Localization of mouse hepatitis virus open reading frame 1A derived proteins

Area of Science:

• Virology
• Cell Biology
• Molecular Biology

Background:

• The 5' portion of mouse hepatitis virus (MHV) gene 1 encodes polyproteins that undergo proteolytic cleavage.
• Understanding the intracellular localization of these cleavage products is crucial for elucidating viral replication mechanisms.

Purpose of the Study:

• To investigate the intracellular localization of proteolytic cleavage products from the N-terminal region of MHV gene 1.
• To determine the association of these viral proteins with cellular organelles like the Golgi complex.

Main Methods:

• Immunofluorescent labeling using antisera against specific MHV gene 1 cleavage products (p28, p65, p240, p290).
• Double immunofluorescent labeling with Golgi-specific markers (anti-mannosidase II).
• Expression of MHV gene 1 sequences in transfected cells using the vaccinia virus-T7 polymerase system.
• Deletion analysis of ORF1a constructs to identify domains responsible for localization.

Main Results:

• The N-terminal cleavage product, p28, showed widespread cytoplasmic distribution.
• Downstream polypeptides (p65, p240, p290) localized to discrete vesicular perinuclear structures.
• p240 and p290 overlapped with the Golgi complex, while p65 also stained the perinuclear cytoplasm.
• Expression of the first 303 amino acids, including the papain-like protease domain 1 (PLP-1), was sufficient for Golgi association.

Conclusions:

• Proteolytic cleavage products of MHV gene 1 exhibit distinct intracellular localization patterns.
• Specific viral proteins are targeted to or associated with the Golgi complex during MHV infection.
• The N-terminal papain-like protease domain (PLP-1) plays a role in the Golgi localization of MHV proteins.