Mutations and deletions in mitochondrial DNA (mtDNA) lead to a number of human diseases characterized by neuromuscular degeneration. Accumulation of truncated mtDNA molecules (∆-mtDNA) lacking a specific 4977-bp fragment, the common deletion, leads to three related mtDNA diseases : Pearson's syndrome; Kearns-Sayre syndrome; and chronic progressive external ophthalmoplegia (CPEO). In addition, the proportion of ∆-mtDNA present increases with age in a range of tissues. Consequently, there is considerable interest in the effects of the accumulation of ∆-mtDNA on cell function. The 4977-bp deletion affects genes encoding 7 polypeptide components of the mitochondrial respiratory chain, and 5 of the 22 tRNAs necessary for mitochondrial protein synthesis. To determine how the accumulation of ∆-mtDNA affects oxidative phosphorylation we constructed a series of cybrids by fusing a human osteosarcoma cell line depleted of mtDNA (ρ 0 ) with enucleated skin fibroblasts from a CPEO patient. The ensuing cybrids contained 0Ϫ86 % ∆-mtDNA and all had volumes, protein contents, plasma-membrane potentials and mitochondrial contents similar to those of the parental cell line. The bioenergetic consequences of accumulating ∆-mtDNA were assessed by measuring the mitochondrial membrane potential, rate of ATP synthesis and ATP/ADP ratio. In cybrids containing less than 50Ϫ55% ∆-mtDNA, these bioenergetic functions were equivalent to those of cybrids with intact mtDNA. However, once the proportion of ∆-mtDNA exceeded this threshold, the mitochondrial membrane potential, rate of ATP synthesis, and cellular ATP/ADP ratio decreased. These bioenergetic deficits will contribute to the cellular pathology associated with the accumulation of ∆-mtDNA in the target tissues of patients with mtDNA diseases.

Ross River virus and Barmah Forest virus are Australian arboviruses of the Alphavirus genus. Features of alphavirus infection include an increased permeability of cells to monovalent cations followed by virion budding. Virally encoded ion channels are thought to have a role in these processes. In this paper, the 6K proteins of Ross River virus and Barmah Forest virus are shown to form cation-selective ion channels in planar lipid bilayers. Using a novel purification method, bacterially expressed 6K proteins were inserted into bilayers with a defined orientation (i.e. N-terminal cis, C-terminal trans). Channel activity was reversibly inhibited by antibodies to the N and C termini of 6K protein added to the cis and trans baths, respectively. Channel conductances varied from 40 -800 picosiemens, suggesting that the protein is able to form channels with a range of possible oligomerization states.

Alphaviruses such as the Sindbis-group viruses, Scandinavian Ockelbo virus, the African Asian chikungunya virus, the African O'nyong-nyong virus, the South American Mayaro virus, and the Australasian Barmah Forest and Ross River viruses, are commonly associated with outbreaks of acute and persistent arthritis and arthralgia in humans. The mechanisms by which these viruses cause arthritis/arthralgia are poorly understood. This chapter summarizes our current understanding of viral arthritides using our newly developed mouse model of Ross River virus-induced joint and muscle inflammation.

The alphaviral 6k gene region encodes the two structural proteins 6K protein and, due to a ribosomal frameshift event, the transframe protein (TF). Here, we characterized the role of the 6k proteins in the arthritogenic alphavirus Ross River virus (RRV) in infected cells and in mice, using a novel 6k in-frame deletion mutant. Comprehensive microscopic analysis revealed that the 6k proteins were predominantly localized at the endoplasmic reticulum of RRV-infected cells. RRV virions that lack the 6k proteins 6K and TF [RRV-(⌬6K)] were more vulnerable to changes in pH, and the corresponding virus had increased sensitivity to a higher temperature. While the 6k deletion did not reduce RRV particle production in BHK-21 cells, it affected virion release from the host cell. Subsequent in vivo studies demonstrated that RRV-(⌬6K) caused a milder disease than wild-type virus, with viral titers being reduced in infected mice. Immunization of mice with RRV-(⌬6K) resulted in a reduced viral load and accelerated viral elimination upon secondary infection with wild-type RRV or another alphavirus, chikungunya virus (CHIKV). Our results show that the 6k proteins may contribute to alphaviral disease manifestations and suggest that manipulation of the 6k gene may be a potential strategy to facilitate viral vaccine development. IMPORTANCE Arthritogenic alphaviruses, such as chikungunya virus (CHIKV) and Ross River virus (RRV), cause epidemics of debilitating rheumatic disease in areas where they are endemic and can emerge in new regions worldwide. RRV is of considerable medical significance in Australia, where it is the leading cause of arboviral disease. The mechanisms by which alphaviruses persist and cause disease in the host are ill defined. This paper describes the phenotypic properties of an RRV 6k deletion mutant. The absence of the 6k gene reduced virion release from infected cells and also reduced the severity of disease and viral titers in infected mice. Immunization with the mutant virus protected mice against viremia not only upon exposure to RRV but also upon challenge with CHIKV. These findings could lead to the development of safer and more immunogenic alphavirus vectors for vaccine delivery.

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