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Heat shock protein 70 inhibits the activity of Influenza A virus ribonucleoprotein and blocks the replication of virus in vitro and in vivo.

Li G, Zhang J, Tong X, Liu W, Ye X - PLoS ONE (2011)

Bottom Line: It was found that Hsp70 was associated with viral RNP by directly interacting with the PB1 and PB2 subunits, and the ATPase domain of Hsp70 was required for the association.Then we found that Hsp70 negatively regulated the expression of viral proteins in infected cells.Real-time PCR analysis revealed that the transcription and replication of all eight viral segments were significantly reduced in Hsp70 overexpressed cells and greatly increased as Hsp70 was knocked down by RNA interference.

View Article: PubMed Central - PubMed

Affiliation: Center for Molecular Immunology, CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences (CAS), Beijing, P. R. China.

ABSTRACT

Background: Heat shock protein 70 (Hsp70) was identified as a cellular interaction partner of the influenza virus ribonucleoprotein (RNP) complex. The biological significance of the interaction between Hsp70 and RNP has not been fully investigated.

Principal findings: Here we demonstrated that Hsp70 was involved in the regulation of influenza A viral transcription and replication. It was found that Hsp70 was associated with viral RNP by directly interacting with the PB1 and PB2 subunits, and the ATPase domain of Hsp70 was required for the association. Immunofluorescence analysis showed that Hsp70 was translocated from the cytoplasm into the nucleus in infected cells. Then we found that Hsp70 negatively regulated the expression of viral proteins in infected cells. Real-time PCR analysis revealed that the transcription and replication of all eight viral segments were significantly reduced in Hsp70 overexpressed cells and greatly increased as Hsp70 was knocked down by RNA interference. Luciferase assay showed that overexpression of Hsp70 could inhibit the viral RNP activity on both vRNA and cRNA promoters. Biochemical analysis demonstrated that Hsp70 interfered with the integrity of RNP. Furthermore, delivered Hsp70 could inhibit the replication of influenza A virus in mice.

Significance: Our study indicated that Hsp70 interacted with PB1 and PB2 of RNP and could interfere with the integrity of RNP and block the virus replication in vitro and in vivo possibly through disrupting the binding of viral polymerase with viral RNA.

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Hsp70 interferes with the integrity of RNP.(A, B) MDCK cells were infected with rWSN, and then subjected to cell fractionation. The nuclear fraction was incubated with His-Hsp70 or with BSA as control, and then applied to a glycerol gradient. Fractions (1 to 12) were collected from the top to the bottom. An aliquot of each fraction was subjected to immunoblotting with indicated antibodies (A). RNA was purified from each fraction, and reverse transcribed into cDNA with primer specific for vRNA or cRNA. The cDNA was then subjected to semiquantitative PCR with primers for NP. The PCR products were subjected to 1% agarose gel and visualized by ethidium bromide (B). These experiments were repeated three times with identical results.
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pone-0016546-g009: Hsp70 interferes with the integrity of RNP.(A, B) MDCK cells were infected with rWSN, and then subjected to cell fractionation. The nuclear fraction was incubated with His-Hsp70 or with BSA as control, and then applied to a glycerol gradient. Fractions (1 to 12) were collected from the top to the bottom. An aliquot of each fraction was subjected to immunoblotting with indicated antibodies (A). RNA was purified from each fraction, and reverse transcribed into cDNA with primer specific for vRNA or cRNA. The cDNA was then subjected to semiquantitative PCR with primers for NP. The PCR products were subjected to 1% agarose gel and visualized by ethidium bromide (B). These experiments were repeated three times with identical results.

Mentions: The viral polymerase complex consists of PA, PB1 and PB2, in which PB1 and PB2 interacted with Hsp70 (Fig. 1). Furthermore the binding regions of PB1 and PB2 with Hsp70 (Fig. 2) were pivotal to the RNP functions. We speculated that Hsp70 might affect the integrity of RNP. To test this idea, the MDCK cells were infected with virus and the nuclear fraction was isolated and incubated with His-Hsp70 or BSA as negative control. Then the mixtures were subjected to glycerol gradient centrifugation. The fractions were collected and subjected to immunoblotting with anti-NP, anti-PA, anti-PB2 and anti-Hsp70 antibodies (Fig. 9A) as well as semiquantitative RT-PCR for vRNA and cRNA of NP segment (Fig. 9B). As shown in Fig. 9A, in BSA-treated RNPs PA sedimented predominantly in fractions 1 to 7, PB2 in fractions 1 to 9, NP in almost all fractions except fraction 12, then in Fig. 9B vRNA and cRNA sedimented in fractions 1 to 8, therefore, it was shown that RNP complex sedimented to fractions 1 to 7, because there existed vRNA (cRNA), NP, PA and PB2. In contrast, in Hsp70-treated RNPs vRNA and cRNA sedimented mainly in fractions 6 to 9, NP in fractions 6 to 10, but PA and PB2 in fractions 1 to 4 where Hsp70 sedimented. We concluded that the addition of Hsp70 disrupted the integrity of RNP, with NP and vRNA (cRNA) binding together, but PA and PB2 were not in the same fractions.


Heat shock protein 70 inhibits the activity of Influenza A virus ribonucleoprotein and blocks the replication of virus in vitro and in vivo.

Li G, Zhang J, Tong X, Liu W, Ye X - PLoS ONE (2011)

Hsp70 interferes with the integrity of RNP.(A, B) MDCK cells were infected with rWSN, and then subjected to cell fractionation. The nuclear fraction was incubated with His-Hsp70 or with BSA as control, and then applied to a glycerol gradient. Fractions (1 to 12) were collected from the top to the bottom. An aliquot of each fraction was subjected to immunoblotting with indicated antibodies (A). RNA was purified from each fraction, and reverse transcribed into cDNA with primer specific for vRNA or cRNA. The cDNA was then subjected to semiquantitative PCR with primers for NP. The PCR products were subjected to 1% agarose gel and visualized by ethidium bromide (B). These experiments were repeated three times with identical results.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3044721&req=5

pone-0016546-g009: Hsp70 interferes with the integrity of RNP.(A, B) MDCK cells were infected with rWSN, and then subjected to cell fractionation. The nuclear fraction was incubated with His-Hsp70 or with BSA as control, and then applied to a glycerol gradient. Fractions (1 to 12) were collected from the top to the bottom. An aliquot of each fraction was subjected to immunoblotting with indicated antibodies (A). RNA was purified from each fraction, and reverse transcribed into cDNA with primer specific for vRNA or cRNA. The cDNA was then subjected to semiquantitative PCR with primers for NP. The PCR products were subjected to 1% agarose gel and visualized by ethidium bromide (B). These experiments were repeated three times with identical results.
Mentions: The viral polymerase complex consists of PA, PB1 and PB2, in which PB1 and PB2 interacted with Hsp70 (Fig. 1). Furthermore the binding regions of PB1 and PB2 with Hsp70 (Fig. 2) were pivotal to the RNP functions. We speculated that Hsp70 might affect the integrity of RNP. To test this idea, the MDCK cells were infected with virus and the nuclear fraction was isolated and incubated with His-Hsp70 or BSA as negative control. Then the mixtures were subjected to glycerol gradient centrifugation. The fractions were collected and subjected to immunoblotting with anti-NP, anti-PA, anti-PB2 and anti-Hsp70 antibodies (Fig. 9A) as well as semiquantitative RT-PCR for vRNA and cRNA of NP segment (Fig. 9B). As shown in Fig. 9A, in BSA-treated RNPs PA sedimented predominantly in fractions 1 to 7, PB2 in fractions 1 to 9, NP in almost all fractions except fraction 12, then in Fig. 9B vRNA and cRNA sedimented in fractions 1 to 8, therefore, it was shown that RNP complex sedimented to fractions 1 to 7, because there existed vRNA (cRNA), NP, PA and PB2. In contrast, in Hsp70-treated RNPs vRNA and cRNA sedimented mainly in fractions 6 to 9, NP in fractions 6 to 10, but PA and PB2 in fractions 1 to 4 where Hsp70 sedimented. We concluded that the addition of Hsp70 disrupted the integrity of RNP, with NP and vRNA (cRNA) binding together, but PA and PB2 were not in the same fractions.

Bottom Line: It was found that Hsp70 was associated with viral RNP by directly interacting with the PB1 and PB2 subunits, and the ATPase domain of Hsp70 was required for the association.Then we found that Hsp70 negatively regulated the expression of viral proteins in infected cells.Real-time PCR analysis revealed that the transcription and replication of all eight viral segments were significantly reduced in Hsp70 overexpressed cells and greatly increased as Hsp70 was knocked down by RNA interference.

View Article: PubMed Central - PubMed

Affiliation: Center for Molecular Immunology, CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences (CAS), Beijing, P. R. China.

ABSTRACT

Background: Heat shock protein 70 (Hsp70) was identified as a cellular interaction partner of the influenza virus ribonucleoprotein (RNP) complex. The biological significance of the interaction between Hsp70 and RNP has not been fully investigated.

Principal findings: Here we demonstrated that Hsp70 was involved in the regulation of influenza A viral transcription and replication. It was found that Hsp70 was associated with viral RNP by directly interacting with the PB1 and PB2 subunits, and the ATPase domain of Hsp70 was required for the association. Immunofluorescence analysis showed that Hsp70 was translocated from the cytoplasm into the nucleus in infected cells. Then we found that Hsp70 negatively regulated the expression of viral proteins in infected cells. Real-time PCR analysis revealed that the transcription and replication of all eight viral segments were significantly reduced in Hsp70 overexpressed cells and greatly increased as Hsp70 was knocked down by RNA interference. Luciferase assay showed that overexpression of Hsp70 could inhibit the viral RNP activity on both vRNA and cRNA promoters. Biochemical analysis demonstrated that Hsp70 interfered with the integrity of RNP. Furthermore, delivered Hsp70 could inhibit the replication of influenza A virus in mice.

Significance: Our study indicated that Hsp70 interacted with PB1 and PB2 of RNP and could interfere with the integrity of RNP and block the virus replication in vitro and in vivo possibly through disrupting the binding of viral polymerase with viral RNA.

Show MeSH
Related in: MedlinePlus