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Involvement of actin microfilaments in the replication of human parainfluenza virus type 3
1Molecular Virology Graduate Program, Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106, USA.
Abstract:
Several studies indicate that paramyxoviruses require a specific cellular factor(s) for transcription of their genomic RNAs. We previously reported that the cellular cytoskeletal protein actin, in its polymeric form, participates in the transcription of human parainfluenza virus type 3 (HPIV3) in vitro. In the present study, we investigated the role of the polymeric form of actin, i.e., the actin microfilaments of the cytoskeletal framework, in the reproduction of HPIV3 in vivo. Pulse-chase labeling analyses indicate that the viral nucleocapsid-associated proteins, NP and P, are present predominantly in the cytoskeletal framework during infection. By in situ hybridization, we found that viral mRNAs and genomic RNA were synthesized from the nucleocapsids that were bound to the cytoskeletal framework. Double immunofluorescent labeling and confocal microscopy of the cytoarchitecture revealed that the viral nucleocapsids are specifically localized on the actin microfilaments. Treatment of cells with the actin-depolymerizing agent, cytochalasin D, resulted in the inhibition of viral RNA synthesis and ribonucleoprotein accumulation. These results strongly suggest that actin microfilaments play an important role in the replication of HPIV3.
Insights
Actin microfilaments are crucial for human parainfluenza virus type 3 (HPIV3) replication. This study shows HPIV3 RNA synthesis and reproduction depend on these cytoskeletal structures in vivo.
Area of Science:
- Virology
- Cell Biology
- Molecular Biology
Background:
- Paramyxoviruses require cellular factors for RNA transcription.
- Polymeric actin was previously shown to participate in human parainfluenza virus type 3 (HPIV3) transcription in vitro.
Purpose of the Study:
- To investigate the role of polymeric actin (actin microfilaments) in HPIV3 reproduction in vivo.
- To elucidate the mechanism by which actin microfilaments influence HPIV3 replication.
Main Methods:
- Pulse-chase labeling to track viral nucleocapsid-associated proteins (NP and P).
- In situ hybridization to detect viral RNA synthesis.
- Double immunofluorescent labeling and confocal microscopy to visualize viral nucleocapsid localization.
- Treatment with cytochalasin D, an actin-depolymerizing agent.
Main Results:
- Viral nucleocapsid proteins (NP and P) were predominantly found in the cytoskeletal framework during infection.
- Viral mRNAs and genomic RNA were synthesized from nucleocapsids bound to the cytoskeletal framework.
- Viral nucleocapsids were specifically localized on actin microfilaments.
- Cytochalasin D treatment inhibited viral RNA synthesis and ribonucleoprotein accumulation.
Conclusions:
- Actin microfilaments play a significant role in HPIV3 replication in vivo.
- The cytoskeletal framework, particularly actin microfilaments, is essential for HPIV3 RNA synthesis and reproduction.