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Human cytomegalovirus capsid assembly protein precursor (pUL80.5) interacts with itself and with the major capsid
L J Wood1, M K Baxter, S M Plafker
1Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Abstract:
We have used the yeast GAL4 two-hybrid system to examine interactions between the human cytomegalovirus (HCMV) major capsid protein (MCP, encoded by UL86) and the precursor assembly protein (pAP, encoded by UL80.5 and cleaved at its carboxyl end to yield AP) and found that (i) the pAP interacts with the MCP through residues located within the carboxy-terminal 21 amino acids of the pAP, called the carboxyl conserved domain (CCD); (ii) the pAP interacts with itself through a separate region, called the amino conserved domain (ACD), located between amino acids His34 and Arg52 near the amino end of the molecule; (iii) the simian CMV (SCMV) pAP and AP can interact with or replace their HCMV counterparts in these interactions, whereas the herpes simplex virus pAP and AP homologs cannot; and (iv) the HCMV and SCMV maturational proteinase precursors (ACpra, encoded by UL80a and APNG1, respectively) can interact with the pAP and MCP. The ACD and CCD amino acid sequences are highly conserved among members of the betaherpesvirus group and appear to have counterparts in the alpha- and gammaherpesvirus pAP homologs. Deleting the ACD from the HCMV pAP, or substituting Ala for a conserved Leu in the ACD, eliminated detectable pAP self-interaction and also substantially reduced MCP binding in the two-hybrid assay. This finding indicates that the pAP self-interaction influences the pAP-MCP interaction. Immunofluorescence studies corroborated the pAP-MCP interaction detected in the GAL4 two-hybrid experiments and showed that nuclear transport of the MCP was mediated by pAP but not AP. We conclude that the pAP interacts with the MCP, that this interaction is mediated by the CCD and is influenced by pAP self-interaction, and that one function of the pAP-MCP interaction may be to provide a controlled mechanism for transporting the MCP into the nucleus.
Insights
Human cytomegalovirus precursor assembly protein (pAP) interacts with major capsid protein (MCP) via its carboxyl conserved domain (CCD). This interaction, influenced by pAP self-binding, is crucial for transporting MCP into the nucleus.
Area of Science:
- Virology
- Molecular Biology
- Structural Biology
Background:
- Human cytomegalovirus (HCMV) assembly involves intricate protein-protein interactions.
- The major capsid protein (MCP) and precursor assembly protein (pAP) are key components in HCMV capsid formation.
- Understanding these interactions is vital for deciphering viral replication mechanisms.
Purpose of the Study:
- To investigate the interaction between HCMV MCP and pAP.
- To identify the specific domains and regions involved in pAP self-interaction and MCP binding.
- To elucidate the role of pAP-MCP interaction in viral nuclear transport.
Main Methods:
- Yeast GAL4 two-hybrid system for protein interaction analysis.
- Site-directed mutagenesis to assess the role of conserved domains (ACD, CCD).
- Immunofluorescence microscopy to visualize protein localization and nuclear transport.
Main Results:
- pAP interacts with MCP via its carboxyl conserved domain (CCD).
- pAP self-interaction occurs through the amino conserved domain (ACD) and influences MCP binding.
- HCMV and Simian CMV (SCMV) pAP/AP interact, unlike Herpes Simplex Virus homologs.
- pAP mediates nuclear transport of MCP, but not cleaved AP.
Conclusions:
- HCMV pAP interacts with MCP through the CCD, with pAP self-interaction modulating this binding.
- The pAP-MCP interaction is essential for the nuclear import of MCP.
- This interaction likely serves as a controlled mechanism for MCP nuclear transport during viral assembly.