Mimivirus Giant Particles Incorporate a Large Fraction of Anonymous and Unique Gene Products

Abstract

Acanthamoeba polyphaga mimivirus is the largest known virus in both particle size and genome complexity. Its 1.2-Mb genome encodes 911 proteins, among which only 298 have predicted functions. The composition of purified isolated virions was analyzed by using a combined electrophoresis/mass spectrometry approach allowing the identification of 114 proteins. Besides the expected major structural components, the viral particle packages 12 proteins unambiguously associated with transcriptional machinery, 3 proteins associated with DNA repair, and 2 topoisomerases. Other main functional categories represented in the virion include oxidative pathways and protein modification. More than half of the identified virion-associated proteins correspond to anonymous genes of unknown function, including 45 "ORFans." As demonstrated by both Western blotting and immunogold staining, some of these "ORFans," which lack any convincing similarity in the sequence databases, are endowed with antigenic properties. Thus, anonymous and unique genes constituting the majority of the mimivirus gene complement encode bona fide proteins that are likely to participate in well-integrated processes.Acanthamoeba polyphaga mimivirus (mimivirus) is the largest virus isolated so far (23). Based on its highly specific characteristics, this double-stranded-DNA icosahedral virus (47) is the first member of the new Mimiviridae family (33, 43). Computational annotation of its 1.2-Mb genome (33) revealed many atypical features, including the presence of key translation enzymes, a full complement of DNA repair pathway components, and the unique presence of three different topoisomerases (of types IA, IB, and II) (2, 33). Another unique characteristic of mimivirus is the presence of nearly identical promoter sequence motifs upstream of half of its 911 proteinencoding genes (42), which are presumably associated with proteins expressed during the early or late-early phase. Only 23% of the predicted coding genes exhibit convincing homology to proteins of known function, and 39% of them do not exhibit a clear (E values, Ͻ10 Ϫ5 ) sequence database match (33). Such coding regions without sequence similarity to other genes in databases are considered orphan open reading frames (ORFs) and termed "ORFans" (12). The origin and function of ORFan genes are still a matter of controversy, with opinions ranging from considering them pieces of junk DNA (1,8,40,44) to seeing them as quickly evolving sequences encoding normally expressed functional proteins (38, 39). Recent clinical evidence raised the possibility that mimivirus might be a human pathogen causing pneumonia (4, 24, 34), as suspected when it was first isolated from a cooling tower following an outbreak of pneumonia (23).Mass spectrometry-based analysis has recently emerged as a technique of choice to identify more comprehensively the set of viral proteins associated with viral particles (19,29,49). We now present the application of this technique to the largest known, and presumably most complex, viral particle,...

mRNA deep sequencing reveals 75 new genes and a complex transcriptional landscape in Mimivirus

“…Accordingly, with the exception of the R453 gene product (a TATA-box binding protein homolog with a predicted pI of 9.8), all of them were detected in the particle proteome (Renesto et al 2006;Claverie et al 2009a). This expression pattern also suggests that the early and intermediate Mimivirus transcripts detected in abundance before T = 3 h (i.e., prior to the appearance of fully mature cytoplasmic virion factories) (Fig.…”

“…The fact that an overwhelming proportion of these genes are maximally expressed at T $ 6 h strongly suggests that these enzymatic activities are not targeting host proteins but are rather involved in the building of Mimivirus particles (the proteomic analysis of which indicated many post-translational modifications) (Renesto et al 2006). …”

“…However, a closer inspection of the genes clustered in the same expression class indicates that their individual transcription profiles are not superimposable ( The biological relevance of the three main classes of transcription profiles was confirmed by examining the expression pattern of several genes of known function. The ''late'' expression class, for instance, includes genes encoding structural components of the Mimivirus particles (such as the main capsid protein L425 or the core protein L410) or genes encoding enzymes carried by the particle (such as the glucose-methanol-choline [GMC]-type oxydoreductase R315) or enzymes most likely involved in the biosynthesis of the lipopolysaccharide (LPS)-like outer layer of the virus particle Claverie et al 2009a;Xiao et al 2009) (L136, R689, L780) ( Accordingly, the proportion of genes from this class, the products of which were also detected in the particle proteome (Renesto et al 2006;Claverie et al 2009a), is much higher than in the other expression classes (Fig. 4E).…”

“…The unique features of Mimivirus revived the debate about the evolution of DNA viruses (Claverie 2006;Claverie et al 2006;Iyer et al 2006) and their position in the Tree of Life (Brüssow 2009;Claverie and Ogata 2009;Moreira and Lopez-Garcia 2009). The complex Mimivirus particle has been the object of detailed proteomic (Renesto et al 2006) and ultrastructural studies (Zauberman et al 2008;Xiao et al 2009), and several individual gene products have been characterized (for review, see Claverie and Abergel 2009); however, a systemic description of the molecular processes at work during the intracellular Mimivirus replication cycle (in particular the eclipse phase) is missing. During this phase, the Acanthamoeba cells are instructed by the infecting Mimivirus to build up a giant organelle-like ''virion factory'' that appears to centralize most of the metabolic processes leading to the synthesis of new Mimivirus particles (Suzan-Monti et al 2007;Claverie and Abergel 2009;Claverie et al 2009b).…”

Sputnik and Mavirus: more than just satellite viruses

“…1). Giant viruses such as APMV and CroV replicate in large cytoplasmic virion factories, and encode an enzymatic complexity that is unrivalled among viruses and includes a presumably complete transcription apparatus, which is packaged in the virion and used to initiate viral gene transcription at the early stage of infection [9][10][11][12] . Although yet to be confirmed experimentally, Sputnik and Mavirus appear to depend on the transcription system of their associated giant viruses, as strongly suggested by the specific promoter and polyadenylation signals that are shared between these virophages and their giant viruses 4,13,14 .…”