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[object Object],The cultured cells used in this study were Hepatitis C Virus that consist of a small positive stranded RNA genome. The outer coat of this genome is covered by “lipid bilayer envelope”. This envelope consist of E1 and E2 glycoproteins and are anchored as heterodimers. “Glycoproteins are proteins that contain oligosaccharide chains covalently attached to polypeptide side chains” 2. What is the difference between HCVcc and HCVpp? HCVcc- Hepatitis C Virus Cell culture—a infection of the “human hepatoma cell line Huh-7 with genomic HCV RNA”. “Viruses that are recovered from infected animals are able to vigorously reproduce in a cell culture”. In this system the whole life cycle of a virus is apparent which includes entry, replication, assembly and release. HCVpp- Hepatitis C Virus pseudoparticles—first in vitro infection system that was made to study the entry and neutralization of major human pathogens. They are “lentiviral vectors that incorporate the HCV glycoproteins E1 and E2 on the viral envelope”. Lentiviral vectors are retro viruses that infect both dividing and nondividing cells. This is due to the preintegration complex that can go through the membrane of the target cell. These viruses work well for gene therapy. 3. Why did Coller et al. (2009) use a siRNA library in their study? What does this siRNA library consist of? we analyzed a siRNA library that targets 140 cellular membrane trafficking genes to identify host genes required for infectious HCV production and HCV pseudoparticle entry. This approach identified 16 host cofactors of HCV entry that function primarily in clathrin-mediated endocytosis, including components of the clathrin endocytosis machinery, actin polymerization, receptor internalization and sorting, and endosomal acidification. 4. What evidence reported by Coller et al. (2009) indicates that HCV gains access to cells via clathrin-mediated endocytosis? Small interfering RNAs (siRNAs) targeting the clathrin machinery inhibit HCV entry, suggesting that HCV uses clathrin-mediated endocytosis, however, HCV has not yet been visualized within a clathrin coated vesicle 5. Which siRNAs significantly inhibit HVCpp luciferase expression? CFL1, CLBT, CLTCL1, SYT1 CFL1 (85%), CTCL1 (73%), SYT1 (64%), CLTB (63%), and CBL (57%). 6. What role are tight junction proteins clausdin-1 and occludin thought to play in HCV entry into cells? Subsequent internalization of HCV requires a complex set of receptor molecules including, the tetraspannin molecule CD81, scavenger receptor B-1 (SR-BI), and the tight junction proteins claudin(s) (CLDN)-1, -6, -9, and occludin (OCDN) to bind and enter into hepatocytes [2]–[14], [22]–[26]. HCV internalization has never been visualized, but is postulated to occur at the tight junction where the entry receptors claudin-1 and occludin localize. Recombinant, soluble E2 re-localizes the CD81 receptor to the tight junction. Engagement of CD81 also activates the Rho GTPase, CDC42, which may result in actin cytoskeletal rearrangements that would allow for trafficking of HCV to the tight junction 7. Do the results presented by the authors support the hypothesis that tight junction proteins are involved in HCV viral entry into cells? Consider results shown in figure 7 and 8. The data presented suggest that HCV entry does not require tight junctinios. This is supported by another study which reports that disruption of tight junctions by calcium depletion enhances HCVpp infection of polarized Caco-2 and HepG2 cells. Nevertheless, they think it is likely that HCV infection in vivo is likey to occur at the tight junction, give that claudin-1 and occluding localization is thought to be limited to the tight junction in primary polarized hepatocytes. 8. How do the authors modify HCV virions for visualization and to ensure infectivity? The authors labeled virions with membrane-permeable lipophilic dyes, DiD or DiL. The infectivity of HCV that is isolated from cell culture supernatants lead to a productive infection. Poor infectivity limits the ability to track fluorescent HCV particles with confidence that they represent a productive infection. Therefore, the authors purified a highly infectious fraction of DiD-labeled HCV (DiD-HCV). They exploited the property that HCV isolated from cell culture supernatants and patients possesses a range of densities. The different densities are attributed to the association of low-density and very-low-density lipoprotein with virions. The majority of HCV RNAs sediment at a density of ~1.15g/ml, while a highly infectious fraction of HCV is associated with a lower density. (~1.06g/ml) 9. What does the viral uncoating assay measure in figure 2? How is uncoating affected by CD81 siRNAs? -In this assay, uncoating is defined as DiD-labeled envelope that no longer co-localizes with the HCV capsid. In other words, Coller et. al measured how many labeled particles (found on the envelope of the virus) were associated with the core after virus entered the Huh-7.5 cell. -CD81 siRNAs inhibited DiD-HCV uncoating. Thus, DiD-HCV entry and uncoating requires CD81. 10. In figure 4 how do the authors examine whether HCV virions are associated with the cytoskeleton? What does MSD analysis suggest is the mechanism of movement for virions associated with the actin cytoskeleton? The authors can tell if the HCV virions are associated with the cytoskeleton based on the movement of the DiD-HCV (the HCV with the membrane-permeable lipophilic dye, DiD). The cells being examined transiently express GFP-actin, meaning the actin glows green. DiD-HCV that were associated with the cytoskeleton moved at a velocity quot;
which is consistent with retrograde actin flow,quot;
meaning about 0.08260.008 mm/sec. DiD-HCV that were not connected to the cytoskeleton moved either randomly or barely at all. 10. Why do the authors use cytochalasin D to study movement of virions following internalization (Figure 5)? To confirm actin-based motility was indeed true, they used cytochalasin D in order to test whether treatment with this mycotoxin would interfere with the entry process of the virions. The treatment stalled HCV migration along the stress fibers for the remainder of the imaging. 11. Which antibodies are used to demonstrate HCV entry occurs via clathrin-mediated endocytosis (Figure 6)? Huh-7.5 cells were infected with DiD-HCV for 1 hour on ice then shifted to 37o C. Cells were fixed and stained for immunofluorescence using clathrin light chain and c-Cbl antibodies. 12. Why do the authors investigate the association of HCV virions with Rab5a immunofluorescence? The authors wanted to determine if the DiD-HCV particles displaying motility on the cell surface were trafficked to an endosome. Following clarthin pit formation, internalized cargoes converge on the early endosome prior to being sorted for recycling or degradation. The early endosome is enriched with the Rab GTPase, Rab5a. Sorting to the early endosome has been implicated in HCV entry because a dominant negative Rab5 inhibits HCVpp entry. This internalization occurred outside the cell-cell junctions, indicating that delivery of the DiD-HCV to the early endosome does not require virion entry at a tight junction. *Coller et al. investigated this association because it showed that DiD-HCV internalization does not need to occur at a tight junction. They treated a Huh-7.5 cell with GFP-Rab5a because the green fluorescing protein allows for visualization of the early endosome that Rab5a is closely associated with. After infecting the cell with DiD-HCV, they saw that a HCV virion entered the cell outside of tight junction. 13. Do you agree with the authors that their data may influence HCV therapeutic developments? We believe that these data may have an impact in HCV therapeutic development. HCV entry is an attractive target for small molecule inhibitors since it precedes the assembly of infectious virus and the maintenance of chronic infection is likely to required continual rounds of re-infection of hepatocytes. The 16 host cofactors of HCV infection identified in this study include a number of enzymes that may be successfully targeted by pharmacological inhibitors. The development of live cell imaging of HCV infection greatly expands our ability to study dynamicentrifugation (80006g, 20 min) and resuspended in 10 mL of spent supernatant. Virus was centrifuged (80006g, 15 min) and resuspended in a final volume of 1 mL of cell culture DMEM. Virus was labeled by adding 5 mL (5 mM final concentration) of lipophilic dye, DiD (Invitrogen, excitation 644 nm/emission 665 nm) or DiI (Invitrogen, excitation 549 nm/emission 565 nm) to the 1 mL of concentrated virus. Virus and dye were incubated for 1 hour with shaking at room temperature while

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