals. By using high-resolution techniques, including electron tomography and cryo-scanning electron microscopy, we show that Mimivirus genome delivery entails a large-scale conformational change of the capsid, whereby five icosahedral faces open up. This opening, which occurs at a unique vertex of the capsid that we coined the ‘‘stargate’’, allows for the formation of a massive membrane conduit through which the viral DNA is released. A transient aperture centered at an icosahedral face distal to the DNA delivery site acts as a non-vertex DNA packaging portal. In conjunction with comparative genomic studies, our observations imply a viral packaging pathway akin to bacterial DNA segregation, which might be shared by diverse internal membrane–containing viruses.
Over the past week, international news stories have concentrated on the devastating cyclone in Burma (Myanmar), and the almost certain consequence of disease outbreaks in the aftermath. But at the same time, there’s another microbiology story unfolding in East Asia. Beginning in March, a large outbreak of hand, foot and mouth disease (HFMD) was reported from Fuyang city in Anhui Province in China. Note that HFMD is a human disease caused by enteroviruses belonging to the picornavirus family, but is not the same as the animal disease foot and mouth (FMD) caused by a different kind of picornavirus.
HFMD usually affects infants and children, is quite common worldwide and can be caused by a number of different enteroviruses. It is highly contagious and is spread through direct contact with the mucus, saliva, or faeces of an infected person. Like other enterovirus infections (including polio), HFMD typically occurs in small epidemics, usually during the summer and autumn months with an incubation period of 3-7 days.
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Enterovirus infections are common and occur worldwide. Although many infections show no symptoms and often go unnoticed, these viruses are also associated with occasional outbreaks in which a larger than usual number of patients develop clinical disease, sometimes with fatal consequences. The current outbreak is one of these. Initial testing for a variety of respiratory diseases did not reveal any conclusive results, but on April 23, the presence of Enterovirus (EV71) was confirmed. As of May 8th, at least 30 deaths had been reported and the disease had spread to 11 cities and several provinces across China. In all the fatal cases, which represent less than 1% of the thousands of children infected, the victims died with serious complications such as neurogenic pulmonary oedema (breathing difficulties reminiscent to those seen in polio victims).
Enterovirus replication begins in the gastrointestinal or respiratory tract but once the virus is present in the bloodstream may affect various tissues and organs, causing a variety of diseases. Clinically, it is difficult to distinguish the specific cause of most enterovirus infections. Diagnostic testing for non-polio enteroviruses requires specialized laboratory facilities. Diagnosis is made by detecting virus in the throat, in faecal samples or, more convincingly, from specimens collected from the affected part of the body, for example, cerebrospinal fluid (CSF) or biopsy material. A four-fold rise in the level of neutralizing antibody in specimens collected during the acute and convalescent phases of illness provides the best evidence for a recent infection. No specific antiviral agents are currently available for treatment of enterovirus infections, although intravenous administration of immune globulin may have a use in preventing severe disease in immunocompromised individuals or those with life-threatening disease.
EV71 was first isolated in an outbreak of neurological disease in California in 1969. One of the nastier enteroviruses, EV71 has been associated with several epidemics of severe neurological disease in children, mostly in East Asia. An outbreak in Taiwan in 1998 resulted in 129,106 reported cases, 405 children hospitalized and more than 80 deaths. EV71 appears to be emerging as an important virulent neurotropic enterovirus just as poliomyelitis is nearing eradication, but little is known about the molecular mechanisms of host response to EV71 infection.
Transmission of enterovirus infections is increased by poor hygiene and overcrowded living conditions. Improved sanitation and general hygiene are important preventive measures. Measures that can be taken to avoid getting infected with enteroviruses include frequent handwashing, especially after nappy (diaper) changes or going to the toilet, disinfection of contaminated surfaces with bleach, and washing soiled articles of clothing. Enteroviruses are quite resistant to many disinfectants so it is important to use chlorinated (bleach) or iodized disinfectants. During recognised epidemics, it may be advised to close institutions such as schools or child care facilities in order to reduce transmission among young children. Chinese public health experts currently predict that the number of cases will continue to increase and peak around June-July.
With two catalytic activities and many substrates, how does HIV’s reverse transcriptase enzyme know what to do to which substrate? Zooming in on the enzyme’s molecular interactions provides tantalizing clues.
Dynamic binding orientations direct activity of HIV reverse transcriptase. Nature 453, 184-189
The reverse transcriptase of human immunodeficiency virus (HIV) catalyses a series of reactions to convert the single-stranded RNA genome of HIV into double-stranded DNA for host-cell integration. This task requires the reverse transcriptase to discriminate a variety of nucleic-acid substrates such that active sites of the enzyme are correctly positioned to support one of three catalytic functions: RNA-directed DNA synthesis, DNA-directed DNA synthesis and DNA-directed RNA hydrolysis. However, the mechanism by which substrates regulate reverse transcriptase activities remains unclear. Here we report distinct orientational dynamics of reverse transcriptase observed on different substrates with a single-molecule assay. The enzyme adopted opposite binding orientations on duplexes containing DNA or RNA primers, directing its DNA synthesis or RNA hydrolysis activity, respectively. On duplexes containing the unique polypurine RNA primers for plus-strand DNA synthesis, the enzyme can rapidly switch between the two orientations. The switching kinetics were regulated by cognate nucleotides and non-nucleoside reverse transcriptase inhibitors, a major class of anti-HIV drugs. These results indicate that the activities of reverse transcriptase are determined by its binding orientation on substrates.
Viruses are small infectious agents responsible for many human diseases, including acquired immunodeficiency syndrome (AIDS). Like other viruses, the human immunodeficiency virus 1 (HIV-1; the cause of AIDS) enters human cells and uses the cellular machinery to replicate before bursting out of its temporary home. During the initial stage of HIV infection, a particular group of cells in the human immune system, CD8+ T cells, are thought to be important in controlling the level of the virus. These immune system cells recognize pieces of viral protein called antigens displayed on the surface of infected cells; different subsets of CD8+ T cells recognize different antigens. When a CD8+ T cell recognizes its specific antigen (or more accurately, a small part of the antigen called an epitope ), it releases cytotoxins (which kill the infected cells) and cytokines, proteins that stimulate CD8+ T cell proliferation and activate other parts of the immune system. With many viruses, when a person first becomes infected (an acute viral infection), antigen-specific CD8+ T cells completely clear the infection. But with HIV-1 and some other viruses, these cells do not manage to remove all the viruses from the body and a chronic (long-term) infection develops, during which the immune system is constantly exposed to viral antigen.
In HIV-1 infections (and other chronic viral infections), virus-specific CD8+ T cells lose their ability to proliferate, to make cytokines, and to kill infected cells as patients progress to the longterm stages of infection. That is, the virus-specific CD8+ T cells gradually lose their effector functions and become functionally impaired or exhausted. Polyfunctional CD8+ T cells (those that release multiple cytokines in response to antigen) are believed to be essential for an effective CD8+ T cell response, so scientists trying to develop HIV-1 vaccines would like to stimulate the production of this type of cell. To do this they need to understand why these polyfunctional cells are lost during chronic infections. Is their loss the cause or the result of viral persistence? In other words, does the constant presence of viral antigen lead to the exhaustion of CD8+ T cells during chronic HIV infection? In this study, the researchers investigate this question by looking at the polyfunctionality of CD8+ cells responding to several different viral epitopes at various times during HIV-1 infection, starting very early after infection with HIV-1 had occurred.
The researchers enrolled 18 patients recently infected with HIV-1 and analyzed their CD8+ T cell responses to specific epitopes at various times after enrollment using a technique called flow cytometry. They found that the epitope-specific CD8+ cells produced several effector proteins after antigen stimulation during the initial stage of HIV-1 infection, but lost their polyfunctionality in the face of persistent viral infection. The CD8+ T cells also increased their production of programmed death 1 (PD-1), a protein that has been shown to be associated with the functional impairment of CD8+ T cells. Some of the patients began antiretroviral therapy during the study, and the researchers found that this treatment, which reduced the viral load, reversed CD8+ T cell exhaustion. Finally, the appearance in the patients blood of viruses that had made changes in the specific epitopes recognized by the CD8+ T cells to avoid being killed by these cells, also reversed the exhaustion of the T cells recognizing these particular epitopes.
These findings suggest that the constant presence of HIV-1 antigen causes the functional impairment of virus-specific CD8+ T cell responses during chronic HIV-1 infections. Treatment with antiretroviral drugs reversed this functional impairment by reducing the amount of antigen in the patients. Similarly, the appearance of viruses with altered epitopes, which effectively reduced the amount of antigen recognized by those epitope-specific CD8+ T cells without reducing the viral load, also reversed T cell exhaustion. These results would not have been seen if the functional impairment of CD8+ cells were the cause rather than the result of antigen persistence. By providing new insights into how the T cell response to viruses evolves during persistent viral infections, these findings should help in the design of vaccines against HIV and other viruses that cause chronic viral infections.
Bacteriophages are among the smallest but most abundant organisms on earth. For most phages, the tail mediates the anchoring of the phage to generally abundant bacterial outer membrane proteins that serve as specific receptors for their substrates. For example, the receptor for the temperate phage λ is the Escherichia coli maltoporin receptor LamB, which functions in amylomaltose uptake. The establishment of a stable phage–host interaction relays signals that allow injection of DNA from the phage capsid through the tail and into the host, leaving the empty capsid (head) attached to the cell surface. Following phage λ DNA injection, a decision between the lytic or lysogenic pathways of bacteriophage λ is made. The poles (ends) of bacterial calls have specialized functions related to the mobilization of DNA and certain proteins. To monitor the infection of Escherichia coli cells by light microscopy, scientists developed procedures for the tagging of mature bacteriophages with quantum dots:
Surprisingly, most of the infecting phages were found attached to the bacterial poles. This was true for a number of temperate and virulent phages of E. coli that use widely different receptors and for phages infecting Yersinia pseudotuberculosis and Vibrio cholerae. The infecting phages colocalized with the polar protein marker IcsA–GFP. ManY, an E. coli protein that is required for phage λ DNA injection, was found to localize to the bacterial poles as well. Furthermore, labelling of λ DNA during infection revealed that it is injected and replicated at the polar region of infection. The evolutionary benefits that lead to this remarkable preference for polar infections may be related to λ’s developmental decision as well as to the function of poles in the ability of bacterial cells to communicate with their environment and in gene regulation.
By labelling different phages with quantum dots and following adsorption using a fluorescence microscope the researchers were able to investigate the initial steps of binding (adsorption) and phage DNA injection. The surprising results showed that at low multiplicities of infection, phages preferentially adsorb, inject and replicate their DNA at the bacterial poles. This spatial preference was independent of host proteins, ManY and Pel, required for phage λ DNA injection. The significance of the pole, the binding of the phage to the pole and its implications in lytic-lysogenic decision are discussed in the paper.
Acanthamoeba polyphaga mimivirus is the largest known ds-DNA virus and its 1.2 Mb-genome sequence has revealed many unique features. Mimivirus occupies an independent lineage among eukaryotic viruses and its known hosts include only species from the Acanthamoeba genus. The existence of Mimivirus relatives was first suggested by the analysis of the Sargasso Sea metagenomic data. We now further demonstrate the presence of numerous “mimivirus-like” sequences using a larger marine metagenomic data set. We also show that the DNA polymerase sequences from three algal viruses (CeV01, PpV01, PoV01) infecting different marine algal species (Chrysochromulina ericina, Phaeocystis pouchetii, Pyramimonas orientalis) are very closely related to their homolog in Mimivirus. The results suggest that the numerous mimivirus-related sequences identified in marine environments are likely to originate from diverse large DNA viruses infecting phytoplankton. Micro-algae thus constitute a new category of potential hosts in which to look for new species of Mimiviridae.
The function of the RNA genome of the human immunodeficiency virus (HIV) is determined both by its sequence and by its ability to fold back on itself to form specific higher-order structures. In order to describe physical structures in a region of the HIV RNA genome known to play multiple, critical roles in viral replication and pathogenesis. In this week’s PLoS Biology scientists from the University of North Carolina show how they have devised a high-throughput, quantitative, and comprehensive structure-mapping approach that locates flexible (unpaired) nucleotides within a folded RNA, assaying hundreds of nucleotides at a time. They find that the first 10% of the HIV-1 genome has a single predominant structure and that regulatory motifs have significantly greater structure than do protein-coding segments. The HIV genome interacts with numerous proteins, including multiple copies of nucleocapsids. They also directly map RNA-protein interactions inside virions and discover that the nucleocapsid interacts with viral RNA in at least three distinct ways, depending on the context within the overall genome structure. The group hopes that further application of the high-throughput RNA-structure analysis tools described will make it possible to address diverse structure-function relationships in intact cellular and viral RNAs.
Replication and pathogenesis of the human immunodeficiency virus (HIV) is tightly linked to the structure of its RNA genome, but genome structure in infectious virions is poorly understood. High-throughput SHAPE (selective 29-hydroxyl acylation analyzed by primer extension) technology uses many of the same tools as DNA sequencing, was used to quantify RNA backbone flexibility at single-nucleotide resolution and from which robust structural information can be immediately derived. We analyze the structure of HIV-1 genomic RNA in four biologically instructive states, including the authentic viral genome inside native particles. Remarkably, given the large number of plausible local structures, the first 10% of the HIV-1 genome exists in a single, predominant conformation in all four states. We also discover that noncoding regions functioning in a regulatory role have significantly lower SHAPE reactivities, and hence more structure, than do viral coding regions that function as the template for protein synthesis. By directly monitoring protein binding inside virions, we identify the RNA recognition motif for the viral nucleocapsid protein. Seven structurally homologous binding sites occur in a well-defined domain in the genome, consistent with a role in directing specific packaging of genomic RNA into nascent virions. In addition, we identify two distinct motifs that are targets for the duplex destabilizing activity of this same protein. The nucleocapsid protein destabilizes local HIV-1 RNA structure in ways likely to facilitate initial movement both of the retroviral reverse transcriptase from its tRNA primer and of the ribosome in coding regions. Each of the three nucleocapsid interaction motifs falls in a specific genome domain, indicating that local protein interactions can be organized by the long-range architecture of an RNA. High-throughput SHAPE reveals a comprehensive view of HIV-1 RNA genome structure, and further application of this technology will make possible newly informative analysis of any RNA in a cellular transcriptome. High-throughput SHAPE analysis reveals structures in HIV-1 genomic RNA strongly conserved across distinct biological states. 2008 PLoS Biol 6: e96
A simplified economical method of giving rabies vaccine is just as effective as the expensive standard vaccination method at stimulating anti-rabies antibodies. A clinical trial in healthy volunteers has found that a simpler and cheaper way of using rabies vaccines proved to be just as effective as the current most widely used method at stimulating antibodies against rabies. The trial is published in this week’s PLoS Neglected Tropical Diseases. Dr Mary Warrell (Centre for Clinical Vaccinology and Tropical Medicine, University of Oxford) and colleagues, who conducted the trial with a vaccine in routine use, say that the simplified method has the advantages of requiring fewer clinic visits, being more practicable, and acceptable, and having a wider margin of safety, especially in inexperienced hands. It would therefore be suitable for use anywhere in the world where there are financial constraints, and especially where two or more patients are likely to be treated on the same day.
All human deaths from rabies result from failure to give adequate prophylaxis. After a rabid animal bite, immediate wound cleaning, rabies vaccine and injections of anti-rabies antibody (immunoglobulin) effectively prevent fatal infection. But anti-rabies immunoglobulin is very rarely available in developing countries, and so prevention relies on giving people bitten by rabid animals effective vaccine treatment. The vaccines that are currently approved by the World Health Organization, which are usually injected into the muscle, are prohibitively expensive, and so are unaffordable in developing countries. In Africa, for example, the average cost of an intramuscular course of vaccine is $US 39.6, equivalent to 50 days wages.
Two more economical regimes, involving injecting small amounts of vaccine into the skin (intradermally) at 2 or 8 sites on the first day of the course with subsequent booster doses, are available in a few places. With the 8-site method, a large dose of vaccine is given on the first day only, whereas with the 2-site method the same dose is divided between the first and third days, entailing an extra visit to the clinic. However, practical or perceived difficulties have restricted widespread uptake of these economical methods. Dr Warrell and colleagues set out to test a new, similar simplified regime, involving injections at 4 sites on the first day. They vaccinated healthy volunteers to compare the antibody levels induced by the 4-site intradermal regimen with those induced by the current 2-site and 8-site intradermal regimes and the “gold standard” intramuscular regimen favored internationally. All of the economical intradermal methods worked just as well as the intramuscular method at stimulating anti-rabies antibodies. The authors conclude that the results provide sufficient evidence that the simplified 4-site regimen now meets all the criteria necessary for its recommendation for use wherever the cost of vaccine is prohibitive.
When measles vaccines were widely introduced in the 1970s, there were concerns that they might cause subacute sclerosing panencephalitis (SSPE), a very rare, late-onset, neurological complication of natural measles infection. Therefore, SSPE registries and routine measles immunization were established in many countries concurrently. We conducted a comprehensive review of the impact of measles immunization on the epidemiology of SSPE and examined epidemiological evidence on whether there was any vaccine-associated risk. Published epidemiological data on SSPE, national SSPE incidence, measles incidence and vaccine coverage, reports of SSPE in pregnancy or shortly post partum were reviewed. Potential adverse relationships between measles vaccines and SSPE were examined using available data. Epidemiological data showed that successful measles immunization programmes protect against SSPE and, consistent with virological data, that measles vaccine virus does not cause SSPE. Measles vaccine does not: accelerate the course of SSPE; trigger SSPE or cause SSPE in those with an established benign persistent wild measles infection. Evidence points to wild virus causing SSPE in cases which have been immunized and have had no known natural measles infection. Perinatal measles infection may result in SSPE with a short onset latency and fulminant course. Such cases are very rare. SSPE during pregnancy appears to be fulminant. Infants born to mothers with SSPE have not been subsequently diagnosed with SSPE themselves. Successful measles vaccination programmes directly and indirectly protect the population against SSPE and have the potential to eliminate SSPE through the elimination of measles. Epidemiological and virological data suggest that measles vaccine does not cause SSPE.
Viroporins are virus-encoded proteins that participate in virus replication, including the promotion of release of virus particles from cells (Viroporins. FEBS Lett 2003 552: 28-34). They also affect cellular functions, including the cell vesicle system, glycoprotein trafficking and membrane permeability. Viroporins are usually not essential for the replication of viruses, but their presence enhances virus growth. Composed of 60-120 amino acids, viroporins have a hydrophobic transmembrane domain that interacts with lipid bilayers, and polymerization of viroporin monomers creates hydrophilic pores in the membranes of virus-infected cells. Viroporins are present in tiny amounts in the virus particles (virions) of many animal RNA viruses, e.g. influenza A virus M2 protein, poliovirus 2B and 3A proteins, HIV Vpu and SARS coronavirus E protein. Viroporins contribute to the pathology of virus diseases by altering membrane permeability and disrupting ion homeostasis in infected cells.
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A paper recently published in Cellular Microbiology reports that viroporins of hepatitis C virus, poliovirus and other animal RNA viruses induce apoptosis in host cells (Viroporins from RNA viruses induce caspase-dependent apoptosis. 2008 Cell Microbiol 10: 437-451). In addition to their capacity to disrupt ionic cellular homeostasis and promote cell lysis, the expressed viroporins were able to induce cell death. Degradation of DNA and generation of apoptotic bodies were observed on viroporin expression. Activation of caspase-3, altered mitochondrial morphology and detection of cytochrome c release from mitochondria suggests involvement of the mitochondrial pathway in viroporin-induced apoptosis and shows that viroporins induce caspase-dependent programmed cell death.
It is possible that viroporins have different effects depending on the level of expression and/or the host-cell type. The induction of apoptosis in host cells by viruses is common and could assist virus spread. The next step in understanding the links between viroporins and apoptosis will be to unravel the mechanisms by which viroporins trigger apoptotic pathways and to demonstrate that these findings are relevant during virus infections.
Two fundamental events in virus replication cycles are the delivery of virus genomes into host cells and the packaging of these genomes into virus protein capsids. In bacteriophages and herpesviruses these processes occur linearly along the genome, base pair after base pair, through a single portal located at a unique site in the viral capsid. New research has addressed the question of whether such a linear translocation through a single portal also takes place for viruses with very large genomes by studying genome delivery and packaging in the amoeba-infecting virus Acanthamoeba polyphaga Mimivirus. With 1.2 million base pairs, this double-stranded DNA genome is the largest documented viral genome. Using electron tomography and cryo-scanning electron microscopy researchers identified a large tunnel in the Mimivirus capsid that is formed shortly after infection, following a large-scale opening of the capsid. The tunnel allows the whole virus genome to exit in a rapid, one-step process. DNA encapsidation is mediated by a transient aperture in the capsid that, they suggest, may promote concomitant entry of multiple segments of the viral DNA molecule. These unprecedented modes of viral genome translocation imply that Mimivirus - and potentially other large viruses - evolved mechanisms that allow them to cope effectively with the exit and entry of particularly large genomes.
