Limits...
Hepatitis C virus-induced cytoplasmic organelles use the nuclear transport machinery to establish an environment conducive to virus replication.

Neufeldt CJ, Joyce MA, Levin A, Steenbergen RH, Pang D, Shields J, Tyrrell DL, Wozniak RW - PLoS Pathog. (2013)

Bottom Line: Moreover, we detected interactions between specific HCV proteins and both Nups and NTFs.We hypothesize that cytoplasmically positioned Nups facilitate formation of the membranous web and contribute to the compartmentalization of viral replication.Accordingly, we show that transport cargo proteins normally targeted to the nucleus are capable of entering regions of the membranous web, and that depletion of specific Nups or Kaps inhibits HCV replication and assembly.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University of Alberta, Edmonton, Alberta, Canada.

ABSTRACT
Hepatitis C virus (HCV) infection induces formation of a membranous web structure in the host cell cytoplasm where the viral genome replicates and virions assemble. The membranous web is thought to concentrate viral components and hide viral RNA from pattern recognition receptors. We have uncovered a role for nuclear pore complex proteins (Nups) and nuclear transport factors (NTFs) in the membranous web. We show that HCV infection leads to increased levels of cytoplasmic Nups that accumulate at sites enriched for HCV proteins. Moreover, we detected interactions between specific HCV proteins and both Nups and NTFs. We hypothesize that cytoplasmically positioned Nups facilitate formation of the membranous web and contribute to the compartmentalization of viral replication. Accordingly, we show that transport cargo proteins normally targeted to the nucleus are capable of entering regions of the membranous web, and that depletion of specific Nups or Kaps inhibits HCV replication and assembly.

Show MeSH

Related in: MedlinePlus

HCV infection increases RNA and protein levels of a subset of Nups.A) Total RNA was isolated from cell lysates at the indicated time points after infection of Huh7.5 cells with HCV, and levels of specific mRNA transcripts were assessed by qPCR. Values for each sample were normalized to HPRT and are expressed relative to uninfected cells (day 0 time point). HCV RNA levels are shown as fold change relative to HPRT. Error bars indicate standard error (based on ≥3 experiments) and statistical significance was evaluated using t-tests comparing each infected sample to an uninfected control sample. p-values less than 0.05 (*), 0.01 (**), and 0.001 (***) are indicated. B) Cell lysates were harvested from HCV infected Huh7.5 cells four days after infection and Nup proteins levels were determined by western blotting using antibodies specific for Nup98, Nup153, Nup155, and Nup358. Protein levels were quantified and normalized to tubulin levels and the fold-change is relative to uninfected cells. Error bars were determined using data from ≥3 experiments.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3814334&req=5

ppat-1003744-g003: HCV infection increases RNA and protein levels of a subset of Nups.A) Total RNA was isolated from cell lysates at the indicated time points after infection of Huh7.5 cells with HCV, and levels of specific mRNA transcripts were assessed by qPCR. Values for each sample were normalized to HPRT and are expressed relative to uninfected cells (day 0 time point). HCV RNA levels are shown as fold change relative to HPRT. Error bars indicate standard error (based on ≥3 experiments) and statistical significance was evaluated using t-tests comparing each infected sample to an uninfected control sample. p-values less than 0.05 (*), 0.01 (**), and 0.001 (***) are indicated. B) Cell lysates were harvested from HCV infected Huh7.5 cells four days after infection and Nup proteins levels were determined by western blotting using antibodies specific for Nup98, Nup153, Nup155, and Nup358. Protein levels were quantified and normalized to tubulin levels and the fold-change is relative to uninfected cells. Error bars were determined using data from ≥3 experiments.

Mentions: The accumulation of Nups in the vicinity of HCV assembly sites could arise from redistribution of cellular pools, increased cellular levels of these proteins, or a combination of both events. To assess the potential contribution of increased Nup synthesis, we examined cellular levels of various Nup mRNA transcripts at time points after HCV infection of Huh7.5 cells (Figure 3A). We found that mRNA levels of Nups composing the cytoplasmic filaments of the NPC (Nup88, Nup214 and Nup358), and one that is part of the nuclear basket (Nup153) were reproducibly elevated 1.5- to 2-fold four days after HCV infection (Figure 3A) in a manner that qualitatively paralleled increasing HCV RNA levels. In addition, Nup358 showed a reproducible biphasic pattern with an additional peak visible at 2 days after infection. By contrast, levels of transcripts encoding for several Nups that make up the scaffold of the NPC (including Nup155, Nup107, Nup53, and Nup205) and two associated Nups, Nup62 and Nup98, showed little or no change during HCV infection. Consistent with the changes in transcript levels, quantitative analysis of a subset of these Nups by western blotting showed increased levels of Nup98, Nup153, and Nup358, but not Nup155 (Figure 3B). These results indicate that a subset of Nups are up-regulated during HCV infection while others show no statistically significant change. Thus, the Nups recruited to sites of viral assembly are likely to arise from both constitutive and HCV-induced Nup expression.


Hepatitis C virus-induced cytoplasmic organelles use the nuclear transport machinery to establish an environment conducive to virus replication.

Neufeldt CJ, Joyce MA, Levin A, Steenbergen RH, Pang D, Shields J, Tyrrell DL, Wozniak RW - PLoS Pathog. (2013)

HCV infection increases RNA and protein levels of a subset of Nups.A) Total RNA was isolated from cell lysates at the indicated time points after infection of Huh7.5 cells with HCV, and levels of specific mRNA transcripts were assessed by qPCR. Values for each sample were normalized to HPRT and are expressed relative to uninfected cells (day 0 time point). HCV RNA levels are shown as fold change relative to HPRT. Error bars indicate standard error (based on ≥3 experiments) and statistical significance was evaluated using t-tests comparing each infected sample to an uninfected control sample. p-values less than 0.05 (*), 0.01 (**), and 0.001 (***) are indicated. B) Cell lysates were harvested from HCV infected Huh7.5 cells four days after infection and Nup proteins levels were determined by western blotting using antibodies specific for Nup98, Nup153, Nup155, and Nup358. Protein levels were quantified and normalized to tubulin levels and the fold-change is relative to uninfected cells. Error bars were determined using data from ≥3 experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3814334&req=5

ppat-1003744-g003: HCV infection increases RNA and protein levels of a subset of Nups.A) Total RNA was isolated from cell lysates at the indicated time points after infection of Huh7.5 cells with HCV, and levels of specific mRNA transcripts were assessed by qPCR. Values for each sample were normalized to HPRT and are expressed relative to uninfected cells (day 0 time point). HCV RNA levels are shown as fold change relative to HPRT. Error bars indicate standard error (based on ≥3 experiments) and statistical significance was evaluated using t-tests comparing each infected sample to an uninfected control sample. p-values less than 0.05 (*), 0.01 (**), and 0.001 (***) are indicated. B) Cell lysates were harvested from HCV infected Huh7.5 cells four days after infection and Nup proteins levels were determined by western blotting using antibodies specific for Nup98, Nup153, Nup155, and Nup358. Protein levels were quantified and normalized to tubulin levels and the fold-change is relative to uninfected cells. Error bars were determined using data from ≥3 experiments.
Mentions: The accumulation of Nups in the vicinity of HCV assembly sites could arise from redistribution of cellular pools, increased cellular levels of these proteins, or a combination of both events. To assess the potential contribution of increased Nup synthesis, we examined cellular levels of various Nup mRNA transcripts at time points after HCV infection of Huh7.5 cells (Figure 3A). We found that mRNA levels of Nups composing the cytoplasmic filaments of the NPC (Nup88, Nup214 and Nup358), and one that is part of the nuclear basket (Nup153) were reproducibly elevated 1.5- to 2-fold four days after HCV infection (Figure 3A) in a manner that qualitatively paralleled increasing HCV RNA levels. In addition, Nup358 showed a reproducible biphasic pattern with an additional peak visible at 2 days after infection. By contrast, levels of transcripts encoding for several Nups that make up the scaffold of the NPC (including Nup155, Nup107, Nup53, and Nup205) and two associated Nups, Nup62 and Nup98, showed little or no change during HCV infection. Consistent with the changes in transcript levels, quantitative analysis of a subset of these Nups by western blotting showed increased levels of Nup98, Nup153, and Nup358, but not Nup155 (Figure 3B). These results indicate that a subset of Nups are up-regulated during HCV infection while others show no statistically significant change. Thus, the Nups recruited to sites of viral assembly are likely to arise from both constitutive and HCV-induced Nup expression.

Bottom Line: Moreover, we detected interactions between specific HCV proteins and both Nups and NTFs.We hypothesize that cytoplasmically positioned Nups facilitate formation of the membranous web and contribute to the compartmentalization of viral replication.Accordingly, we show that transport cargo proteins normally targeted to the nucleus are capable of entering regions of the membranous web, and that depletion of specific Nups or Kaps inhibits HCV replication and assembly.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University of Alberta, Edmonton, Alberta, Canada.

ABSTRACT
Hepatitis C virus (HCV) infection induces formation of a membranous web structure in the host cell cytoplasm where the viral genome replicates and virions assemble. The membranous web is thought to concentrate viral components and hide viral RNA from pattern recognition receptors. We have uncovered a role for nuclear pore complex proteins (Nups) and nuclear transport factors (NTFs) in the membranous web. We show that HCV infection leads to increased levels of cytoplasmic Nups that accumulate at sites enriched for HCV proteins. Moreover, we detected interactions between specific HCV proteins and both Nups and NTFs. We hypothesize that cytoplasmically positioned Nups facilitate formation of the membranous web and contribute to the compartmentalization of viral replication. Accordingly, we show that transport cargo proteins normally targeted to the nucleus are capable of entering regions of the membranous web, and that depletion of specific Nups or Kaps inhibits HCV replication and assembly.

Show MeSH
Related in: MedlinePlus