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The five complexes (complexes I-V) of the oxidative phosphorylation (OXPHOS) system of mitochondria can be extracted in the form of active supercomplexes. Single-particle electron microscopy has provided 2D and 3D data describing the interaction between complexes I and III, among I, III and IV and in a dimeric form of complex V, between two ATP synthase monomers. The stable interactions are called supercomplexes which also form higher-ordered oligomers. Cryo-electron tomography provides new insights on how these supercomplexes are arranged within intact mitochondria. The structure and function of OXPHOS supercomplexes are discussed.

Foamy viruses (FV) are unusual among retroviruses since they require both Gag and Env structural proteins for particle egress. Recently significant progress has been made towards the mechanistic understanding of the viral release process, in particular that of retroviruses, and the viral domains and cellular pathways involved. However little is currently known about domains of FV structural proteins and cellular proteins engaged in this process. By mutational analysis of sequence motifs in prototype FV (PFV) Gag, bearing homology to known late assembly (L) domains, a PSAP motif with L domain function that was functionally interchangeable by heterologous L domains was identified. In contrast the inactivation of a PPPI motif had no significant influence on PFV particle release, although mutant viral particles displayed reduced infectivity. Similarly mutation of an evolutionary conserved YXXL motif revealed no classical L-domain function but resulted in release of noninfectious viruslike particles. Biochemical and electron microscopy analysis demonstrated that these mutant particles incorporated all viral structural proteins but contained aberrantly capsid structures, suggesting a role in capsid assembly for this PFV Gag sequence motif. In line with the mutational analysis, overexpression of dominant negative (DN) mutants and wild-type TSG101 but not the DN mutant of AIP-1/ALIX reduced PFV particle release and infectivity. Furthermore, DN mutants of Vps4A, Vps4B, and CHMP3 inhibited PFV egress and infectivity. Taken together these results demonstrate that PFV, like other viruses, requires components of the vacuolar protein sorting (VPS) machinery for egress and enters the VPS pathway through interaction with TSG101.Particle release from infected cells is one of the last steps in the retroviral replication cycle, which is accomplished by budding of viral particles across cellular membranes. On the viral side the orthoretroviral Gag polyprotein contains all the essential structural information for particle egress, and Gag expression by itself is sufficient for the release of viruslike particles (VLP). Although for some orthoretroviruses, such as Mason-Pfizer monkey virus (MPMV), coexpression of Env enhances viral particle release (37). In recent years significant advances have been made in the understanding of the mechanistic processes involved in the retroviral budding process. In particular the interaction domains of the viral Gag protein with essential cellular factors required during late stages of the budding process and involved in pinching off the viral particle from the cellular membrane have been identified. To date three consensus sequences of so-called viral late-budding or late-assembly (L) domains have been characterized. A P(T/ S)AP L-domain motif originally identified in the human immunodeficiency virus type 1 (HIV-1) Gag p6 domain (13), a PPXY L-domain motif first found in the Rous sarcoma virus (RSV) Gag p2b cleavage product (47) and a YPXL motif present in the equine infectious anemia virus (EIAV) G...

Foamy viruses (FVs) generate their Pol protein precursor molecule independently of the Gag protein from a spliced mRNA. This mode of expression raises the question of the mechanism of Pol protein incorporation into the viral particle (capsid). We previously showed that the packaging of (pre)genomic RNA is essential for Pol encapsidation (M. Heinkelein, C. Leurs, M. Rammling, K. Peters, H. Hanenberg, and A. Rethwilm, J. Virol. 76: [10069][10070][10071][10072][10073] 2002). Here, we demonstrate that distinct sequences in the RNA, which we termed Pol encapsidation sequences (PES), are required to incorporate Pol protein into the FV capsid. Two PES were found, which are contained in the previously identified cis-acting sequences necessary to transfer an FV vector. One PES is located in the U5 region of the 5 long terminal repeat and one at the 3 end of the pol gene region. Neither element has any significant effect on RNA packaging. However, deletion of either PES resulted in a significant reduction in Pol encapsidation. On the protein level, we show that only the Pol precursor, but not the individual reverse transcriptase (RT) and integrase (IN) subunits, is incorporated into FV particles. However, enzymatic activities of the protease (PR), RT, or IN are not required. Our results strengthen the view that in FVs, (pre)genomic RNA functions as a bridging molecule between Gag and Pol precursor proteins.Spumaretroviruses, or foamy viruses (FVs), are one of two subfamilies of retroviruses (30). A hallmark of the replication strategy of FVs is the Gag-independent expression of a Pol precursor protein from a spliced RNA (3,6,18,25,40). The consequences of this highly unusual strategy for the generation of a retroviral Pol protein are poorly understood. It has been shown that FV reverse transcriptase (RT) is much more processive than that of orthoretroviruses (31). Furthermore, it has been suggested that a very few molecules of Pol precursor are packaged into the FV capsid (4, 31). The FV means of expressing and encapsidating Pol protein has to be viewed in the context of reverse transcription, which appears to occur to a large extent in the virus-producing cell prior to budding (26,32,41). This leads to linear DNA, which is probably the virion nucleic acid and more relevant for infection than RNA (30).The actual mechanism of FV Pol protein particle incorporation despite lack of a Gag-Pol precursor has remained obscure. It is evident that FVs must have found a means of Pol encapsidation that acknowledges that a Gag-Pol precursor protein, which in orthoretroviruses facilitates Pol incorporation into the viral particle, is not generated (35). We previously showed that the incorporation of (pre)genomic RNA is essential for Pol protein encapsidation (12). Here, we took the experiments a step further and asked whether specific sequences on the RNA which allow Pol protein incorporation can be identified and whether such sequences are different from signals needed to package RNA. Furthermore, we wanted to know what requirements e...

Clofazimine nanosuspensions were produced by high pressure homogenization and the formulation was optimized for lyophilization. Characterization of the product by photon correlation spectroscopy, laser diffraction and Coulter counter analysis showed that the clofazimine nanosuspensions were suitable for iv injection with a particle size permitting passive targeting to the reticuloendothelial system. Following iv administration to mice of either the nanocrystalline or a control liposomal formulation at a dose of 20 mg clofazimine/kg bodyweight, drug concentrations in livers, spleens and lungs reached comparably high concentrations, well in excess of the MIC for most Mycobacterium avium strains. When C57BL/6 mice were experimentally infected with M. avium strain TMC 724, nanocrystalline clofazimine was as effective as liposomal clofazimine in reducing bacterial loads in the liver, spleen and lungs of infected mice. Nanocrystalline suspensions of poorly soluble drugs such as riminophenazines are easy to prepare and to lyophilize for extended storage and represent a promising new drug formulation for intravenous therapy of mycobacterial infections.

The foamy virus (FV) Pol polyprotein is translated independently of Gag from a spliced mRNA. This method of expression raises the question of how Pol is associated with the viral particle. Using a transient FV vector transfection system, it is shown that pregenomic RNA is required for efficient virion incorporation of functionally active Pol and that protein-protein interactions of Pol with Gag are not sufficient to complete particle assembly.

Complex I has a unique structure in plants and includes extra subunits. Here, we present a novel study to define its protein constituents. Mitochondria were isolated from Arabidopsis thaliana cell cultures, leaves, and roots. Subunits of complex I were resolved by 3D blue-native (BN)/SDS/SDS-PAGE and identified by mass spectrometry. Overall, 55 distinct proteins were found, seven of which occur in pairs of isoforms. We present evidence that Arabidopsis complex I consists of 49 distinct types of subunits, 40 of which represent homologs of bovine complex I. The nine other subunits represent special proteins absent in the animal linage of eukaryotes, most prominently a group of subunits related to bacterial gamma-type carbonic anhydrases. A GelMap is presented for promoting future complex I research in Arabidopsis thaliana.

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