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.
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.
Trypanosoma brucei is a pathogen which causes epidemics of human and animal sleeping sickness in central and Southern Africa, a fatal disease if untreated. A new paper published this week in PLoS Biology investigates the formation of a trypanosome structure called the Flagellar Pocket (FP). The paper describes a newly identified protein called BILBO1 that is crucial for Flagellar Pocket formation. Experimental inhibition of the protein BILBO1 is fatal to Trypanosoma brucei. The Flagellar Pocket is a part of the single-celled pathogen that has several functions. As well as being the site where the flagellum attaches the flagellum being a structure that enables the bug to propel itself the FP is the site of endo- and exocytosis, processes that transport material in and out of the pathogen from the outside world. The FP also plays an important role in allowing the trypanosome to avoid the immune system of the host. The FP permits the trypanosomes surface proteins (the structures seen by the immune system) to be changed, so that the bug can remain camouflaged from the host’s defences.
The Flagellar Pocket is a multi-tasking organelle but we can now consider it as the parasite’s Achilles heel. How the trypanosome forms this important structure has previously been unknown. The new paper shows that BILBO1 is crucial for the formation of the FP, specifically, it is an important component of a part of the FP called the Flagellar Pocket Collar. BILBO1 is a cytoskeletal protein (the cytoskeleton being a network of proteins that provide internal structure for a cell), thus BILBO1 is involved in creating the cytoskeletal framework that supports the FP. This paper describes the effect of preventing BILBO1 expression in the trypanosome. Without BILBO1, the trypanosome is unable to create a new FP; with the FP disrupted, it is unable to regulate endo- and exocytosis, therefore preventing it from taking up nutrients. These factors prove fatal to the trypanosome. Therefore, this work provides a target for anti-sleeping sickness drugs: knock out BILBO1 function and you will kill the trypanosome.
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.
Humans are hosts to nearly 300 species of parasitic worms and over 70 species of protozoa, some derived from our primate ancestors and some acquired from the animals we have domesticated or come into contact with during our history (History of human parasitology. Clin Microbiol Rev 2002 15: 595-612). The best-documented parasitic disease known from ancient times is caused by the nematode worm Dracunculus medinensis. The earliest description is from an Egyptian papyrus from 1500 BC that refers to both the nature of the infection and to techniques for removing the worm. Confirmation of the presence of this worm in ancient Egypt comes from the finding of a well-preserved worms in Egyptian mummies. Dracunculiasis, or Guinea worm disease, is one of the few diseases unambiguously described in the Bible, and most parasitologists accept that the “fiery serpents” that struck down the Israelites in the region of the Red Sea after the Exodus from Egypt somewhere between 1250 to 1200 BC were actually Guinea worms.
The adult worms live in the subcutaneous connective tissues of their victims, from which the females emerge to release thousands of larvae into water, where they are taken up by intermediate hosts, tiny aquatic crustaceans called Cyclops. In these hosts they mature into infectious larvae that infect humans when the crustaceans are accidentally swallowed in contaminated drinking water. On maturity, the large female worm, up to nearly a metre in length, protrudes from the skin, usually of the leg, and causes intense inflammation and irritation. The effects of the disease are crippling. Its victims develop large ulcers, usually in the lower leg. The ulcers swell, sometimes to the size of a tennis ball, and burst, releasing the spaghetti-like parasitic worm. Victims experience a pain so excruciating that they say it feels as if their leg is on fire. The searing pain compels people to jump into water, often the community’s only source of drinking water, to relieve the pain. When the infected person immerses his or her leg in the water, the worm in the leg releases thousands of larvae. The larvae are then ingested by Cyclops that live in the water. Thus the cycle begins again - when people drink the water, they are in effect drinking in the disease.
The most common way to treat Guinea worm disease involves wrapping the worm around a stick. This treatment has been employed for millennia and may have inspired the Rod of Asclepius which historically has symbolized the medical profession. As the adult worm begins to emerge from the patient’s skin, it is wound around a stick, then further extracted by a few centimeters per day. This slow process can take days or even weeks, but it is required to avoid breakage and leaving behind a portion of the worm. Leaving a portion of the dead worm remain within the host’s body increases the risk of infection, and can trigger immune responses resulting in pain and swelling. In many countries, a broken worm is immediately removed surgically, or the worm can be excised surgically from the very beginning if health care facilities are available. Antihelminthic drugs such as metronidazole or thiabendazole are sometimes used in conjunction with physical extraction. However, one study found that antihelminthic therapy was associated with aberrant migration of worms, resulting in infection in areas other than the lower extremity.
Dracunculiasis is a classic example of a neglected tropical disease, a symptom of poverty and disadvantage. Those most affected are the poorest populations often living in remote, rural areas, urban slums or in conflict zones. With little political voice, neglected tropical diseases have a low profile and status in public health priorities. In 1997 the World Health Assembly pledged to completely eradicate Guinea worm disease. This is no small task, but there are several factors which make eradication a possibility. Dracunculiasis is the first parasitic disease targeted for eradication because:
Diagnosis is easy and unambiguous (presence of an emerging adult worm).
The transmission agent, Cyclops, is not a mobile vector as is a mosquito.
The incubation period in both Cyclops and humans is of limited duration.
Interventions are effective, low cost, and relatively simple to implement.
The disease has a limited geographic distribution and is seasonal in nature.
Success in eliminating the disease has been demonstrated in several countries in Asia and the Middle East.
There is no known animal reservoir.
Is Dracunculiasis eradication close? In 2007 the WHO announced that Guinea worm disease now affects around 25,000 people in nine countries, compared with an estimated 3 million people were infected in over 20 countries in the early 1980s. Twelve countries were declared Guinea worm-free in early March. If progress continues at this rate, the disease could be eradicated in less than two years. It is probable that complete eradication will take quite a few years yet, although it should be possible to eliminate the disease from seven countries in a couple of years, leaving only two endemic countries, Sudan and Ghana (Dracunculiasis eradication by 2009: will endemic countries meet the target? Tropical Medicine & International Health 2007 12: 1403-1408). One lesson to be drawn from the problems of local ownership and the experience of cash rewards is that there are dangers in throwing money at the problem. While the eradication initiative badly needs additional resources, it needs them at such a level and managed in such a way that they do not distort the priorities of the health care system, or exceed the capacity of local staff to manage them. The amounts needed are not large, but their continuity and flexibility is important. Given the highly seasonal transmission of dracunculiasis, the resources must be available at very specific times of the year, which is not always achieved. In spite of the difficulties, complete worldwide eradication of this ancient disease is drawing nearer.
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.
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.
