Posts Tagged ‘RNA’

Over at Principles of Molecular Virology: RNAi – miRNA – siRNA – do you know the difference?

Thursday, November 21st, 2013

Principles of Molecular Virology, Chapter 6, discusses RNA interference and virus infection.

 

RNA Interference RNA interference (RNAi) is an important set of pathways that are used to regulate gene expression. RNAi is a blanket term which can refer to both small interfering RNAs (siRNAs) and microRNAs (miRNAs). There are important differences between siRNAs and miRNAs but I’ll get to those later…

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Tricks an IRES uses to enslave ribosomes

Sunday, October 21st, 2012

Poliovirus IRES In eukaryotes, mRNAs are primarily translated through a cap-dependent mechanism whereby initiation factors recruit the 40S ribosomal subunit to a cap structure at the 5′ end of the mRNA. However, some viral and cellular messages initiate protein synthesis without a cap. They use a structured RNA element termed an internal ribosome entry site (IRES) to recruit the 40S ribosomal subunit. IRESs were discovered over 20 years ago, but only recently have studies using a model IRES from dicistroviruses expanded our understanding of how a 3D RNA structure can capture and manipulate the ribosome to initiate translation.

 

Tricks an IRES uses to enslave ribosomes. Trends Microbiol. 31 Aug 2012

Self-amplifying RNA vaccines

Monday, August 27th, 2012

Vaccination Vaccines are a triumph of medicine and science – but is the pipeline running dry? What about all those viruses we have not been able to make effective vaccines against – HIV, RSV, Ebola, etc? DNA vaccines have generally proved to be disappointing in clinical trials. This interesting new paper in PNAS suggests possible future strategies.

 

Nonviral delivery of self-amplifying RNA vaccines. PNAS USA 20 August2012, doi: 10.1073/pnas.12093671
Despite more than two decades of research and development on nucleic acid vaccines, there is still no commercial product for human use. Taking advantage of the recent innovations in systemic delivery of short interfering RNA (siRNA) using lipid nanoparticles (LNPs), we developed a self-amplifying RNA vaccine. Here we show that nonviral delivery of a 9-kb self-amplifying RNA encapsulated within an LNP substantially increased immunogenicity compared with delivery of unformulated RNA. This unique vaccine technology was found to elicit broad, potent, and protective immune responses, that were comparable to a viral delivery technology, but without the inherent limitations of viral vectors. Given the many positive attributes of nucleic acid vaccines, our results suggest that a comprehensive evaluation of nonviral technologies to deliver self-amplifying RNA vaccines is warranted.

Inhibition and avoidance of mRNA degradation by RNA viruses

Friday, June 8th, 2012

RNA The cellular mRNA decay machinery plays a major role in regulating the quality and quantity of gene expression in cells. This machinery involves multiple enzymes and pathways that converge to promote the exonucleolytic decay of mRNAs. The transcripts made by RNA viruses are susceptible to degradation by this machinery and, in fact, can be actively targeted. To maintain gene expression and replication, RNA viruses have evolved a number of strategies to avoid and/or inactivate aspects of the cellular mRNA decay machinery. Recent work uncovering the mechanisms used by RNA viruses to maintain the stability of their transcripts is described in this review.

 

Inhibition and avoidance of mRNA degradation by RNA viruses. Curr Opin Microbiol. 23 May 2012

Rates of Virus Evolution Are Linked to Host Geography in Bats

Friday, May 18th, 2012

Myotis lucifugus Rapid evolution of RNA viruses is intimately linked to their success in overcoming the defenses of their hosts. Several studies have shown that rates of viral evolution can vary dramatically among distantly related viral families. Variability in the speed of evolution among closely related viruses has received less attention, but could be an important determinant of the geographic or host species origins of viral emergence if certain species or regions promote especially rapid evolution.

A new paper uses a dataset of rabies virus sequences collected from bat species throughout the Americas to test the role of inter-specific differences in reservoir host biology on the tempo of viral evolution. This shows the annual rate of molecular evolution to be a malleable trait of viruses that is accelerated in subtropical and tropical bats compared to temperate species. The association between geography and the speed of evolution appears to reflect differences in the seasonality of rabies virus transmission in different climatic zones.

These results illustrate that the viral mechanisms commonly invoked to explain heterogeneous rates of evolution among viral families may be insufficient to explain evolution in multi-host viruses and indicate a role for host biology in shaping the speed of viral evolution.

 

Rates of Viral Evolution Are Linked to Host Geography in Bat Rabies. (2012) PLoS Pathog 8(5):e1002720. doi:10.1371/journal.ppat.1002720
Rates of evolution span orders of magnitude among RNA viruses with important implications for viral transmission and emergence. Although the tempo of viral evolution is often ascribed to viral features such as mutation rates and transmission mode, these factors alone cannot explain variation among closely related viruses, where host biology might operate more strongly on viral evolution. Here, we analyzed sequence data from hundreds of rabies viruses collected from bats throughout the Americas to describe dramatic variation in the speed of rabies virus evolution when circulating in ecologically distinct reservoir species. Integration of ecological and genetic data through a comparative Bayesian analysis revealed that viral evolutionary rates were labile following historical jumps between bat species and nearly four times faster in tropical and subtropical bats compared to temperate species. The association between geography and viral evolution could not be explained by host metabolism, phylogeny or variable selection pressures, and instead appeared to be a consequence of reduced seasonality in bat activity and virus transmission associated with climate. Our results demonstrate a key role for host ecology in shaping the tempo of evolution in multi-host viruses and highlight the power of comparative phylogenetic methods to identify the host and environmental features that influence transmission dynamics.

First RNA virus-encoded miRNAs

Tuesday, February 14th, 2012

miRNAs

Although the first miRNA was identified 18 years ago, it was only in 2001, with the development of technologies that allowed the efficient cDNA cloning and sequencing of small RNA species, that it became apparent that all multicellular eukaryotes encode numerous members of this class of small regulatory RNAs. Shortly after the identification of the first human miRNAs, the first virally encoded miRNAs were reported in the human herpesvirus Epstein–Barr virus (EBV). Although this initial discovery suggested that viruses in general might use miRNAs to down-regulate cellular factors that inhibit viral replication, the subsequent analysis of a wide range of RNA viruses failed to identify any viral miRNAs. However, virally encoded miRNAs are expressed by many members of the herpesvirus family of nuclear DNA viruses and are also found in a small number of other nuclear DNA viruses, particularly polymaviruses.

Although >250 viral microRNAs (miRNAs) are expressed by a range of nuclear DNA viruses, efforts to identify miRNAs expressed by RNA viruses have so far been in vain. In PNAS, Kincaid et al. report the identification of five miRNAs encoded by the delta retrovirus bovine leukemia virus (BLV) that are expressed in BLV-transformed B cells. It appears likely that these viral miRNAs play an important role in BLV pathogenesis:

MicroRNA expression by an oncogenic retrovirus. PNAS USA, 30 Jan 2012

RNA virus microRNA that mimics a B-cell oncomiR. PNAS USA, 30 Jan 2012; doi: 10.1073/pnas.1116107109

The transcriptome of the adenovirus infected cell

Wednesday, February 1st, 2012

Adenovirus transcription 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.

Negative strand RNA viruses – the state of the art

Wednesday, January 18th, 2012

Virus Research 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

 

Go FISH

Friday, November 25th, 2011

FISH 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.