Abstract:The primary deficiency in the membrane cytoskeletal protein dystrophin results in complex changes in dystrophic muscles. In order to compare the degree of secondary alterations in differently affected subtypes of skeletal muscles, we have conducted a global analysis of proteome-wide changes in various dystrophin-deficient muscles. In contrast to the highly degenerative mdx diaphragm muscle, which showed considerable alterations in 35 distinct proteins, the spectrum of mildly to moderately dystrophic skeletal muscles, including interosseus, flexor digitorum brevis, soleus, and extensor digitorum longus muscle, exhibited a smaller number of changed proteins. Compensatory mechanisms and/or cellular variances may be responsible for differing secondary changes in individual mdx muscles. Label-free mass spectrometry established altered expression levels for diaphragm proteins associated with contraction, energy metabolism, the cytoskeleton, the extracellular matrix and the cellular stress response. Comparative immunoblotting verified the differences in the degree of secondary changes in dystrophin-deficient muscles and showed that the up-regulation of molecular chaperones, the compensatory increase in proteins of the intermediate OPEN ACCESSMolecules 2015, 20 11318 filaments, the fibrosis-related increase in collagen levels and the pathophysiological decrease in calcium binding proteins is more pronounced in mdx diaphragm as compared to the less severely affected mdx leg muscles. Annexin, lamin, and vimentin were identified as universal dystrophic markers.

The almost complete loss of the membrane cytoskeletal protein dystrophin and concomitant drastic reduction in dystrophin-associated glycoproteins are the underlying mechanisms of the highly progressive neuromuscular disorder Duchenne muscular dystrophy. In order to identify new potential binding partners of dystrophin or proteins in close proximity to the sarcolemmal dystrophin complex, proteomic profiling of the isolated dystrophin-glycoprotein complex was carried out. Subcellular membrane fractionation and detergent solubilisation, in combination with ion exchange, lectin chromatography and density gradient ultracentrifugation, was performed to isolate a dystrophin complex-enriched fraction. Following gradient gel electrophoresis and on-membrane digestion, the protein constituents of the dystrophin fraction were determined by peptide mass spectrometry. This proteomic strategy resulted in the novel identification of desmoglein and desmoplakin, which act as cytolinker proteins and possibly exist in close proximity to the dystrophin complex in the sarcolemma membrane. Interestingly, comparative immunoblotting showed a significant reduction in desmoglein in dystrophin-deficient mdx skeletal muscles, reminiscent of the pathobiochemical fate of the dystrophin-associated core proteins in muscular dystrophy. Comparative membrane proteomics was used to correlate this novel finding to large-scale changes in the dystrophic phenotype. A drastic increase in the extracellular stabilizers biglycan and fibronectin was shown by both mass spectrometric analysis and immunoblotting. The reduced expression of desmoglein in dystrophin-deficient skeletal muscles, and simultaneous increase in components of the extracellular matrix, suggest that muscular dystrophy is associated with plasmalemmal disintegration, loss of cellular linkage and reactive myofibrosis.

To facilitate tracing of virion movement, the non-essential capsid proteins pUL35 of herpes simplex virus type 1 and pseudorabies virus (PrV) have been tagged with green fluorescent protein (GFP). However, the biological relevance of PrV pUL35 and the functionality of the fusion proteins have not yet been investigated in detail. We generated PrV mutants either lacking the 12 kDa UL35 gene product, or expressing GFP fused to the N terminus of pUL35. Remarkably, both mutants exhibited significant replication defects in rabbit kidney cells, which could be corrected in pUL35-expressing cells. After intranasal infection of mice both mutants showed delayed neuroinvasion, and survival times of the animals were extended to 3 days, compared with 2 days after wild-type infection. Thus, fusion of pUL35 with GFP resulted in a non-functional protein, which has to be considered for the use of corresponding mutants in tracing studies.

After fusion of the envelope of herpesvirus particles with the host cell plasma membrane, incoming nucleocapsids are transported to nuclear pores. Inner tegument proteins pUL36, pUL37, and pUS3 remain attached to the nucleocapsid after entry and therefore might mediate interactions between the nucleocapsid and cellular microtubule-associated motor proteins during transport. To assay for the role of pUL37 in this process, we constructed a pUL37-deleted pseudorabies virus mutant, PrV-⌬UL37/UL35GFP, which expresses a fusion protein of green fluorescent protein (GFP) and the nonessential small capsid protein pUL35, resulting in the formation of fluorescently labeled capsids. Confocal laser-scanning microscopy of rabbit kidney cells infected with PrV-⌬UL37/UL35GFP revealed that, whereas penetration was not affected in the absence of pUL37, nuclear translocation of incoming particles was delayed by approximately 1 h compared to PrV-UL35GFP, but not abolished. In contrast, phenotypically complemented pUL37-containing virions of PrV-⌬UL37/UL35GFP exhibited wild type-like entry kinetics. Thus, the presence of pUL37 is required for rapid nuclear translocation of incoming nucleocapsids.The herpesvirus replication cycle starts with attachment of extracellular virions to cellular receptors, which is followed by fusion of the virion envelope either with the cellular plasma membrane (20) or, after endocytosis, with the endosomal membrane (49), resulting in release of the nucleocapsid into the cytosol. During this process, most proteins of the tegument, which in the herpes virion resides between the envelope and nucleocapsid, are released from the incoming particle. Several tegument proteins prime the cell for virus infection by shutting off cellular protein synthesis (virion host shut-off factor, pUL41) (29) or, after translocation into the nucleus, boost viral transcription (␣-transinducing factor, pUL48) (2). Other tegument proteins, however, remain bound to the incoming nucleocapsid during its transit to the nuclear pore, where the viral genome is released into the nucleus to start viral gene expression. Thus, efficient transport of nucleocapsids to the nuclear pore is crucial for herpesvirus infectivity.For efficient nuclear targeting of incoming nucleocapsids, herpesviruses recruit cellular motor proteins to move along microtubules (MT) (9,11,17,43,45,48). This process is particularly relevant to overcome even long distances, such as between neuronal axon termini and the nucleated cell bodies. MT are polar cytoskeletal filaments with a fast-growing plus end, extended toward the cell periphery, and a less dynamic minus end, attached to the MT organizing center (38). Minus end-directed transport catalyzed by the huge dynein-dynactin motor complex (44, 53) is used by a variety of viruses (9,15,30,35,40,41,45,48,50). Studies of epithelial and neuronal cells revealed that incoming herpes simplex virus type 1 (HSV-1) capsids colocalize with cytoplasmic dynein and dynactin and that capsid transport to the nucleus is inhibited by...