Revised Mimivirus major capsid protein sequence reveals intron-containing gene structure and extra domain

Saïd Azza,Didier Raoult,Marie Suzan-Monti,Christian Cambillau

doi:10.1186/1471-2199-10-39

Abstract

BackgroundAcanthamoebae polyphaga Mimivirus (APM) is the largest known dsDNA virus. The viral particle has a nearly icosahedral structure with an internal capsid shell surrounded with a dense layer of fibrils. A Capsid protein sequence, D13L, was deduced from the APM L425 coding gene and was shown to be the most abundant protein found within the viral particle. However this protein remained poorly characterised until now. A revised protein sequence deposited in a database suggested an additional N-terminal stretch of 142 amino acids missing from the original deduced sequence. This result led us to investigate the L425 gene structure and the biochemical properties of the complete APM major Capsid protein.ResultsThis study describes the full length 3430 bp Capsid coding gene and characterises the 593 amino acids long corresponding Capsid protein 1. The recombinant full length protein allowed the production of a specific monoclonal antibody able to detect the Capsid protein 1 within the viral particle. This protein appeared to be post-translationnally modified by glycosylation and phosphorylation. We proposed a secondary structure prediction of APM Capsid protein 1 compared to the Capsid protein structure of Paramecium Bursaria Chlorella Virus 1, another member of the Nucleo-Cytoplasmic Large DNA virus family.ConclusionThe characterisation of the full length L425 Capsid coding gene of Acanthamoebae polyphaga Mimivirus provides new insights into the structure of the main Capsid protein. The production of a full length recombinant protein will be useful for further structural studies.

Highlights

Acanthamoebae polyphaga Mimivirus (APM) is the largest known dsDNA virus
The Capsid protein 1 cDNA was synthsesized from APM infected A. polyphaga RNA by using the Superscript One-Step RT-PCR with Platinum Taq kit (Invitrogen) with the following primers: RT-PCR analysis RNA was extracted from uninfected or APM-infected amoebae at 0, 2, 4, 8 and 16 hours post infection. 100 ng of each RNA was submitted to RT-PCR amplification using the SuperScript One-Step RT-PCR kit (Invitrogen) with the Q5UQL7NcoIF/Q5UQL7SmaIR primer pair. cDNA synthesis was performed in one cycle of 30 minutes at 50°C, 3 minutes at 94°C and subsequent PCR reaction with 35 cycles of 30 seconds at 94°C, 30 seconds at 60°C, 30 seconds at 72°C, and one cycle of 10 minutes at 72°C
RNA was extracted from APM-infected A. polyphaga and used to synthetise full length L425 cDNA

Summary

Introduction

Acanthamoebae polyphaga Mimivirus (APM) is the largest known dsDNA virus. The viral particle has a nearly icosahedral structure with an internal capsid shell surrounded with a dense layer of fibrils. A Capsid protein sequence, D13L, was deduced from the APM L425 coding gene and was shown to be the most abundant protein found within the viral particle. This protein remained poorly characterised until now. A revised protein sequence deposited in a database suggested an additional N-terminal stretch of 142 amino acids missing from the original deduced sequence. This result led us to investigate the L425 gene structure and the biochemical properties of the complete APM major Capsid protein.

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Conclusion