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

Depletion of Nups and Kaps inhibits HCV replication.A–D) Huh7.5 cells were coinfected with HCV and lentivirus encoding shRNAs directed against Nup98, Nup155, Nup153, Kap β3/IPO5, NDC1, or a scrambled control sequence for four days. The effects of Nup or Kap depletion on HCV titers were evaluated by qPCR analysis of HCV RNA levels in cell extracts (panel A) or in the culture supernatant (panel B) from HCV infected Huh7.5 cells co-infected with and without lentivirus. In addition, intracellular levels of the HCV core protein were examined by quantitative western blotting using antibodies specific for HCV core (panel C). Values for each sample are normalized to HPRT mRNA (Panel A and B) or α-tubulin (Panel C) and are expressed as fold-change relative to HCV infected cells not treated with lentivirus. Error bars indicate standard error (based on ≥3 experiments) and statistics are based on t-tests comparing each Nup or Kap specific shRNA treated sample to samples expressing the scrambled shRNA control. D) Huh7.5 cells were grown as described in panel A and the infectious titers of HCV particles present in the media of cells depleted of the indicated proteins were determined. Focus-forming units were determined using indirect immunofluorescence microscopy. Values shown represent focus-forming units per mL of medium (FFU/mL). E–F) Huh7.5 cells were infected with HCV and 4 hrs post infection a penetratin peptide, a Kap β3-NLS peptide, or a penetratin peptide containing a N-terminal Kap α NLS (Kap α-NLS) was added to the media. Four days later the effects of these peptides on HCV RNA levels in intracellular (panel E) and extracellular (panel F) compartments were assessed by qPCR analysis. Values for each sample are normalized to HPRT mRNA levels and expressed as fold change relative to cells receiving no peptide. Error bars indicate standard error (based on ≥3 experiments) and statistics based on t-tests comparing cells treated with penetratin alone to those treated with the Kap β3-NLS peptide or the Kap α-NLS containing peptide. p-values less than 0.05 (*), 0.01 (**), and 0.001 (***) are indicated.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3814334&req=5

ppat-1003744-g005: Depletion of Nups and Kaps inhibits HCV replication.A–D) Huh7.5 cells were coinfected with HCV and lentivirus encoding shRNAs directed against Nup98, Nup155, Nup153, Kap β3/IPO5, NDC1, or a scrambled control sequence for four days. The effects of Nup or Kap depletion on HCV titers were evaluated by qPCR analysis of HCV RNA levels in cell extracts (panel A) or in the culture supernatant (panel B) from HCV infected Huh7.5 cells co-infected with and without lentivirus. In addition, intracellular levels of the HCV core protein were examined by quantitative western blotting using antibodies specific for HCV core (panel C). Values for each sample are normalized to HPRT mRNA (Panel A and B) or α-tubulin (Panel C) and are expressed as fold-change relative to HCV infected cells not treated with lentivirus. Error bars indicate standard error (based on ≥3 experiments) and statistics are based on t-tests comparing each Nup or Kap specific shRNA treated sample to samples expressing the scrambled shRNA control. D) Huh7.5 cells were grown as described in panel A and the infectious titers of HCV particles present in the media of cells depleted of the indicated proteins were determined. Focus-forming units were determined using indirect immunofluorescence microscopy. Values shown represent focus-forming units per mL of medium (FFU/mL). E–F) Huh7.5 cells were infected with HCV and 4 hrs post infection a penetratin peptide, a Kap β3-NLS peptide, or a penetratin peptide containing a N-terminal Kap α NLS (Kap α-NLS) was added to the media. Four days later the effects of these peptides on HCV RNA levels in intracellular (panel E) and extracellular (panel F) compartments were assessed by qPCR analysis. Values for each sample are normalized to HPRT mRNA levels and expressed as fold change relative to cells receiving no peptide. Error bars indicate standard error (based on ≥3 experiments) and statistics based on t-tests comparing cells treated with penetratin alone to those treated with the Kap β3-NLS peptide or the Kap α-NLS containing peptide. p-values less than 0.05 (*), 0.01 (**), and 0.001 (***) are indicated.

Mentions: To examine the relevance of interactions between HCV proteins and Nups or Kaps, we investigated the consequences of reducing cellular levels of specific Nups and Kap β3/IPO5 on HCV replication. Lentivirus expressing shRNAs were used to reduce levels of targeted proteins. Using this approach, mRNA and protein levels for each of the targeted genes were decreased by >60% in Huh7.5 cells by 4 days after lentivirus transduction (Figure S9A and S9B) with little effect on cell viability (Figure S9C). Cells were coinfected with lentivirus and HCV and, 4 days post infection, intracellular and extracellular HCV RNA levels were determined using quantitative real-time PCR (qPCR)(Figure 5A, 5B, and S9E). The results of these experiments revealed that intracellular levels of viral RNA were significantly decreased upon depletion of Nup98 or Nup153 (Figure 5A and S9E), while reduced levels of Nup155, NDC1, or Kap β3/IPO5, or treatment with lentivirus encoding a scrambled control sequence, had no effect. Consistent with these observations, quantitative western blotting revealed a decrease in HCV core protein levels in Nup98- or Nup153-depleted cells (Figure 5C). In accordance with decreased intracellular viral RNA levels, cells depleted of Nup98 or Nup153 also showed similar decreases in the levels of secreted virus (Figure 5B). Although Nup155- or Kap β3/IPO5-depleted cells showed no change in intracellular levels of HCV RNA, extracellular levels of secreted virus were decreased in these cells (Figure 5B), suggesting a requirement for Nup155 and Kap β3/IPO5 at a post-replication stage of virus assembly or in viral egress. These divergent effects of Nup depletions on intracellular versus extracellular RNA levels suggest functions for Nups at different stages of the HCV infectious cycle.


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)

Depletion of Nups and Kaps inhibits HCV replication.A–D) Huh7.5 cells were coinfected with HCV and lentivirus encoding shRNAs directed against Nup98, Nup155, Nup153, Kap β3/IPO5, NDC1, or a scrambled control sequence for four days. The effects of Nup or Kap depletion on HCV titers were evaluated by qPCR analysis of HCV RNA levels in cell extracts (panel A) or in the culture supernatant (panel B) from HCV infected Huh7.5 cells co-infected with and without lentivirus. In addition, intracellular levels of the HCV core protein were examined by quantitative western blotting using antibodies specific for HCV core (panel C). Values for each sample are normalized to HPRT mRNA (Panel A and B) or α-tubulin (Panel C) and are expressed as fold-change relative to HCV infected cells not treated with lentivirus. Error bars indicate standard error (based on ≥3 experiments) and statistics are based on t-tests comparing each Nup or Kap specific shRNA treated sample to samples expressing the scrambled shRNA control. D) Huh7.5 cells were grown as described in panel A and the infectious titers of HCV particles present in the media of cells depleted of the indicated proteins were determined. Focus-forming units were determined using indirect immunofluorescence microscopy. Values shown represent focus-forming units per mL of medium (FFU/mL). E–F) Huh7.5 cells were infected with HCV and 4 hrs post infection a penetratin peptide, a Kap β3-NLS peptide, or a penetratin peptide containing a N-terminal Kap α NLS (Kap α-NLS) was added to the media. Four days later the effects of these peptides on HCV RNA levels in intracellular (panel E) and extracellular (panel F) compartments were assessed by qPCR analysis. Values for each sample are normalized to HPRT mRNA levels and expressed as fold change relative to cells receiving no peptide. Error bars indicate standard error (based on ≥3 experiments) and statistics based on t-tests comparing cells treated with penetratin alone to those treated with the Kap β3-NLS peptide or the Kap α-NLS containing peptide. p-values less than 0.05 (*), 0.01 (**), and 0.001 (***) are indicated.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1003744-g005: Depletion of Nups and Kaps inhibits HCV replication.A–D) Huh7.5 cells were coinfected with HCV and lentivirus encoding shRNAs directed against Nup98, Nup155, Nup153, Kap β3/IPO5, NDC1, or a scrambled control sequence for four days. The effects of Nup or Kap depletion on HCV titers were evaluated by qPCR analysis of HCV RNA levels in cell extracts (panel A) or in the culture supernatant (panel B) from HCV infected Huh7.5 cells co-infected with and without lentivirus. In addition, intracellular levels of the HCV core protein were examined by quantitative western blotting using antibodies specific for HCV core (panel C). Values for each sample are normalized to HPRT mRNA (Panel A and B) or α-tubulin (Panel C) and are expressed as fold-change relative to HCV infected cells not treated with lentivirus. Error bars indicate standard error (based on ≥3 experiments) and statistics are based on t-tests comparing each Nup or Kap specific shRNA treated sample to samples expressing the scrambled shRNA control. D) Huh7.5 cells were grown as described in panel A and the infectious titers of HCV particles present in the media of cells depleted of the indicated proteins were determined. Focus-forming units were determined using indirect immunofluorescence microscopy. Values shown represent focus-forming units per mL of medium (FFU/mL). E–F) Huh7.5 cells were infected with HCV and 4 hrs post infection a penetratin peptide, a Kap β3-NLS peptide, or a penetratin peptide containing a N-terminal Kap α NLS (Kap α-NLS) was added to the media. Four days later the effects of these peptides on HCV RNA levels in intracellular (panel E) and extracellular (panel F) compartments were assessed by qPCR analysis. Values for each sample are normalized to HPRT mRNA levels and expressed as fold change relative to cells receiving no peptide. Error bars indicate standard error (based on ≥3 experiments) and statistics based on t-tests comparing cells treated with penetratin alone to those treated with the Kap β3-NLS peptide or the Kap α-NLS containing peptide. p-values less than 0.05 (*), 0.01 (**), and 0.001 (***) are indicated.
Mentions: To examine the relevance of interactions between HCV proteins and Nups or Kaps, we investigated the consequences of reducing cellular levels of specific Nups and Kap β3/IPO5 on HCV replication. Lentivirus expressing shRNAs were used to reduce levels of targeted proteins. Using this approach, mRNA and protein levels for each of the targeted genes were decreased by >60% in Huh7.5 cells by 4 days after lentivirus transduction (Figure S9A and S9B) with little effect on cell viability (Figure S9C). Cells were coinfected with lentivirus and HCV and, 4 days post infection, intracellular and extracellular HCV RNA levels were determined using quantitative real-time PCR (qPCR)(Figure 5A, 5B, and S9E). The results of these experiments revealed that intracellular levels of viral RNA were significantly decreased upon depletion of Nup98 or Nup153 (Figure 5A and S9E), while reduced levels of Nup155, NDC1, or Kap β3/IPO5, or treatment with lentivirus encoding a scrambled control sequence, had no effect. Consistent with these observations, quantitative western blotting revealed a decrease in HCV core protein levels in Nup98- or Nup153-depleted cells (Figure 5C). In accordance with decreased intracellular viral RNA levels, cells depleted of Nup98 or Nup153 also showed similar decreases in the levels of secreted virus (Figure 5B). Although Nup155- or Kap β3/IPO5-depleted cells showed no change in intracellular levels of HCV RNA, extracellular levels of secreted virus were decreased in these cells (Figure 5B), suggesting a requirement for Nup155 and Kap β3/IPO5 at a post-replication stage of virus assembly or in viral egress. These divergent effects of Nup depletions on intracellular versus extracellular RNA levels suggest functions for Nups at different stages of the HCV infectious cycle.

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