MicrobiologyBytes

Bacteriophage lysis is a precisely scheduled process controlled by proteins of the holin family. Holins are a diverse class of small phage-encoded membrane proteins. The best studied holin is S105, a 105-residue polypeptide with three transmembrane domains (TMDs) encoded by the S gene of phage λ. Throughout the period of late gene expression and particle assembly, S105 accumulates in the cytoplasmic membrane of Escherichia coli without any effect on its integrity. Suddenly, at a programmed time, S105 triggers to form a lesion, or hole, in the membrane; this allows the λ-endolysin, R, to escape from the cytoplasm and attack the cell wall. In phages of Gram-negative hosts, there is a third step to complete the lysis pathway involving a protein or protein complex, the spanin, which connects the cytoplasmic and outer membranes. In λ, the spanin complex consists of the cytoplasmic membrane protein, Rz, and the outer membrane lipoprotein, Rz1. This complex is thought to act by disrupting the outer membrane, possibly by fusion with the inner membrane. The large holes observed can be viewed as supporting the notion that at the time of lethal triggering, the S105 holin exists in such large aggregates, leading to one or a small number of holes rather than many smaller holes distributed throughout the membrane.

Micron-scale holes terminate the phage infection cycle. PNAS USA January 11 2010. doi: 10.1073/pnas.0914030107
Holins are small phage-encoded proteins that accumulate harmlessly in the cytoplasmic membrane during the phage infection cycle until suddenly, at an allele-specific time, triggering to form lethal lesions, or “holes.” In the phages λ and T4, the holes have been shown to be large enough to allow release of prefolded active endolysin from the cytoplasm, which results in destruction of the cell wall, followed by lysis within seconds. Here, the holes caused by S105, the λ-holin, have been captured in vivo by cryo-EM. Surprisingly, the scale of the holes is at least an order of magnitude greater than any previously described membrane channel, with an average diameter of 340 nm and some exceeding 1 μm. Most cells exhibit only one hole, randomly positioned in the membrane, irrespective of its size. Moreover, on coexpression of holin and endolysin, the degradation of the cell wall leads to spherically shaped cells and a collapsed inner membrane sac. To obtain a 3D view of the hole by cryo-electron tomography, we needed to reduce the average size of the cells significantly. By taking advantage of the coupling of bacterial cell size and growth rate, we achieved an 80% reduction in cell mass by shifting to succinate minimal medium for inductions of the S105 gene. Cryotomographic analysis of the holes revealed that they were irregular in shape and showed no evidence of membrane invagination. The unexpected scale of these holes has implications for models of holin function.

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• How can lytic viruses be evolutionarily competitive?

Aedes aegypti is the urban vector of dengue viruses worldwide. While climate influences the geographical distribution of this mosquito species, other factors also determine the suitability of the physical environment for this mosquito. Importantly, the close association of Ae. aegypti with humans and the domestic environment allows this species to persist in regions that may otherwise be unsuitable based on climatic factors alone. This review highlights the need to incorporate the impact of the urban environment in attempts to model the potential distribution of Ae. aegypti and briefly discuss the potential for future technology to aid management and control of this widespread vector species.

The dengue vector Aedes aegypti: What comes next. Microbes Infect. Jan 20 2010. doi:10.1016/j.micinf.2009.12.011

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• Population movement a critical factor in dengue virus spread
• Dengue Virus
• Rethinking dengue hemorrhagic fever
• Pathogenic Flaviviruses

There is strong evidence to suggest that climate change has, and will continue to affect the occurrence, distribution and prevalence of livestock diseases in Great Britain (GB). This paper reviews how climate change could affect livestock diseases in GB. Factors influenced by climate change and that could affect livestock diseases include the molecular biology of the pathogen itself; vectors (if any); farming practice and land use; zoological and environmental factors; and the establishment of new microenvironments and microclimates. The interaction of these factors is an important consideration in forecasting how livestock diseases may be affected. Risk assessments should focus on looking for combinations of factors that may be directly affected by climate change, or that may be indirectly affected through changes in human activity, such as land use (e.g. deforestation), transport and movement of animals, intensity of livestock farming and habitat change. A risk assessment framework is proposed, based on modules that accommodate these factors. This framework could be used to screen for the emergence of unexpected disease events.

The effect of climate change on the occurrence and prevalence of livestock diseases in Great Britain: a review. J Appl Microbiol. (2009) 106(5): 1409-1423

Related:

• Bluetongue virus
• MicrobiologyBytes: Climate Change

MicroRNAs (miRNAs) are ~22-nt regulatory RNAs expressed by all multicellular eukaryotes. Humans encode >700 miRNAs and similar numbers are likely to exist in other mammalian species. Almost all cellular miRNAs are initially transcribed by RNA polymerase II (Pol II) as part of a long, capped, polyadenylated primary miRNA (pri-miRNA) precursor. The miRNA forms part of one arm of an RNA stem-loop that consists of an ~32-bp imperfect stem flanked by unstructured RNA sequences. This stem-loop is recognized by the nuclear RNase III enzyme Drosha, which cleaves the stem to liberate an ~60-nt pre-miRNA hairpin. The pre-miRNA is then transported to the cytoplasm where it is cleaved by a second RNase III enzyme, called Dicer, which removes the terminal loop to generate the miRNA duplex intermediate. One strand of this duplex is incorporated into the RNA-induced silencing complex (RISC), where it acts as a guide RNA to direct RISC to complementary mRNA species. Depending on the level of complementarity, RISC can either cleave bound mRNAs and/or inhibit their translation. Inhibition of mRNA translation generally requires full complementarity of the mRNA to nucleotides 2 through 7 or 8 from the miRNA 5′ end – the miRNA seed region. The primary, and possibly sole, function of mammalian miRNAs is therefore to act as specific post-transcriptional inhibitors of mRNA function.

1. Do All Viruses Encode miRNAs? In general, we can divide mammalian viruses into three categories, i.e., the herpesviruses, which encode multiple viral miRNAs; other nuclear DNA viruses, which may encode one or two miRNAs; and the RNA viruses and cytoplasmic DNA viruses, which appear to lack any miRNAs.
2. What Do Viral miRNAs Do? Virus miRNAs may serve two major functions. First, to inhibit the expression of cellular factors that play a role in cellular innate or adaptive antiviral immune responses. Second, to downregulate the expression of virus regulatory proteins.
3. Are Virus miRNAs Conserved between Related Virus Species? No, but the miRNAs of individual viruses are conserved.
4. Do Viruses Regulate miRNA Transcription, Processing, or Function? At present, there is no evidence indicating that viruses modify the cellular environment to selectively favor the processing and expression of viral miRNAs, although viral miRNAs may be expressed at such high levels that they have the potential to competitively inhibit cellular miRNA function.
5. Can Viruses Use Cellular miRNAs to Promote Their Replication? At least one clear-cut example of a cellular miRNA that facilitates virus replication is known, activation of HCV replication by the liver-specific miRNA miR-122.

Five Questions about Viruses and MicroRNAs. PLoS Pathog 6(2): e1000787. doi:10.1371/journal.ppat.1000787

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• Virus-encoded microRNAs in herpesvirus biology
• Human Cytomegalovirus-encoded microRNA regulates expression of multiple genes involved in replication
• Cellular Genes Targeted by KSHV MicroRNAs
• RNAi and Cold Sores

Metagenomics is a discipline that enables the genomic study of uncultured microorganisms. Faster, cheaper sequencing technologies and the ability to sequence uncultured microbes sampled directly from their habitats are expanding and transforming our view of the microbial world. Distilling meaningful information from the millions of new genomic sequences presents a serious challenge to bioinformaticians. In cultured microbes, the genomic data come from a single clone, making sequence assembly and annotation tractable. In metagenomics, the data come from heterogeneous microbial communities, sometimes containing more than 10,000 species, with the sequence data being noisy and partial. From sampling, to assembly, to gene calling and function prediction, bioinformatics faces new demands in interpreting voluminous, noisy, and often partial sequence data. Although metagenomics is a relative newcomer to science, the past few years have seen an explosion in computational methods applied to metagenomic-based research. It is therefore not within the scope of this article to provide an exhaustive review. Rather, we provide here a concise yet comprehensive introduction to the current computational requirements presented by metagenomics, and review the recent progress made. We also note whether there is software that implements any of the methods presented here, and briefly review its utility. Nevertheless, it would be useful if readers of this article would avail themselves of the comment section provided by this journal, and relate their own experiences. Finally, the last section of this article provides a few representative studies illustrating different facets of recent scientific discoveries made using metagenomics.

A Primer on Metagenomics. 2010 PLoS Comput Biol 6(2): e1000667. doi:10.1371/journal.pcbi.1000667

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• Vineyardomics: SOS – Save our Shiraz!
• New dimensions of the virus world discovered through metagenomics
• A Glimpse Into the World of Metagenomics

Hepatitis delta virus (HDV) was first discovered in 1977 among a group of patients infected with hepatitis B virus (HBV). Subsequent studies revealed that HDV is a replication-defective virus which requires a helper virus, HBV, to supply the hepatitis B surface antigen (HBsAg) for virion assembly and infectivity. Being a human pathogen, HDV infection may lead to progressive chronic liver disease and occasional fulminant hepatitis in patients coinfected or superinfected with HBV. In recent years, the incidence of new HDV infections has significantly declined in some parts of the world due to HBV vaccination. However, investigation of the HDV replication cycle has raised many issues which molecular biologists are interested in.

Unlike other RNA satellite viruses which rely on the RNA-dependent RNA polymerase (RdRp) provided by the coexisting helper virus for genome replication, the dependence of HDV on HBV is limited to the supply of HBsAg for the production of HDV viral particle. Similar to plant viroids which do not encode RdRp, HDV undergoes robust RNA replication autonomously once inside the cells. Thus, it is certain that HDV and plant viroids have to replicate their RNA genome using a cellular enzyme(s). Unlike plant viroids which do not encode any protein, HDV encodes a protein, hepatitis delta antigen (HDAg), which is intimately involved in its RNA replication. In addition, HDV RNA not only has to replicate itself but also needs to transcribe a subgenomic mRNA species coding for HDAg. The transcription of the HDAg-encoding mRNA has all of the hallmarks of the cellular mRNA transcription except for the nature of the template (DNA versus RNA). Therefore, distinct from plant viroids, HDV represents a hybrid of the conventional DNA-dependent transcription and the unique RNA-dependent RNA synthesis in the absence of an RdRP. To coordinate with this sophisticated and unique RNA amplification process in mammalian cells, HDAg plays important regulatory roles which will be reviewed herein. Additionally, the nature of the enzyme involved in HDV RNA replication will also be addressed.

This review article examines the HDV RNA replication cycle, with emphasis on the function of HDAg in modulating RNA replication and the nature of the enzyme involved:

Hepatitis Delta Virus RNA Replication. Viruses 2009, 1(3), 818-831 doi:10.3390/v1030818

Related:

• Hepatitis B and C viruses
• Update on HBV and HCV Therapy

With an amazingly small genome size (250–400 nt) and simple structure, viroids are giants in terms of functional versatility in the world of RNA. Without encoding proteins to provide specific functions and without being protected in a protein or membrane shell, these single-stranded, circular RNAs can triumph over the cellular RNA degradation machineries and further enlist host-encoded factors for replicating themselves in specific subcellular compartments and trafficking throughout a plant. They can also cause devastating plant diseases.

Viroids are single-stranded, circular, and noncoding RNAs that infect plants. They replicate in the nucleus or chloroplast and then traffic cell-to-cell through plasmodesmata and long distance through the phloem to establish systemic infection. They also cause diseases in certain hosts. All functions are mediated directly by the viroid RNA genome or genome-derived RNAs. This review summarizes recent advances in the understanding of viroid structures and cellular factors enabling these functions, emphasizing conceptual developments, major knowledge gaps, and future directions. Newly emerging experimental systems and research tools are discussed that are expected to enable significant progress in a number of key areas. It highlights examples of groundbreaking contributions of viroid research to the development of new biological principles and offer perspectives on using viroid models to continue advancing some frontiers of life science.

The Biology of Viroid-Host Interactions. 2009 Annual Review of Phytopathology 47: 105-131 doi: 10.1146/annurev-phyto-080508-081927

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• Viroids and Virusoids
• Smallest Things Considered – Viroids
• Resistance to Plant Viruses

Grapevines are an important global crop which are widely planted throughout temperate regions. Viruses are a significant factor in reducing the quality and quantity of the yield and are known to reduce the productive life of vineyards. Grapevines are subject to infection by more than 60 different viruses, the most known for any crop plant. Most important grapevine virus diseases are caused by complexes of viruses, with up to nine different viruses having been identified in a single vine. In South Africa, as in most grape-growing regions of the world, grapevine leafroll is regarded to be the most significant virus disease affecting grapevine, with Shiraz disease and Shiraz decline becoming more prominent as emerging diseases in the industry.

Present disease diagnostics rely on ELISA or RT-PCR and target the viruses that have historically been associated with these diseases. While these tests are highly specific, they may not result in an accurate reflection of the etiological status of the tested plant, or of the particular disease, since none of the current diagnostic techniques address the potential contribution of other known or unknown viruses that may be involved in the etiology of a particular disease. Moreover, the error prone replication of RNA viruses leads to quasispecies, which can further complicate PCR-based detection assays as not all variants of the virus may be detected.

New and powerful technologies which are able to sequence viruses from environmental samples without the need for laborious and costly purification, cloning and screening techniques can result in the generation of sequence information for the complete virome in an unbiased fashion. This paper describes the use of sequencing-by-synthesis technology on the massively parallel Illumina Genome Analyzer II, to sequence an environmental sample composed of 44 randomly selected vines, to determine the virus profile of a severely diseased vineyard.

Deep sequencing analysis of viruses infecting grapevines: Virome of a vineyard. Virology. Feb 19 2010
Double stranded RNA, isolated from 44 pooled randomly selected vines from a diseased South African vineyard, has been used in a deep sequencing analysis to build a census of the viral population. The dsRNA was sequenced in an unbiased manner using the sequencing-by-synthesis technology offered by the Illumina Genome Analyzer II and yielded 837 megabases of metagenomic sequence data. Four known viral pathogens were identified. It was found that Grapevine leafroll-associated virus 3 (GLRaV-3) is the most prevalent species, constituting 59% of the total reads, followed by Grapevine rupestris stem pitting-associated virus and Grapevine virus A. Grapevine virus E, a virus not previously reported in South African vineyards, was identified in the census. Viruses not previously identified in grapevine were also detected. The second most prevalent virus detected was a member of the Chrysoviridae family similar to Penicillium chrysogenum virus. Sequences aligning to two other mycoviruses were also detected.

Related:

• New dimensions of the virus world discovered through metagenomics
• A Glimpse Into the World of Metagenomics
• Genetic and environmental factors affect gene expression in yeast

The flavivirus genus includes viruses with a remarkable ability to produce disease on a large scale. The expansion and increased endemicity of dengue and West Nile viruses in the Americas exemplifies their medical and epidemiological importance. The rapid detection of virus infection and induction of the innate antiviral response are crucial to determining the outcome of infection. The intracellular pathogen receptors RIG-I and MDA5 play a central role in detecting flavivirus infections and initiating a robust antiviral response. Yet, these viruses are still capable of producing acute illness in humans. It is now clear that flaviviruses utilize a variety of mechanisms to modulate the interferon response. The non-structural proteins of the various flaviviruses reduce expression of interferon dependent genes by blocking phosphorylation, enhancing degradation or down-regulating expression of major components of the JAK/STAT pathway. Recent studies indicate that interferon modulation is an important factor in the development of severe flaviviral illness. This suggests that an increased understanding of viral-host interactions will facilitate the development of novel therapeutics to treat these viral infections and improved biological models to study flavivirus pathogenesis.

How Flaviviruses Activate and Suppress the Interferon Response. Viruses 2010, 2(2), 676-691; doi:10.3390/v2020676

Related:

• MicrobiologyBytes: Flaviviruses
• MicrobiologyBytes: Interferon