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Intracellular accumulation of Punta Toro virus glycoproteins expressed from cloned cDNA
Y Matsuoka1, T Ihara, D H Bishop
1Department of Microbiology, University of Alabama, Birmingham 35294.
Abstract:
The Punta Toro virus (PTV) middle size (M) RNA encodes two glycoproteins, G1 and G2, and possibly a nonstructural protein, NSM. A partial cDNA clone of the M segment which contains G1 and G2 glycoprotein coding sequences but lacks most of the NSM sequences was inserted into the genome of vaccinia virus under the control of an early vaccinia promoter. Cells infected with the recombinant virus were found to synthesize two polypeptides with molecular weights of 65,000 (G1) and 55,000 (G2) that reacted specifically with antibody against PTV. Studies using indirect immunofluorescence microscopy revealed that these proteins accumulated intracellularly in the perinuclear region. The results of endoglycosidase H digestion of these glycoproteins suggested that both G1 and G2 glycoproteins were transported from the RER to the Golgi complex. These proteins were not chased out from the Golgi region during a 6-hr incubation in the presence of cycloheximide. Surface immune precipitation and 125I-protein A binding assays also demonstrated that the majority of the G1 and G2 glycoproteins are retained intracellularly. These results indicate that the PTV glycoproteins contain the necessary information for retention in the Golgi apparatus.
Insights
Punta Toro virus (PTV) glycoproteins G1 and G2 are synthesized and processed in the Golgi apparatus. These viral proteins are retained intracellularly, indicating specific retention signals within the PTV glycoproteins.
Area of Science:
- Virology
- Molecular Biology
- Cell Biology
Background:
- Punta Toro virus (PTV) is an RNA virus with a middle-sized (M) RNA segment encoding glycoproteins G1 and G2.
- Understanding the intracellular trafficking and localization of viral glycoproteins is crucial for viral pathogenesis and vaccine development.
Purpose of the Study:
- To investigate the synthesis, processing, and intracellular localization of PTV glycoproteins G1 and G2.
- To determine if PTV glycoproteins contain signals for intracellular retention.
Main Methods:
- A recombinant vaccinia virus was constructed expressing PTV G1 and G2 glycoproteins.
- Immunofluorescence microscopy was used to track protein localization.
- Endoglycosidase H digestion assessed glycoprotein processing.
- Surface immune precipitation and 125I-protein A binding assays evaluated cell surface expression.
Main Results:
- Recombinant vaccinia virus infected cells synthesized PTV G1 (65 kDa) and G2 (55 kDa) glycoproteins.
- Glycoproteins were detected intracellularly, primarily in the perinuclear region, and processed through the RER and Golgi complex.
- The majority of G1 and G2 glycoproteins were retained within the Golgi apparatus and not detected at the cell surface.
Conclusions:
- PTV glycoproteins G1 and G2 undergo proper folding and transport to the Golgi complex.
- These glycoproteins contain intrinsic signals that mediate their retention within the Golgi apparatus.
- This intracellular retention may play a role in PTV replication or pathogenesis.