Involvement of p32 and Microtubules in Alteration of Mitochondrial Functions by Rubella Virus

Abstract

The interaction of the rubella virus (RV) capsid (C) protein and the mitochondrial p32 protein is believed to participate in virus replication. In this study, the physiological significance of the association of RV with mitochondria was investigated by silencing p32 through RNA interference. It was demonstrated that downregulation of p32 interferes with microtubule-directed redistribution of mitochondria in RV-infected cells. However, the association of the viral C protein with mitochondria was not affected. When cell lines either pretreated with respiratory chain inhibitors or cultivated under (mild) hypoxic conditions were infected with RV, viral replication was reduced in a time-dependent fashion. Additionally, RV infection induces increased activity of mitochondrial electron transport chain complex III, which was associated with an increase in the mitochondrial membrane potential. These effects are outstanding among the examples of mitochondrial alterations caused by viruses. In contrast to the preferential localization of p32 to the mitochondrial matrix in most cell lines, RV-permissive cell lines were characterized by an almost exclusive membrane association of p32. Conceivably, this contributes to p32 function(s) during RV replication. The data presented suggest that p32 fulfills an essential function for RV replication in directing trafficking of mitochondria near sites of viral replication to meet the energy demands of the virus.Rubella virus (RV), a single-stranded RNA virus, is the sole member of the genus Rubivirus in the family Togaviridae and causes a generally mild exanthematous childhood disease. However, severe malformations known as congenital rubella syndrome may result from the infection of seronegative women, especially during the first trimester of pregnancy. The mechanisms contributing to RV teratogenesis remain largely unknown. The 5Ј-proximal open reading frame (ORF) of the genome encodes the two replicase proteins P150 and P90, while the 3Ј ORF encodes the structural proteins, the capsid (C) protein and two envelope glycoproteins (E1 and E2). Viral RNA synthesis occurs on replication complexes, which are membrane bound to a structure called the cytopathic vacuole (CPV). CPVs are of endolysosomal origin and surrounded by rough endoplasmic reticulum (RER) cisternae, the Golgi apparatus, and mitochondria (13,14). CPVs are replication factories and provide a protected environment for virus replication and assembly.The C protein of RV represents one of the few RNA virusencoded structural proteins that interact with mitochondria and is so far the only known viral protein that impairs protein transport into mitochondria (17). Additionally, the C protein participates in viral RNA synthesis (42), which is emphasized by its accumulation around CPVs (14). The C protein is also involved in the process of mitochondrial redistribution to a perinuclear region in proximity to CPVs (3,28) and interacts with the p32 protein (3). Besides its predominant localization to the matrix of mitochondria, p32 is a...

LIM Kinase 1 (LIMK1) Interacts with Tropomyosin-related Kinase B (TrkB) and Mediates Brain-derived Neurotrophic Factor (BDNF)-induced Axonal Elongation

“…Neuronal lysates were separated into membrane and cytosol fractions to assess the distribution of LIMK1. We used calnexin and ␣-tubulin as membrane and cytosolic protein markers, respectively, to examine the cross-contamination and extraction efficiency as described previously (34). Notably, we detected a significant increase in membrane-associated LIMK1 levels after BDNF stimulation.…”

“…Cell Fractionation-Cytoplasmic and membrane-associated proteins were isolated from primary hippocampal neurons using the ProteoJET TM membrane protein extraction kit (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's instructions (34). In brief, plated neurons in a 6-cm culture plate (5 ϫ 10 6 cells) were stimulated with BDNF (50 ng/ml) for 30 min; then the culture medium was removed, and the cells were rinsed once with 4 ml of ice-cold Cell Wash Solution.…”

Activity Increase in Respiratory Chain Complexes by Rubella Virus with Marginal Induction of Oxidative Stress

“…After blocking of the membrane with 5% dry milk, probing was performed with a 1:200 dilution of rabbit polyclonal antibody against Mfn2 and rabbit polyclonal antibody against VDAC1 (Santa Cruz Biotechnology, Inc., Heidelberg, Germany), a 1:200 dilution of polyclonal antibody to VDAC, and a 1:500 dilution of rabbit SDHA (see below) polyclonal antibody (Acris Antibodies, Herford, Germany) overnight at 4°C. Incubation with secondary antibody and detection by chemiluminescence were done as described previously (9).…”

“…The RV capsid protein localizes to mitochondria and associates with p32 (7), which is required for RV replication (8). Additionally, the perinuclear accumulation of mitochondria in the proximity of RV replication complexes requires p32 (9). Moreover, the RV capsid protein is so far the only known RNA virus-encoded protein that interferes with protein import into mitochondria (10).…”

“…While many viral infections affect the mitochondrial energy and oxidative balance, RV appears to exert an overall positive influence on mitochondrial and bioenergetic function. This is reflected by an increase in respiratory chain (RC) complex III activity, in the ⌬ m , and by a high level of intracellular ATP in RV-infected cells in comparison to the mock-infected control even at late stages of infection (9).…”