MicrobiologyBytes

By convention, the human adenovirus replication cycle is divided into two phases, an early and a late phase, which are separated by the onset of viral DNA replication. Based on temporal changes of the gene expression pattern as revealed by DNA microarray analysis, adenovirus type 2 (Ad2) infection in human primary lung fibroblasts can be divided into four periods. The first period is from 0 to 12 h after infection before or shortly after adenoviral gene expression has commenced. During this time, changes in cellular gene expression are likely to be triggered by the virus entry process, such as attachment of virus to cell surface receptors, and its intracellular transport along microtubules.

The second period covers the time from 12 to 24 h after infection and follows activation of the immediate early E1A gene. During this period, there is an increase in the number of differentially expressed cellular genes. About 50% of these genes are involved in cell cycle regulation, cell proliferation and antiviral response. The third period extends from 24 to 42 h after infection. By this time, the virus has gained control of the cellular metabolic machinery, resulting in an efficient replication of the viral genome. Additional changes in cellular gene expression are modest during this phase. During the fourth and last period, when the cytopathic effect becomes apparent, the number of down-regulated genes increases dramatically including many genes involved in intra- and extracellular structure.

The most intensive battle between the adenovirus and its host takes place during the second period after adenovirus genes expression has started. The major functions of the early gene products are to force the host cell to enter the S phase in order to provide optimal conditions for viral DNA replication and to suppress the host antiviral response. Adenoviruses encode several regulatory proteins within the early regions E1A, E1B, E3, and E4. The immediate-early E1A gene encodes two regulators of viral and cellular gene expression, the E1A-243R and E1A-289R proteins. The E1A proteins act as promiscuous transcriptional activators or repressors of cellular genes. E1A proteins are essential for promoting the host cell to enter the S phase. This is achieved by the binding of the E1A proteins to members of the retinoblastoma tumor suppressor (pRB) family, thereby releasing the E2F transcription factors, which are activators of genes required in the S-phase.

The transcriptome of the adenovirus infected cell. Virology. 9 Jan 2012
Alternations of cellular gene expression following an adenovirus type 2 infection of human primary cells were studied by using superior sensitive cDNA sequencing. In total, 3791 cellular genes were identified as differentially expressed more than 2-fold. Genes involved in DNA replication, RNA transcription and cell cycle regulation were very abundant among the up-regulated genes. On the other hand, genes involved in various signaling pathways including TGF-β, Rho, G-protein, Map kinase, STAT and NF-κB stood out among the down-regulated genes. Binding sites for E2F, ATF/CREB and AP2 were prevalent in the up-regulated genes, whereas binding sites for SRF and NF-κB were dominant among the down-regulated genes. It is evident that the adenovirus has gained a control of the host cell cycle, growth, immune response and apoptosis at 24h after infection. However, efforts from host cell to block the cell cycle progression and activate an antiviral response were also observed.

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It was my priveledge to work with Brian Mahy many years ago. Brian has just retired as long-serving Editor of Virus Research, and his swansong is an excellent special issue on negative strand RNA viruses – an important read for all virologists and an even more impirtant one for all aspiring virologists.

Virus Research: Negative Strand RNA Viruses Special Issue

• Insights on influenza pathogenesis from the grave
• Taming influenza viruses
• Induction and evasion of type I interferon responses by influenza viruses
• Immune responses to influenza virus infection
• Novel vaccines against influenza viruses
• Prospects for controlling future pandemics of influenza
• New concepts in measles virus replication: Getting in and out in vivo and modulating the host cell environment
• Recombinant vaccines against the mononegaviruses—What we have learned from animal disease controls
• Biological feasibility of measles eradication
• Progress in understanding and controlling respiratory syncytial virus: Still crazy after all these years
• An unconventional pathway of mRNA cap formation by vesiculoviruses
• Rhabdovirus accessory genes
• Structural insights into the rhabdovirus transcription/replication complex
• Hantavirus pulmonary syndrome
• Progress in recombinant DNA-derived vaccines for Lassa virus and filoviruses
• Borna disease virus – Fact and fantasy
• A review of Nipah and Hendra viruses with an historical aside
• Negative-strand RNA viruses: The plant-infecting counterparts
• Quasispecies as a matter of fact: Viruses and beyond

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The introduction of rRNA-targeted fluorescence in situ hybridization (FISH) using oligonucleotide probes for the cultivation-independent identification of microbes more than 20 years ago marked the beginning of a new era for environmental and medical microbiology. When integrated into the so-called full-cycle rRNA approach, FISH enables microbiologists to decipher complete structures of microbial communities in a quantitative manner. Furthermore, this phylogenetic staining technique in its basic format is easy to apply and once probes have been designed and evaluated, the detection of their target organisms in environmental or medical samples is straightforward and can be completed in a few hours. In its original format, fluorescent monolabeled oligonucleotide probes are used for FISH, but as the signal intensity of this technique is insufficient for cells with low ribosome contents, FISH detection efficiencies in oligotrophic environments are generally rather low. For such systems, catalyzed reporter deposition (CARD)-FISH, which exploits horseradish peroxidase (HRP)-labeled oligonucleotide probes and tyramide signal amplification is the method of choice to capture most microbial community members.

rRNA-targeting FISH techniques are continuously developed further and major improvements regarding increased cell permeability, accessibility of probe target sites, probe specificity, signal intensity, and so on have been achieved. A second rapidly evolving FISH-related research area is the combination of rRNA-FISH with other techniques, which provide additional information on (i) the presence of specific genes or mRNA molecules of the target cell, (ii) its specific metabolic activity or (iii) important environmental parameters such as the concentration of chemical compounds in the vicinity of the detected cell. For this purpose rRNA-FISH or CARD-FISH have been combined with various other FISH techniques and staining procedures as well as with microautoradiography, microelectrode measurements, Raman microspectroscopy, and NanoSIMS. This review provides an overview on the most recent developments in the FISH field.

New trends in fluorescence in situ hybridization for identification and functional analyses of microbes. Curr Opin Biotechnol. Nov 11 2011
Fluorescence in situ hybridization (FISH) has become an indispensable tool for rapid and direct single-cell identification of microbes by detecting signature regions in their rRNA molecules. Recent advances in this field include new web-based tools for assisting probe design and optimization of experimental conditions, easy-to-implement signal amplification strategies, innovative multiplexing approaches, and the combination of FISH with transmission electron microscopy or extracellular staining techniques. Further emerging developments focus on sorting FISH-identified cells for subsequent single-cell genomics and on the direct detection of specific genes within single microbial cells by advanced FISH techniques employing various strategies for massive signal amplification.

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Inflammatory responses generated during virus infections are critical for antiviral immune responses. The exact virus recognition elements that activate cells to induce pro-inflammatory signals are not completely characterized. Virus recognition elements such as dsRNA and 5′-triphosphate single-stranded RNA are recognized by several cellular pathways. The intracellular or extracellular interaction of cells with virus recognition elements results in activation of innate immune responses as indicated by expression of inflammatory cytokines and chemokines. In addition to the innate immune inflammation, virus recognition elements trigger establishment of an antiviral state, under which each cell resists virus infection. The resistance to virus infection is in part through inhibition of virus replication by perturbation of RNA and protein synthesis. Determining the exact mechanisms by which immune and antivirus responses are activated is essential for understanding virus pathogenesis.

RNA with high inosine content is not commonly found in normally growing eukaryotic cells but it is present during infections with DNA and RNA viruses such as polyomavirus, Rous-associated virus, vesicular stomatitis virus, measles virus, and respiratory syncytial virus. Extracellular Ino-RNA is generated during virus infections. Cell lysis occurs frequently during virus infections, which results in the release of cell content, including intracellular generated Ino-RNA, into the extracellular space. Extracellular dsRNA has been shown to be able to stimulate antiviral responses in neighboring, uninfected cells.

Using RSV infection as a model, this paper reports that the presence of inosines in ssRNA is a potent inducer of inflammatory cytokines and the antiviral state during virus infection and suggests that Ino-RNA, of virus or cellular origin, in the surrounding tissue after release from infected cells is a signal for the presence of virus infections.

Inosine-Containing RNA Is a Novel Innate Immune Recognition Element and Reduces RSV Infection. (2011) PLoS ONE 6(10): e26463. doi:10.1371/journal.pone.0026463
During viral infections, single- and double-stranded RNA (ssRNA and dsRNA) are recognized by the host and induce innate immune responses. The cellular enzyme ADAR-1 (adenosine deaminase acting on RNA-1) activation in virally infected cells leads to presence of inosine-containing RNA (Ino-RNA). Here we report that ss-Ino-RNA is a novel viral recognition element. We synthesized unmodified ssRNA and ssRNA that had 6% to16% inosine residues. The results showed that in primary human cells, or in mice, 10% ss-Ino-RNA rapidly and potently induced a significant increase in inflammatory cytokines, such as interferon (IFN)-β (35 fold), tumor necrosis factor (TNF)-α (9.7 fold), and interleukin (IL)-6 (11.3 fold) (p

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A hallmark of negative-strand RNA viruses is that their genomes never exist as free RNA, but instead are always assembled with many copies of a single nucleoprotein, N, to form highly stable nucleocapsids. Moreover, viral genomes are the only RNAs in infected cells that are assembled with N. The mechanism by which this specific association occurs, for both the segmented and non-segmented viruses, has recently become clearer due to our expanding knowledge of N protein and nucleocapsid structures.

Nucleoproteins and nucleocapsids of negative-strand RNA viruses. Curr Opin Microbiol. Aug 6 2011

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Synthesis of subgenomic (SG) messenger RNAs (mRNAs) by (+)strand RNA viruses allows the differential expression of specific virus genes, both quantitatively and temporally. SG RNAs have the following properties:

• They are made in infected cells but do not interfere with the normal course of virus replication
• The SG RNA sequences are shorter than their cognate genomic RNAs
• Their sequences are usually co-terminal with the 3′ genomic sequence but sometimes are co-terminal with the 5′ sequences. Yet other viruses make SG RNAs which contain a 5′ co-terminal leader joined to a 3′ co-terminal sequence
• Typically, whether a messenger SG RNA contains only one ORF, or multiple ORFs, with some rare exceptions, only the 5′ ORF is translated. Although most SG RNAs function as messengers and are translated, other SG RNAs, generally those with 5′ co-terminal sequences, have other functions.

Subgenomic messenger RNAs: mastering regulation of (+)-strand RNA virus life cycle. Virology. 2011 412(2):245-255
Many (+)-strand RNA viruses use subgenomic (SG) RNAs as messengers for protein expression, or to regulate their viral life cycle. Three different mechanisms have been described for the synthesis of SG RNAs. The first mechanism involves internal initiation on a (-)-strand RNA template and requires an internal SGP promoter. The second mechanism makes a prematurely terminated (-)-strand RNA which is used as template to make the SG RNA. The third mechanism uses discontinuous RNA synthesis while making the (-)-strand RNA templates. Most SG RNAs are translated into structural proteins or proteins related to pathogenesis: however other SG RNAs regulate the transition between translation and replication, function as riboregulators of replication or translation, or support RNA-RNA recombination. In this review we discuss these functions of SG RNAs and how they influence viral replication, translation and recombination.

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Eucaryotic organisms depend on networks of gene regulatory pathways. Small RNAs (sRNAs), are key components of these networks. sRNAs are short (21–24 nt in length), endogenously expressed, and are processed from double stranded (ds)RNAs or dsRNA-like precursors. In both plants and animals, sRNAs exert their functions upon incorporation into ribonucleoprotein silencing complexes and through their base-paring capacity. They are implicated in a variety of processes, including post-transcriptional regulation of mRNA, mRNA stability and availability for translation, establishment of heterochromatin and silencing of transposons. Different classes of sRNAs differ in the proteins required in their biogenesis, the constitution of ribonucleoprotein complexes that mediate their regulatory functions, their type of gene regulation, and the biological functions in which they are implicated. Plants display a remarkable diversity of sRNA types and sRNA pathways, likely needed for managing multiple environmental stimuli, including biotic and abiotic stresses. Several lines of evidence suggest that plant sRNAs play critical roles in plant–pathogen interactions. Indeed, upon infection, most plant pathogens can interfere with the expression of endogenous sRNAs, thus altering the expression of specific host factors implicated in the suppression or in the activation of plant defences. Evidence for these phenomena has been reported for bacterial and fungal pathogens.

Viruses are obligate infectious agents, whose life cycle (expression of viral proteins, viral genome replication and virion assembly) is integrated with host cell functions. Plant viruses can both modify the profiles of endogenous sRNAs (in common with bacteria and fungi) and induce the production of additional sRNAs derived from their own genomes (viral sRNAs; vsiRNAs). The latter gives a clear indication of the activation of RNA silencing-based responses of the plant. In some cases, this results in reduction of the titre of the invading virus and, in recovery of upper, non-inoculated leaves. To counteract RNA silencing, many plant viruses have evolved proteins (viral suppressors of RNA silencing: VSR) that target various components of the plant silencing machinery. Viruses can induce specific symptoms resembling developmental anomalies and affecting organs and tissues such as leaves, flowers and fruits. These anomalies are often reconcilable with virus-induced alterations of RNA silencing-based endogenous pathways, due to: i) the direct activity of VSRs on endogenous sRNAs or on silencing related effectors; ii) the abundance of vsiRNAs in competition with endogenous sRNAs; iii) the action of specific vsiRNAs entering into RNA silencing complexes and targeting specific host genes.

This review provides an overview of the major cellular RNA silencing pathways in plants with particular reference to those involved in antiviral functions and highlights examples of the complex interactions between viral molecular processes and host RNA processes.

Viral induction and suppression of RNA silencing in plants. Biochim Biophys Acta. Apr 30 2011
RNA silencing in plants and insects can function as a defence mechanism against invading viruses. RNA silencing-based antiviral defence entails the production of virus-derived small interfering RNAs which guide specific antiviral effector complexes to inactivate viral genomes. As a response to this defence system, viruses have evolved viral suppressors of RNA silencing (VSRs) to overcome the host defence. VSRs can act on various steps of the different silencing pathways. Viral infection can have a profound impact on the host endogenous RNA silencing regulatory pathways; alterations of endogenous short RNA expression profile and gene expression are often associated with viral infections and their symptoms. Here we discuss our current understanding of the main steps of RNA-silencing responses to viral invasion in plants and the effects of VSRs on endogenous pathways. This article is part of a Special Issue entitled: MicroRNAs in viral gene regulation.

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The role of RNA localization in controlling gene expression and RNA stability in eukaryotes has been well established, but until recently there were no bacterial RNAs known to localize to specific subcellular sites. Studies in Caulobacter crescentus and Escherichia coli have now demonstrated that both regulatory RNAs and mRNAs can be concentrated at fixed positions in bacterial cells. tmRNA, a regulatory RNA involved in trans-translation, is localized to a helical structure in C. crescentus, and some mRNAs are localized to discrete spots in C. crescentus and E. coli. Given that bacteria also localize proteins, plasmids, and regions of the chromosome, the localization of RNA may not be particularly surprising. Nonetheless, the possibility that RNA activity and abundance can be controlled by subcellular localization represents a new paradigm for regulation of RNA, and suggests that many new discoveries of bacterial RNA localization lie ahead. Why are RNAs localized, and how is RNA localization achieved? Our understanding is far from complete because these questions are just beginning to be addressed, yet it is now reasonable to review what is known about the mechanisms and physiological rationales for localization of RNAs in bacteria, and to speculate about what additional pathways remain to be discovered.

RNA localization in bacteria. Curr Opin Microbiol. 2011 14(2):155-159
Bacteria localize proteins and DNA regions to specific subcellular sites, and several recent publications show that RNAs are localized within the cell as well. Localization of tmRNA and some mRNAs indicates that RNAs can be sequestered at specific sites by RNA binding proteins, or can be trapped at the location where they are transcribed. Although the functions of RNA localization are not yet completely understood, it appears that one function of RNA localization is to regulate RNA abundance by controlling access to nucleases. New techniques for visualizing RNAs will likely lead to increased examination of spatial control of RNAs and the role this control plays in the regulation of gene expression and bacterial physiology.

The fact that all other viruses encapsidate single copy genomes begs the question of why retroviruses co-package two genomics RNSa (gRNAs). One reason may be economy of scale: RNA dimerization allows formation of a unique structure – the dimer linkage – that distinguishes gRNAs from mRNAs. Monomeric HIV-1 RNAs are packaged when engineered with tandem dimer linkages, underscoring the importance of this RNA structure, and not gRNA counting per se, to packaging.

Co-packaging gRNAs allows retroviruses to generate intact proviruses despite pervasive gRNA nicking. Template switching during reverse transcription is probably why retroviruses maintain infectivity when their gRNAs are damaged by gamma rays, and why retroviruses are much less radiation-sensitive than RNA viruses like vescicular stomatitis virus. Researchers previously thought retroviral recombination might be mutagenic, but these notions have been dispelled. HIV-1 particles with two gRNAs generate full-length proviruses more efficiently than virions engineered to contain single gRNAs. Thus, another advantage of gRNA dimers appears to be increased replication fidelity.

Co-packaging gRNAs promotes higher recombination frequencies for retroviruses than all other viruses, allowing rapid loss of deleterious alleles and re-assortment of genome segments. With approximately three to ten crossovers occurring during the synthesis of every provirus, recombination is perhaps 10-fold more frequent than reverse transcriptase base substitution rates, and is an evolutionary driving force for retroviruses such as HIV-1 that display high levels of replication and multi-strain infection.

These observations help seal the case for likely evolutionary advantages of dimeric genome packaging. The DLS in its immature form, which results only upon association of two gRNAs, likely provides the means for selective gRNA packaging. The need to generate an intact provirus provides a strong motive for packaging redundant genetic information. And because retroviruses encapsidate gRNA dimers, recombination can provide the opportunity for almost limitless combinatorial genetic sampling.

Retroviral RNA Dimerization and Packaging: The What, How, When, Where, and Why. (2010) PLoS Pathog 6(10): e1001007. doi:10.1371/journal.ppat.1001007

Related:

• HIV-1 Genomic RNA Trafficking
• The shape of HIV RNA