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NS2 is dispensable for efficient assembly of hepatitis C virus-like particles in a bipartite trans-encapsidation system.

Bentham MJ, Marraiki N, McCormick CJ, Rowlands DJ, Griffin S - J. Gen. Virol. (2014)

Bottom Line: This closely resembled replicon-mediated NS2 trans-complementation, confirming that baculovirus expression of HCV proteins did not unduly affect particle production.Furthermore, this suggests that separation of structural protein expression from replicating HCV RNAs that are destined to be packaged alleviates an early stage requirement for NS2 during particle formation.This highlights our current lack of understanding of how NS2 mediates assembly, yet comparison of full-length and bipartite systems may provide further insight into this process.

View Article: PubMed Central - PubMed

Affiliation: Leeds Institute of Cancer & Pathology (LICAP), and Leeds CRUK Clinical Centre, Faculty of Medicine and Health, St James's University Hospital, University of Leeds, Beckett St., Leeds, West Yorkshire LS9 7TF, UK.

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Direct analysis of intracellular HCV-LP on sucrose density gradients. (a) Anti-E2 ELISA using ALP98 mouse monoclonal antibody conducted on fractions from a 10–60 % (w/v) sucrose density gradient from C-p7-transduced FK5.1neo cell lysates. Western blots for HCV proteins and RT-PCR for HCV RNA are shown underneath for each fraction, along with colony formation assays for the top 12 fractions. (b) Negative-stain TEM of peak gradient fraction (4) revealing HCV-LP. Bars, 100 nm.
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f3: Direct analysis of intracellular HCV-LP on sucrose density gradients. (a) Anti-E2 ELISA using ALP98 mouse monoclonal antibody conducted on fractions from a 10–60 % (w/v) sucrose density gradient from C-p7-transduced FK5.1neo cell lysates. Western blots for HCV proteins and RT-PCR for HCV RNA are shown underneath for each fraction, along with colony formation assays for the top 12 fractions. (b) Negative-stain TEM of peak gradient fraction (4) revealing HCV-LP. Bars, 100 nm.

Mentions: Intracellular RNA-containing HCV-LP were next analysed by sucrose density gradient ultracentrifugation (Fig. 3a). HCV E2 ELISA indicated a broad peak of particulate material towards the top of the gradient, which we showed to contain E2 and core (by Western blot), replicon RNA [by reverse transcriptase (RT)-PCR], and pseudo-infectivity (colony formation). Furthermore, TEM analysis of peak gradient fractions revealed the presence of enveloped particles with diameters of around 50 nm (Fig. 3b). Some HCV-LP contained a clearly identifiable core as well as prominent envelope spike proteins. Equivalent particles were not observed in control gradients from LacZ-transduced cells, or within non-peak fractions from the BacC-p7(1b)-transduced cells (data not shown). Thus, pseudo-infectious genotype 1b HCV-LP assemble within cells in the absence of NS2 but are not secreted, and are reminiscent of particles produced in analogous systems as well as authentic wtHCV virions.


NS2 is dispensable for efficient assembly of hepatitis C virus-like particles in a bipartite trans-encapsidation system.

Bentham MJ, Marraiki N, McCormick CJ, Rowlands DJ, Griffin S - J. Gen. Virol. (2014)

Direct analysis of intracellular HCV-LP on sucrose density gradients. (a) Anti-E2 ELISA using ALP98 mouse monoclonal antibody conducted on fractions from a 10–60 % (w/v) sucrose density gradient from C-p7-transduced FK5.1neo cell lysates. Western blots for HCV proteins and RT-PCR for HCV RNA are shown underneath for each fraction, along with colony formation assays for the top 12 fractions. (b) Negative-stain TEM of peak gradient fraction (4) revealing HCV-LP. Bars, 100 nm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4202265&req=5

f3: Direct analysis of intracellular HCV-LP on sucrose density gradients. (a) Anti-E2 ELISA using ALP98 mouse monoclonal antibody conducted on fractions from a 10–60 % (w/v) sucrose density gradient from C-p7-transduced FK5.1neo cell lysates. Western blots for HCV proteins and RT-PCR for HCV RNA are shown underneath for each fraction, along with colony formation assays for the top 12 fractions. (b) Negative-stain TEM of peak gradient fraction (4) revealing HCV-LP. Bars, 100 nm.
Mentions: Intracellular RNA-containing HCV-LP were next analysed by sucrose density gradient ultracentrifugation (Fig. 3a). HCV E2 ELISA indicated a broad peak of particulate material towards the top of the gradient, which we showed to contain E2 and core (by Western blot), replicon RNA [by reverse transcriptase (RT)-PCR], and pseudo-infectivity (colony formation). Furthermore, TEM analysis of peak gradient fractions revealed the presence of enveloped particles with diameters of around 50 nm (Fig. 3b). Some HCV-LP contained a clearly identifiable core as well as prominent envelope spike proteins. Equivalent particles were not observed in control gradients from LacZ-transduced cells, or within non-peak fractions from the BacC-p7(1b)-transduced cells (data not shown). Thus, pseudo-infectious genotype 1b HCV-LP assemble within cells in the absence of NS2 but are not secreted, and are reminiscent of particles produced in analogous systems as well as authentic wtHCV virions.

Bottom Line: This closely resembled replicon-mediated NS2 trans-complementation, confirming that baculovirus expression of HCV proteins did not unduly affect particle production.Furthermore, this suggests that separation of structural protein expression from replicating HCV RNAs that are destined to be packaged alleviates an early stage requirement for NS2 during particle formation.This highlights our current lack of understanding of how NS2 mediates assembly, yet comparison of full-length and bipartite systems may provide further insight into this process.

View Article: PubMed Central - PubMed

Affiliation: Leeds Institute of Cancer & Pathology (LICAP), and Leeds CRUK Clinical Centre, Faculty of Medicine and Health, St James's University Hospital, University of Leeds, Beckett St., Leeds, West Yorkshire LS9 7TF, UK.

Show MeSH
Related in: MedlinePlus