MicrobiologyBytes

Biologists have uncovered virus fragments from the same family of the modern Hepatitis B virus locked inside the genomes of songbirds such as the modern-day zebra finch. This article marks the first time that endogenous hepadnaviruses have been found in any organism. An endogenous virus is one that deposits itself or fragments of itself into the chromosome of an organism, allowing it to be passed from generation-to-generation. Previously, most of these known “fossilized” virus sequences have come from retroviruses. These fragments have been sitting in the bird’s genomes for at least 19 million years, far longer than anyone previously thought this family of viruses had been in existence.

The researchers dated the hepadnavirus fragments by locating them in the same spot on the genome of five species of passerine birds and then tracing those species to a common ancestor that lived more than 19 million years ago. This work provides a glimpse into an ancient viral world that we never knew existed. The results are remarkable – hepadnaviruses, and likely many other viruses as well, are far older than we previously thought. Another surprise is that the older versions of the hepadnaviruses are remarkably similar to today’s viruses. This suggests that the slow evolution of the hepadnaviruses observed in birds indicates that the viruses are, in the long run, better adapted to their hosts than what is suggested by study of the disease-causing Hepatitis B viruses. Genomic fossils like these remarkable hepadnaviral fossils have the prospect of completely revising our preconceived notions about the age and evolution of such viruses.

This study also opens new avenues for research that might help predict and prevent human viral pandemics originating in bird species. Given that they were infected in the past, it is legitimate to think that some of these birds may still carry such viruses today. We can use this discovery as a guide to screen targeted groups of bird species for the presence of new circulating Hepatitis B-like viruses.

Genomic Fossils Calibrate the Long-Term Evolution of Hepadnaviruses. (2010) PLoS Biol 8(9): e1000495. doi:10.1371/journal.pbio.1000495
Because most extant viruses mutate rapidly and lack a true fossil record, their deep evolution and long-term substitution rates remain poorly understood. In addition to retroviruses, which rely on chromosomal integration for their replication, many other viruses replicate in the nucleus of their host’s cells and are therefore prone to endogenization, a process that involves integration of viral DNA into the host’s germline genome followed by long-term vertical inheritance. Such endogenous viruses are highly valuable as they provide a molecular fossil record of past viral invasions, which may be used to decipher the origins and long-term evolutionary characteristics of modern pathogenic viruses. Hepadnaviruses (Hepadnaviridae) are a family of small, partially double-stranded DNA viruses that include hepatitis B viruses. Here we report the discovery of endogenous hepadnaviruses in the genome of the zebra finch. We used a combination of cross-species analysis of orthologous insertions, molecular dating, and phylogenetic analyses to demonstrate that hepadnaviruses infiltrated repeatedly the germline genome of passerine birds. We provide evidence that some of the avian hepadnavirus integration events are at least 19 My old, which reveals a much deeper ancestry of Hepadnaviridae than could be inferred based on the coalescence times of modern hepadnaviruses. Furthermore, the remarkable sequence similarity between endogenous and extant avian hepadnaviruses (up to 75% identity) suggests that long-term substitution rates for these viruses are on the order of 10^-8 substitutions per site per year, which is a 1,000-fold slower than short-term rates estimated based on the sequences of circulating hepadnaviruses. Together, these results imply a drastic shift in our understanding of the time scale of hepadnavirus evolution, and suggest that the rapid evolutionary dynamics characterizing modern avian hepadnaviruses do not reflect their mode of evolution on a deep time scale.

Related:

1. Phoenix from the ashes: The 5 million year old virus
2. The Island of Fossil Viruses

miRNAs are small single-stranded RNA species of approximately 20–24 bases in length that regulate gene expression through post transcriptional mechanisms. Expression of miRNAs is thought to be ubiquitous among multicellular eukaryotes. In addition to eukaryotic miRNAs, more than 100 viral miRNAs have been identified, almost all of which are expressed by herpesviruses. Targets for the majority of viral miRNAs are currently unknown due to the difficulty involved in identifying novel target transcripts. This remains one of the major challenges in elucidating the function of miRNAs. However, recent reports have begun to elucidate the various roles of viral miRNAs. These include blocking apoptosis, immune evasion and regulation of virus replication through targeting of both cellular and virus gene expression.

Regulation of gene expression is as important as the genes themselves in determining the diverse array of living creatures we see in nature. Recently, scientists have discovered a whole new level of gene regulation through the actions of small molecules called microRNAs (miRNAs). It is currently thought that miRNAs regulate gene expression primarily through binding to target sites within the 3′ untranslated region (UTR) of mRNAs. This paper identifies a population of cellular genes that are targeted by a virally-encoded miRNA. Many of the genes are related to cell cycle control, suggesting that the viral miRNA is targeting genes within a related pathway. In contrast to most miRNAs, this miRNA inhibits gene expression through binding to target sites within the 5′ UTRs, suggesting that viral miRNAs may target genes through mechanisms divergent from cellular miRNAs.

A Viral microRNA Down-Regulates Multiple Cell Cycle Genes through mRNA 5′ UTRs. (2010) PLoS Pathog 6(6): e1000967. doi:10.1371/journal.ppat.1000967
Global gene expression data combined with bioinformatic analysis provides strong evidence that mammalian miRNAs mediate repression of gene expression primarily through binding sites within the 3′ untranslated region (UTR). Using RNA induced silencing complex immunoprecipitation (RISC-IP) techniques we have identified multiple cellular targets for a human cytomegalovirus (HCMV) miRNA, miR-US25-1. Strikingly, this miRNA binds target sites primarily within 5′UTRs, mediating significant reduction in gene expression. Intriguingly, many of the genes targeted by miR-US25-1 are associated with cell cycle control, including cyclin E2, BRCC3, EID1, MAPRE2, and CD147, suggesting that miR-US25-1 is targeting genes within a related pathway. Deletion of miR-US25-1 from HCMV results in over expression of cyclin E2 in the context of viral infection. Our studies demonstrate that a viral miRNA mediates translational repression of multiple cellular genes by targeting mRNA 5′UTRs.

Related:

1. Five Questions about Viruses and MicroRNAs
2. Virus-encoded microRNAs in herpesvirus biology
3. MicroRNAs in Picornavirus Infection

Bacterial virulence results from the interaction between bacteria and their hosts. This interaction provides selection pressure for bacterial adaptation towards increased fitness or virulence. Basic mechanisms involved in bacterial adaptation at the genetic level are point mutations and recombination. As bacterial genome plasticity is higher in vivo than in vitro, host-pathogen interaction may facilitate bacterial adaptation. Comparative genomics has so far been almost entirely focused on genomic changes upon prolonged bacterial growth in vitro.

To achieve a better comprehension of bacterial genome plasticity and the capacity to adapt in response to their host, researchers studied bacterial genome evolution in vivo. They analyzed the impact of individual hosts on genome-wide bacterial adaptation under controlled conditions, by administration of an asymptomatic E. coli isolate to several hosts. Interestingly, the different hosts appeared to personalize their microflora. Adaptation at the genomic level included point mutations in several metabolic and virulence-related genes, often affecting pleiotropic regulators, but re-isolates from each patient showed a distinct pattern of genetic alterations in addition to random changes. These results provide new insights into bacterial traits under selection during E. coli in vivo growth, further explaining the mechanisms of bacterial adaptation to specific host environments.

Host Imprints on Bacterial Genomes – Rapid, Divergent Evolution in Individual Patients. (2010) PLoS Pathog 6(8): e1001078. doi:10.1371/journal.ppat.1001078
Bacterial virulence results from the interaction between bacteria and their hosts. This interaction provides selection pressure for bacterial adaptation towards increased fitness or virulence. Basic mechanisms involved in bacterial adaptation at the genetic level are point mutations and recombination. As bacterial genome plasticity is higher in vivo than in vitro, host-pathogen interaction may facilitate bacterial adaptation. Comparative genomics has so far been almost entirely focused on genomic changes upon prolonged bacterial growth in vitro. To achieve a better comprehension of bacterial genome plasticity and the capacity to adapt in response to their host, we studied bacterial genome evolution in vivo. We analyzed the impact of individual hosts on genome-wide bacterial adaptation under controlled conditions, by administration of asymptomatic bacteriuria E. coli isolate 83972 to several hosts. Interestingly, the different hosts appeared to personalize their microflora. Adaptation at the genomic level included point mutations in several metabolic and virulence-related genes, often affecting pleiotropic regulators, but re-isolates from each patient showed a distinct pattern of genetic alterations in addition to random changes. Our results provide new insights into bacterial traits under selection during E. coli in vivo growth, further explaining the mechanisms of bacterial adaptation to specific host environments.

Related:

• The long now – 40,000 generations of E. coli
• Spread of Pathogenicity Islands in Escherichia coli
• The continuing evolution of E. coli O157:H7

An epigenetic trait is a stably heritable phenotype resulting from changes in a chromosome without alterations in the DNA sequence. Such changes are mediated by chemical modifications to chromatin on both DNA and DNA-associated histones. Post-translational covalent modifications to the flexible NH2 terminus (tail) of histones include methylation, acetylation, phosphorylation and ubiquitylation, and these are associated with the structural organization of chromatin and its transcriptional status. However, not all histone modifications are truly epigenetic, as very few satisfy the heritable part of the definition. To establish and mediate epigenetic memory, such modifications must be transmitted during DNA replication. Methylation of cytosine in CpG dinucleotides (often referred to as DNA methylation) also contributes to the epigenetic status of a gene locus. When this occurs in a CpG island adjacent to a transcription initiation site, it is generally associated with repression or silencing of transcription. Histone modification, DNA methylation and the resulting reorganisation of chromatin are closely interlinked enzyme-driven processes that determine the transcriptional status of genes, gene clusters and noncoding RNAs such as micro (mi)RNAs. Most of the epigenetic markers mentioned above are associated with transcriptional repression. Multiple additional covalent modifications to histones exist in parallel to these, resulting in a complex and context-influenced ‘histone code’ that dictates transcriptional state.

One of the key questions in the study of mammalian gene regulation is how epigenetic methylation patterns on histones and DNA are initiated and established. These stable, heritable, covalent modifications are largely associated with the repression or silencing of gene transcription, and when deregulated can be involved in the development of human diseases such as cancer. This article reviews examples of viruses and bacteria known or thought to induce epigenetic changes in host cells, and how this might contribute to disease. The heritable nature of these processes in gene regulation suggests that they could play important roles in chronic diseases associated with microbial persistence; they might also explain so-called ‘hit-and-run’ phenomena in infectious disease pathogenesis.

Epigenetic reprogramming of host genes in viral and microbial pathogenesis. Trends Microbiol. Aug 17 2010

Related:

• Antigenic Variation in African Trypanosomes
• Hepatitis B virus X protein (HBx)
• Bacteria hedge their bets

Viruses with genomes greater than 300 kb and up to 1200 kb are being discovered with increasing frequency. These large viruses (often called giruses) can encode up to 900 proteins and also many tRNAs. Consequently, these viruses have more protein-encoding genes than many bacteria, and the concept of small particle/small genome that once defined viruses is no longer valid. Giruses infect bacteria and animals although most of the recently discovered ones infect protists. Thus, genome gigantism is not restricted to a specific host or phylogenetic clade. To date, most of the giruses are associated with aqueous environments. Many of these large viruses (phycodnaviruses and Mimiviruses) probably have a common evolutionary ancestor with the poxviruses, iridoviruses, asfarviruses, ascoviruses, and a recently discovered Marseillevirus. One issue that is perhaps not appreciated by the microbiology community is that large viruses, even ones classified in the same family, can differ significantly in morphology, lifestyle, and genome structure. This review focuses on some of these differences rather than provides extensive details about individual viruses.

DNA Viruses: The Really Big Ones (Giruses). Annu Rev Microbiol. May 12 2010 | PDF

Related:

• DNA Viruses
• Marine mimivirus relatives are large algal viruses
• Marvellous mimivirus

The evolutionary dynamics of RNA viruses are complex and their high mutation rates, rapid replication kinetics, and large population sizes present a challenge to traditional population genetics. Quasispecies theory is a mathematical framework that was initially formulated to explain the evolution of life in the “precellular RNA world”. It builds on classical population genetics, but seeks to explore the consequences of error-prone replication and near-infinite population sizes for genome evolution. More recently, quasispecies theory has been used to describe the evolutionary dynamics of RNA viruses, and many of its predictions have been validated experimentally in model systems. Some of these observations challenge more traditional views of evolution and have profound implications for the control and treatment of viral diseases.

This article explains basic aspects of quasispecies theory, describe key experiments that define “quasispecies effects” and highlights how these results may shape our view of viral pathogenesis, antiviral drug development, and vaccine design. It stresses three clinically relevant principles. First, the fitness of a particular virus sequence may be determined more by its freedom to mutate into related sequences than by its own replicative capacity. Second, many viruses operate near a threshold of “error catastrophe” and may be combated by increasing their replication error rates. Third, increasing the fidelity of genome replication may paradoxically attenuate viruses.

Quasispecies Theory and the Behavior of RNA Viruses. PLoS Pathog 6(7): e1001005. doi:10.1371/journal.ppat.1001005
A large number of medically important viruses, including HIV, hepatitis C virus, and influenza, have RNA genomes. These viruses replicate with extremely high mutation rates and exhibit significant genetic diversity. This diversity allows a viral population to rapidly adapt to dynamic environments and evolve resistance to vaccines and antiviral drugs. For the last 30 years, quasispecies theory has provided a population-based framework for understanding RNA viral evolution. A quasispecies is a cloud of diverse variants that are genetically linked through mutation, interact cooperatively on a functional level, and collectively contribute to the characteristics of the population. Many predictions of quasispecies theory run counter to traditional views of microbial behavior and evolution and have profound implications for our understanding of viral disease. Here, we discuss basic principles of quasispecies theory and describe its relevance for our understanding of viral fitness, virulence, and antiviral therapeutic strategy.

Related:

• Do viruses evolve to protect their hosts?
• Does the outcome of HCV infection vary with the infecting virus type?

Pathogenicity islands have a major role in spreading virulence genes among bacterial populations. A notable example are the phage-related pathogenicity islands of staphylococci, the SaPIs, which are responsible for the inter- as well as intrageneric spread of toxins – such as TSST-1 (toxic shock syndrome toxin) and other superantigens – through the exploitation of specific staphylococcal helper phages for high-frequency transfer within phage-encoded particles. Toxic shock syndrome is a rare, potentially fatal illness that can be caused by the release of toxins from Staphylococcus. The toxic particles are encoded by discrete genetic units called pathogenicity islands, which reside passively in the host chromosome, under the control of the global repressor Stl, unless activated by a helper phage. This paper shows that a non-essential and specific protein from the helper phage 80α is responsible for de-repression of the pathogenicity island, providing the mechanism for the first step of its mobilization. The proteins involved are ‘moonlighters’, because they have two different and genetically distinct activities. Through a remarkable evolutionary adaptation, various related pathogenicity islands co-opt entirely unrelated phage proteins to aid in their mobilization.

Moonlighting bacteriophage proteins derepress staphylococcal pathogenicity islands. 2010 Nature. 465(7299): 779-782
Staphylococcal superantigen-carrying pathogenicity islands (SaPIs) are discrete, chromosomally integrated units of ~15 kilobases that are induced by helper phages to excise and replicate. SaPI DNA is then efficiently encapsidated in phage-like infectious particles, leading to extremely high frequencies of intra- as well as intergeneric transfer. In the absence of helper phage lytic growth, the island is maintained in a quiescent prophage-like state by a global repressor, Stl, which controls expression of most of the SaPI genes4. Here we show that SaPI derepression is effected by a specific, non-essential phage protein that binds to Stl, disrupting the Stl–DNA complex and thereby initiating the excision-replication-packaging cycle of the island. Because SaPIs require phage proteins to be packaged, this strategy assures that SaPIs will be transferred once induced. Several different SaPIs are induced by helper phage 80α and, in each case, the SaPI commandeers a different non-essential phage protein for its derepression. The highly specific interactions between different SaPI repressors and helper-phage-encoded antirepressors represent a remarkable evolutionary adaptation involved in pathogenicity island mobilization.

Related:

• Genomic islands in bacterial evolution
• Spread of Pathogenicity Islands in Escherichia coli

A bacteria that lives in hot springs in Japan may help solve one of the mysteries of the early evolution of complex organisms. It may also be the key to 21st century biofuel production. Group II introns are bacterial mobile elements thought to be ancestors of introns and retroelements in higher organisms. They comprise a catalytically active intron RNA and an intron-encoded reverse transcriptase, which promotes splicing of the intron from precursor RNA and integration of the excised intron into new genomic sites. While most bacteria have small numbers of group II introns, in the thermophilic cyanobacterium Thermosynechococcus elongatus, a single intron has proliferated and constitutes 1.3% of the genome. Researchers investigated how the T. elongatus introns proliferated to such high copy numbers. They found divergence of DNA target specificity, evolution of reverse transcriptases that splice and mobilize multiple degenerate introns, and preferential insertion into other mobile introns or insertion elements, which provide new integration sites in non-essential regions of the genome. Unlike mesophilic group II introns, the thermophilic T. elongatus introns rely on higher temperatures to help promote DNA strand separation, facilitating access to DNA target sites. They discuss how these mechanisms, including elevated temperature, might have contributed to intron proliferation in early eukaryotes.

Mechanisms Used for Genomic Proliferation by Thermophilic Group II Introns. 2010 PLoS Biol 8(6): e1000391. doi:10.1371/journal.pbio.1000391
Mobile group II introns, which are found in bacterial and organellar genomes, are site-specific retroelments hypothesized to be evolutionary ancestors of spliceosomal introns and retrotransposons in higher organisms. Most bacteria, however, contain no more than one or a few group II introns, making it unclear how introns could have proliferated to higher copy numbers in eukaryotic genomes. An exception is the thermophilic cyanobacterium Thermosynechococcus elongatus, which contains 28 closely related copies of a group II intron, constituting ,1.3% of the genome. Here, by using a combination of bioinformatics and mobility assays at different temperatures, we identified mechanisms that contribute to the proliferation of T. elongatus group II introns. These mechanisms include divergence of DNA target specificity to avoid target site saturation; adaptation of some intron-encoded reverse transcriptases to splice and mobilize multiple degenerate introns that do not encode reverse transcriptases, leading to a common splicing apparatus; and preferential insertion within other mobile introns or insertion elements, which provide new unoccupied sites in expanding non-essential DNA regions. Additionally, unlike mesophilic group II introns, the thermophilic T. elongatus introns rely on elevated temperatures to help promote DNA strand separation, enabling access to a larger number of DNA target sites by base pairing of the intron RNA, with minimal constraint from the reverse transcriptase. Our results provide insight into group II intron proliferation mechanisms and show that higher temperatures, which are thought to have prevailed on Earth during the emergence of eukaryotes, favor intron proliferation by increasing the accessibility of DNA target sites. We also identify actively mobile thermophilic introns, which may be useful for structural studies, gene targeting in thermophiles, and as a source of thermostable reverse transcriptases.

Chlamydia pneumoniae is an intracellular bacterial pathogen with an extremely diverse host range (humans, amphibians, reptiles and marsupials). C. pneumoniae exposure is widespread in humans, with sero-prevalence studies reporting 50% infection levels by age 20 and reaching 80% in the elderly. In humans, C. pneumoniae infections can range from asymptomatic to severe respiratory disease, including pneumonia. Less common presentations include bronchitis, pharyngitis, laryngitis and sinusitis, making up 5% of cases. In addition to respiratory infections in humans, C. pneumoniae has also been associated with atherosclerosis and stroke, myocarditis, multiple sclerosis and Alzheimer’s disease.

Despite the widespread prevalence of C. pneumoniae in humans, all isolates studied to date are extremely similar at the DNA level. Four C. pneumoniae human isolates have had their full genome sequenced. Genomic comparisons revealed a highly conserved (>99.9%) gene order and organisation, with few deletions and less than 300 single nucleotide polymorphisms (SNPs) distinguishing the isolates. This near clonality of C. pneumoniae human isolates that are temporally and geographically separate, has been taken to indicate that human infections are a relatively recent event and that the efficient respiratory spread of the agent explains how 60–80% of adults worldwide have been infected at least once in their lifetime .

Researchers selected 23 target genes in this organism to investigate genetic diversity: seven of these had been lost or gained by C. pneumoniae, a further six were conserved, four were polymorphic, and six were truncated or length polymorphic in one strain or the other. The results highlights that C. pneumoniae animal isolates are much more genetically diverse than C. pneumoniae human isolates, and have crossed the host barrier to humans on at least two occasions. This study provides new insights into the evolution of this complex pathogen.

Chlamydia pneumoniae Is Genetically Diverse in Animals and Appears to Have Crossed the Host Barrier to Humans on (At Least) Two Occasions. 2010 PLoS Pathog 6(5): e1000903. doi:10.1371/journal.ppat.1000903

Related:

• Chlamydia use proteolysis to evade host defences