Sindbis virus usurps the cellular HUR protein to stabilize its transcripts and promote infections of mammalian and mosquito cells

2010 Summer.Includes bibliographic references (pages 174-208).Covers not scanned.Print version deaccessioned 2022.Members of the genus Alphavirus are recognized as significant human pathogens. Infection of vertebrate hosts often results in febrile illness and occasionally severe encephalitis. The archetypical alphavirus is Sindbis virus, which we have utilized in these studies. The genomic and subgenomic RNAs of Sindbis virus strongly resemble cellular mRNAs as they are capped at their 5’ ends and polyadenylated at their 3’ termini. These features allow the viral RNAs to act like cellular mRNAs and make them prime substrates for the cellular mRNA decay machinery. Sindbis virus RNAs are indeed subject to degradation by the cellular mRNA decay machinery in cell culture models of infection. Nevertheless, they decay by a mechanism that is different from the majority of cellular mRNAs as the decay of Sindbis virus transcripts is predominantly deadenylation-independent. As cellular mRNAs are often regulated by elements present in their 3’ untranslated regions (UTR), we hypothesized that these viral 3’UTR elements were functioning similarly to cellular mRNA stability elements resulting in the enhancement of viral infection. The primary goal of the research described in this dissertation was to characterize in mechanistic detail how the Sindbis virus 3’UTR represses deadenylation. To this end we used both cell free extracts and tissue culture systems to assay the effects of the viral 3’UTR on transcript stability. Interestingly, multiple elements were found to be independently repressing deadenylation in mosquito cytoplasmic extracts. Further examination revealed that a major stability determinant was the U-rich element (URE) observed in the 3’UTR of many alphaviruses. The ability to repress deadenylation in our cell free extract system was similarly observed with the UREs of Venezuelan equine, eastern equine, western equine and Semliki Forest viruses. Taken together, these data strongly assert that the repression of deadenylation via the URE is evolutionarily conserved. Prior to this study, the URE had no ascribed function. The repression of deadenylation imparted by the URE correlated with the binding of a cellular 38kDa factor. This 38kDa factor was determined to be the cellular HuR protein. Both the human and mosquito HuR proteins were found to bind with high affinity to the Sindbis virus 3’UTR. Reduction of cellular HuR protein levels using RNAi resulted in an increase in the rate of viral RNA decay. Furthermore, a significant decrease in the titer of progeny virus was observed. A similar effect on viral titer was observed when the predominant HuR binding site, the URE, was deleted from the viral 3’UTR. Taken together these observations identify a novel Alphavirus/ host interface that significantly impacts viral biology. Furthermore these studies have confirmed our hypothesis that the members of genus Alphavirus have indeed evolved RNA stability elements that resemble cellular mRNA stability elements for the purpose of enhancing viral infection. Furthermore these studies identify a potential therapeutic anti-viral target - the cellular HuR protein

A study of the mass loss rates of symbiotic star systems

The amount of mass loss in symbiotic systems is investigated, specifically mass loss via the formation of jets in R Aquarii (R Aqr). The jets in R Aqr have been observed in the X-ray by Chandra over a four year time period. The jet changes on times scales of a year and new outflows have been observed. Understanding the amount of mass and the frequency of ejection further constrain the ability of the white dwarf in the system to accrete enough mass to become a Type 1a supernova progenitor. The details of multi-wavelength studies, such as speed, density and spatial extent of the jets will be discussed in order to understand the mass balance in the binary system. We examine other symbiotic systems to determine trends in mass loss in this class of objects.Comment: To be published in the proceedings of "The Multicoloured Landscape of Compact Objects and their Explosive Origins

Symbiotic Stars as Laboratories for the Study of Accretion and Jets: A Call for Optical Monitoring

Symbiotic binary stars typically consist of a white dwarf (WD) that accretes material from the wind of a companion red giant. Orbital periods for these binaries are on the order of years, and their relatively small optical outbursts tend to occur every few years to decades. In some symbiotics, material that is transferred from the red giant to the WD forms a disk around the WD. Thus, symbiotic stars are a bit like overgrown cataclysmic variables (CVs), but with less violent eruptions. Symbiotic stars are not as well understood as CVs, in part because their longer variability time scales mean that observations over many years are required to cover different outburst states and orbital phases. The recent discovery of collimated outflows ("jets") from a number of symbiotics, provides a new motivation for such long-term study of these objects. Astrophysical jets are observed in almost every type of accretion-powered system, and symbiotic stars may help us understand these structures. In symbiotics, most jets appear to be associated with optical eruptions. Optical monitoring by amateurs can identify systems in outburst, and also help to build a comprehensive database of outburst and quiescent symbiotic light curves. Together with radio through X-ray observations that will be performed when new outbursts are found, long-term optical light curves will improve understanding of symbiotic outbursts, jet production, and the connection between outbursts, jets, and accretion disks in symbiotic stars.Comment: 14 pages, 3 figures. Invited review paper to appear in the Journal of the American Association of Variable Star Observers (AAVSO

Swift observations of the 2015 outburst of AG Peg -- from slow nova to classical symbiotic outburst

Symbiotic stars often contain white dwarfs with quasi-steady shell burning on their surfaces. However, in most symbiotics, the origin of this burning is unclear. In symbiotic slow novae, however, it is linked to a past thermonuclear runaway. In June 2015, the symbiotic slow nova AG Peg was seen in only its second optical outburst since 1850. This recent outburst was of much shorter duration and lower amplitude than the earlier eruption, and it contained multiple peaks -- like outbursts in classical symbiotic stars such as Z And. We report Swift X-ray and UV observations of AG Peg made between June 2015 and January 2016. The X-ray flux was markedly variable on a time scale of days, particularly during four days near optical maximum, when the X-rays became bright and soft. This strong X-ray variability continued for another month, after which the X-rays hardened as the optical flux declined. The UV flux was high throughout the outburst, consistent with quasi-steady shell burning on the white dwarf. Given that accretion disks around white dwarfs with shell burning do not generally produce detectable X-rays (due to Compton-cooling of the boundary layer), the X-rays probably originated via shocks in the ejecta. As the X-ray photo-electric absorption did not vary significantly, the X-ray variability may directly link to the properties of the shocked material. AG Peg's transition from a slow symbiotic nova (which drove the 1850 outburst) to a classical symbiotic star suggests that shell burning in at least some symbiotic stars is residual burning from prior novae.Comment: Accepted by MNRAS 23 June 2016. Manuscript submitted in original form 5 April 201

A NuSTAR observation of the fast symbiotic nova V745 Sco in outburst

The fast recurrent nova V745 Sco was observed in the 3-79 keV X-rays band with NuSTAR 10 days after the optical discovery. The measured X-ray emission is consistent with a collisionally ionized optically thin plasma at temperature of about 2.7 keV. A prominent iron line observed at 6.7 keV does not require enhanced iron in the ejecta. We attribute the X-ray flux to shocked circumstellar material. No X-ray emission was observed at energies above 20 keV, and the flux in the 3-20 keV range was about 1.6 $\times$ 10$^{-11}$ erg cm$^{-2}$ s$^{-1}$. The emission measure indicates an average electron density of order of 10$^7$ cm$^{-3}$. The X-ray flux in the 0.3-10 keV band almost simultaneously measured with Swift was about 40 times larger, mainly due to the luminous central supersoft source emitting at energy below 1 keV. The fact that the NuSTAR spectrum cannot be fitted with a power law, and the lack of hard X-ray emission, allow us to rule out Comptonized gamma rays, and to place an upper limit of the order of 10$^{-11}$ erg cm$^{-2}$ s$^{-1}$ on the gamma-ray flux of the nova on the tenth day of the outburst.Comment: in press in Monthly Notices of the Royal Astronomical Society, 201