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- A. Te Velthuis (United States of America)
Mechanisms of Replication and Assembly
- A. Te Velthuis (United States of America)
Abstract
Abstract Body
Influenza A viruses are important human pathogens that cause moderate to severe disease. The molecular processes that determine the outcome of influenza virus infection in humans are multifactorial and involve a complex interplay between host, viral and bacterial factors. It is generally accepted that a strong innate immune dysregulation known as the ‘cytokine storm’ contributes to the pathology of infections with the 1918 H1N1 pandemic or the highly pathogenic avian influenza viruses of the H5N1 subtype. The RNA sensor retinoic acid-inducible gene I (RIG-I) plays an important role in sensing viral RNA and initiating a signalling cascade that leads to interferon expression. During infection, the influenza A virus RNA polymerase produces both full-length and aberrant RNA molecules, such as defective viral genomes (DVG) and mini viral RNAs (mvRNA). Subsequent innate immune activation involves the binding of the viral RNAs to RIG-I. However, it is not clear what factors determine which influenza A virus RNAs are RIG-I agonists and what molecular steps lead to the detection of the infection by RIG-I. We will discuss how innate immune activation by mvRNAs is determined, in part, by transient RNA structures, called template loops (t-loop), that stall the viral RNA polymerase. Impairment of replication by t-loops depends on the formation of an RNA duplex near the template entry and exit channels of the RNA polymerase, and this effect is enhanced by mutation of the template exit path from the RNA polymerase active site. Overall, these findings provide a mechanism that links aberrant viral replication to the activation of the innate immune response.
ROLE OF PLATELET FACTOR 4 IN FLAVIVIRUS REPLICATION
- A. SINGH (India)
Abstract
Background and Aims
Recently we described that platelet factor 4 (PF4) is pro-viral for Dengue virus (DV) and Japanese Encephalitis virus (JEV). PF4 bound to receptor CXCR3 on monocytes and downregulated the interferon (IFN) production by targeting p38-MAPK-STAT2-IRF9 axis. We investigated the role of PF4 on viral replication using wild type (WT), PF4-knockout (PF4-ko) and PF4-overexpressed (PF4-oe) mice.
Methods
We infected monocyte and microglia cells with DV and JEV respectively in presence of PF4 and assessed virus replication in vitro. We assessed the virus replication in vivo in JEV infected PF4-oe and PF4-ko mice.
Results
A significantly elevated replication of both DV and JEV was observed in cells in vitro. A decreased replication of JEV was observed in PF4-ko mice brain. A similar observation of decreased replication of mouse-adapted DV2 strain was noted in monocytes isolated from PF4-ko mice. Decreased colocalization of JEV Capsid and LAMP1 was observed in vitro in cell lines in presence of PF4, similarly, in microglia isolated from PF4-oe mice, indicating a less interaction of virus containing endosomes with lysosomal vesicles. Besides, a decreased LTR stained acidic vacuoles existed in presence of PF4, indicating its role in cytosolic acidification. Thus, suggesting an involvement of endosomal-lysosomal pathway in the replication of these flaviviruses in presence of PF4. Elevation of PF4 is associated with DV infection.
Conclusions
PF4 is helping the DV and JEV propagation by affecting acidification and delaying lysosomal degradation of virus containing endosomes.
CHARACTERIZATION OF HOST FACTORS ASSOCIATED WITH THE INTERNAL RIBOSOMAL ENTRY SITES OF FOOT-AND-MOUTH DISEASE AND CLASSICAL SWINE FEVER VIRUSES
- K. Tsukiyama-Kohara (Japan)
Abstract
Background and Aims
Foot-and-mouth disease virus (FMDV) and classical swine fever virus (CSFV) possess positive-sense single-stranded RNA genomes and an internal ribosomal entry site (IRES) element within their 5¢-untranslated regions.
Methods
To investigate the common host factors associated with these IRESs, we established cell lines expressing a bicistronic luciferase reporter plasmid containing an FMDV-IRES or CSFV-IRES element between the Renilla and firefly luciferase genes.
Results
First, we treated FMDV-IRES cells with the French maritime pine extract, Pycnogenol (PYC), and examined its suppressive effect on FMDV-IRES activity, as PYC has been reported to have antiviral properties. Next, we performed microarray analysis to identify the host factors that modified their expression upon treatment with PYC, and confirmed their function using specific siRNAs. We found that polycystic kidney disease 1-like 3 (PKD1L3) and ubiquitin-specific peptidase 31 (USP31) were associated with FMDV-IRES activity. Moreover, silencing of these factors significantly suppressed CSFV-IRES activity.
Conclusions
Thus, PKD1L3 and USP31 are host factors associated with the functions of FMDV- and CSFV-IRES elements.
ACTIVATION OF UNFOLDED PROTEIN RESPONSE DURING HIV-1 INFECTION AND REGULATION OF VIRION INFECTIVITY BY IRE1
- A. Tripathi (India)
Abstract
Background and Aims
HIV-1 and other viruses, depend on cellular Endoplasmic Reticulum (ER) for translation, protein folding and maturation. This causes ER stress leading to the Unfolded Protein Response (UPR). Viruses have evolved strategies to manipulate the UPR for their own benefit. However, the UPR modulation by HIV-1 and its functional significance has not been explored in depth. Our study aims to assess the effect of HIV-1 on UPR and its role in HIV-1 replication and infectivity.
Methods
HIV-1 infected CD4+T-cells were profiled for various UPR markers. Knockdown of UPR markers was conducted using sh/siRNAs and the viral gene expression and infectivity was analyzed using luciferase reporter assay, p24 ELISA and β-gal staining assay. Effect of selected viral protein on UPR was assessed by utilizing mutants and selective UPR inhibitor. Pharmaceutical ER stress inducers were tested for their anti-HIV activity.
Results
HIV-1 induces ER stress and activates UPR in CD4+T-cells with differential expression of phospho-IRE1α, phospho-PERK, cleaved ATF6α and their downstream targets. Knockdown of UPR markers leads to decrease in viral replication. IRE1α knockdown results in reduced virion infectivity through gp120. Moreover, HIV-1 Nef was found to regulate IRE1α. Also, pharmacological inducers which overstimulate ER stress, shows significant anti-HIV activity.
Conclusions
HIV-1 differentially regulates various UPR markers, suggesting a virus-specific UPR modulation. The results demonstrate that the UPR activation works in favor of HIV-1 replication. Nef mediated regulation of IRE1α and gp120, provides a novel mechanism involved in virion infectivity. Finally, overstimulation of ER stress using chemical inducers, which can negatively regulate UPR, shows anti-HIV activity.