Antagonism of Innate Immunity by Paramyxovirus Accessory Proteins

Paramyxovirinae, a subfamily of Paramyxoviridae, are negative strand RNA viruses comprised of many important human and animal pathogens, which share a high degree of genetic and structural homology. The accessory proteins expressed from the P/V/C gene are major factors in the pathogenicity of the viruses, because of their ability to abrogate various facets of type I interferon (IFN) induction and signaling. Most of the paramyxoviruses exhibit a commonality in their ability to antagonize innate immunity by blocking IFN induction and the Jak/STAT pathway. However, the manner in which the accessory proteins inhibit the pathway differs among viruses. Similarly, there are variations in the capability of the viruses to counteract intracellular detectors (RNA helicases, mda-5 and RIG-I). Furthermore, a functional specificity in the antagonism of the IFN response has been reported, suggesting that specificity in the circumvention of innate immunity restricts viral host range. Available evidence indicates that paramyxoviruses employ specific strategies to antagonize the IFN response of their specific hosts, which is one of the major factors that determine viral pathogenicity and host range

Trafficking of Sendai Virus Nucleocapsids Is Mediated by Intracellular Vesicles

Paramyxoviruses are assembled at the plasma membrane budding sites after synthesis of all the structural components in the cytoplasm. Although viral ribonuclocapsid (vRNP) is an essential component of infectious virions, the process of vRNP translocation to assembly sites is poorly understood.To analyze real-time trafficking of vRNPs in live infected cells, we created a recombinant Sendai virus (SeV), rSeVLeGFP, which expresses L protein fused to enhanced green fluorescent protein (eGFP). The rSeVLeGFP showed similar growth kinetics compared to wt SeV, and newly synthesized LeGFP could be detected as early as 8 h postinfection. The majority of LeGFP co-localized with other components of vRNPs, NP and P proteins, suggesting the fluorescent signals of LeGFP represent the locations of vRNPs. Analysis of LeGFP movement using time-lapse digital video microscopy revealed directional and saltatory movement of LeGFP along microtubules. Treatment of the cells with nocodazole restricted vRNP movement and reduced progeny virion production without affecting viral protein synthesis, suggesting the role of microtubules in vRNP trafficking and virus assembly. Further study with an electron microscope showed close association of vRNPs with intracellular vesicles present in infected cells. In addition, the vRNPs co-localized with Rab11a protein, which is known to regulate the recycling endocytosis pathway and Golgi-to-plasma membrane trafficking. Simultaneous movement between LeGFP and Rab11a was also observed in infected cells, which constitutively express mRFP-tagged Rab11a. Involvement of recycling endosomes in vRNP translocation was also suggested by the fact that vRNPs move concomitantly with recycling transferrin labeled with Alexa 594.Collectively, our results strongly suggest a previously unrecognized involvement of the intracellular vesicular trafficking pathway in vRNP translocation and provide new insights into the transport of viral structural components to the assembly sites of enveloped viruses

Target genes, variants, tissues and transcriptional pathways influencing human serum urate levels.

Elevated serum urate levels cause gout and correlate with cardiometabolic diseases via poorly understood mechanisms. We performed a trans-ancestry genome-wide association study of serum urate in 457,690 individuals, identifying 183 loci (147 previously unknown) that improve the prediction of gout in an independent cohort of 334,880 individuals. Serum urate showed significant genetic correlations with many cardiometabolic traits, with genetic causality analyses supporting a substantial role for pleiotropy. Enrichment analysis, fine-mapping of urate-associated loci and colocalization with gene expression in 47 tissues implicated the kidney and liver as the main target organs and prioritized potentially causal genes and variants, including the transcriptional master regulators in the liver and kidney, HNF1A and HNF4A. Experimental validation showed that HNF4A transactivated the promoter of ABCG2, encoding a major urate transporter, in kidney cells, and that HNF4A p.Thr139Ile is a functional variant. Transcriptional coregulation within and across organs may be a general mechanism underlying the observed pleiotropy between urate and cardiometabolic traits.The Genotype-Tissue Expression (GTEx) Project was supported by the Common Fund of the Office of the Director of the National Institutes of Health, and by NCI, NHGRI, NHLBI, NIDA, NIMH, and NINDS. Variant annotation was supported by software resources provided via the Caché Campus program of the InterSystems GmbH to Alexander Teumer

Defining the active site of 2-(2'-hydroxyphenyl) benzenesulfinate desulfinase

Diminishing resources of light crude oils containing low sulfur fractions have compelled petroleum refineries to process sour crude oils. Current sulfur removal processes include the use of hydrodesulfurization. Hydrodesulfurization uses an inorganic catalyst, hydrogen, high temperature and pressure to remove sulfur from petroleum leaving the sulfur in the form of hydrogen sulfide, resulting in environmental problems. Since larger volumes of sour crude oils, in the form of middle distillates, are being used in processing, the use of hydrodesulfurization has become costly and inefficient. Additionally, governmental regulation of the allowable sulfur content in refined fossil fuels has become more stringent. These trends have led scientists to research new desulfurization methods. One such method is biodesulfurization, which utilizes microbes that possess carbon-sulfur bond cleavage capabilities to remove the sulfur. These microbes utilize multiple enzyme pathways to perform the desulfurization of compounds. An example of a carbon-sulfur bond cleaving organism is Rhodococcus erythropolis IGTS8. R. erythropolis uses a metabolic pathway consisting of four enzymes. This pathway catalyzes the desulfurization of dibenzothiophene to sulfite and hydroxybiphenol. The research conducted for this project has focused on the enzyme found in the final step of the pathway. The enzyme, known as 2-(2' -hydroxyphenyl) benzenesulfinate desulfinase (HPBS desulfinase), is considered the rate limiting step in the pathway. The initial phase of this study included the purification ofHPBS desulfinase. The enzyme was purified 260-fold from R. erythropolis. Purification of the enzyme was monitored using UV -Vis, spectrofluorimetric assays, and SDS-PAGE. In order to define the active site of HPBS desulfinase, it was necessary to determine what types of compounds could be used as substrates or inhibitors. Twenty-one commercially available analogs of both the substrate (HPBS) and the product (HBP) were tested, along with two synthesized substrate analogs, and none were found to act as substrates. However, several behaved as competent inhibitors, and the two synthesized analogs increased the activity of the enzyme. Analysis of the two synthesized analogs was performed using 13C NMR, 1H NMR, mass spectroscopy, TLC, and pKa determination. Further studies were conducted using thiourea dioxide and CuCh, which had both previously been shown to inhibit HPBS desulfinase. The results from the current study agree. In addition, it was determined that CuCh binds to the cysteine in the active site and not the sulfonic acid on the substrate. Chemical modification of the cysteine in the active site using DTNB showed that once modified, the enzyme showed no activity in the presence of substrate. Finally, a fluorescence method of determining inhibitor binding was developed and a Ko of 1.6 µM was calculated for 1, 8-naphthosultam' s ( an inhibitor) binding to the enzyme.Chemistry and Biochemistr

Mechanisms involved in the host range restriction of parainfluenza

Thesis (Ph. D.)--University of Rochester. School of Medicine and Dentistry. Dept. of Microbiology and Immunology, 2009.An essential means to understanding virus-host interaction is to determine what makes a specific cell type or host permissive or non-permissive for virus replication. Many viruses are able to productively infect only a limited number of host species. The molecular basis behind this restricted host range involves the most fundamental aspects of the virus-host interaction. With the recent emergence of highly pathogenic animal pathogens, which have evolved the ability to infect human hosts, it has become extremely important to understand the mechanism behind host range restriction. As a model to study host range, the paramyxoviruses Sendai virus (SeV) and human parainfluenza virus type 1 (hPIV1) are ideal since they are highly homologous and structurally similar, but maintain distinct host ranges, murine versus human. The gene that is likely to determine the host range of parainfluenza viruses is the P/C gene. It expresses accessory proteins, which are known to counteract the IFN activities of host cells. In addition to the C proteins, SeV, but not hPIV1, expresses V protein from the gene. To evaluate the role and specificity of the anti-IFN activities of the P/C gene products in host range restriction, we determined the infectivity and anti-IFN activities of the viruses in human and murine cultured lung cells. SeV and hPIV1 infection were able to block the Jak/STAT pathway in both murine and human cells, through the inhibition of STAT1 nuclear localization. However, the viruses could not overcome antiviral activities induced by IFN pre-treatment in their non-native hosts, suggesting specificity in anti-IFN activity. We further characterized the specificity of the P/C gene products in antagonizing the signaling pathway that leads to IFN production, using a recombinant SeV, rSeVhP, whose P/C gene was replaced with that of hPIV1. Our data indicate that the ability of the P/C gene products to inhibit IFN induction is species specific, and the SeV V protein plays a central role in the antagonism of IFN induction in murine cells. We also found that the hPIV1 P/C gene product cannot prevent infected murine cells from apoptotic cell death, and the SeV V protein is required for this protection. Our study indicates specificity in the activity of hPIV1 and SeV P/C gene products in antagonizing IFN induction and apoptosis, which is likely to be one of the factors that affect the host range of these viruses. In addition to anti-innate activity, C protein has been suggested to have a role in viral assembly. We found that production of hPIV1 or rSeVhP from murine cells was limited, and large aggregates of viral nucleocapsids (vRNP) were detected in infected cells. Upon addition of the SeV C proteins, vRNP distribution was restored similar to SeV, suggesting interplay of the C proteins with specific host cellular machinery to mediate transport of vRNPs and virus assembly. It is not known, however, how vRNPs are transported from areas of synthesis to sites of assembly at the plasma membrane. We created a recombinant SeV, rSeVLeGFP, which expresses the L protein fused to enhanced green fluorescent protein (eGFP), and visualized trafficking of vRNP in live infected cells, using time-lapse digital video microscopy. We captured directional, saltatory movement suggestive of cargo transport along the microtubule network. Disruption of microtubules by nocodazole-treatment inhibited vRNP movement, and reduced the progeny virion production without affecting viral protein synthesis. Additional study using electron microscopy and membrane flotation assays revealed vRNP association with vesicles present in the infected cells. Our results suggest that SeV utilize cellular vesicles for transport of vRNPs on microtubules to plasma membrane budding sites. The factors involved in viral host range restriction are multi-faceted. Here we have shown specificity of the P/C gene products in counteracting the anti-viral response of host cells. These gene products also play a role in viral assembly and production of progeny virions. Thus, the interactions of the P/C gene products with host cellular machinery are an essential determinant in host range restriction

Target genes, variants, tissues and transcriptional pathways influencing human serum urate levels

Elevated serum urate levels cause gout and correlate with cardio-metabolic diseases via poorly understood mechanisms. We performed a trans-ethnic genome-wide association study of serum urate among 457,690 individuals, identifying 183 loci (147 novel) that improve prediction of gout in an independent cohort of 334,880 individuals. Serum urate showed significant genetic correlations with many cardio-metabolic traits, with genetic causality analyses supporting a substantial role for pleiotropy. Enrichment analysis, fine-mapping of urate-associated loci, and co-localization with gene expression in 47 tissues implicated kidney and liver as main target organs and prioritized potentially causal genes and variants, including the transcriptional master regulators in liver and kidney, HNF1A and HNF4A. Experimental validation showed that HNF4A trans-activated the promoter of the major urate transporter ABCG2 in kidney cells, and that HNF4A p.Thr139Ile is a functional variant. Transcriptional co-regulation within and across organs may be a general mechanism underlying the observed pleiotropy between urate and cardio-metabolic traits.Accepted Manuscrip