Cellular origin and ultrastructure of membranes induced during poliovirus infection

Poliovirus RNA replicative complexes are associated with cytoplasmic membranous structures that accumulate during viral infection. These membranes were immunoisolated by using a monoclonal antibody against the viral nonstructural protein 2C. Biochemical analysis of the isolated membranes revealed that several organelles of the host cell (lysosomes, trans-Golgi stack and trans-Golgi network, and endoplasmic reticulum) contributed to the virus-induced membranous structures. Electron microscopy of infected cells preserved by high-pressure freezing revealed that the virus-induced membranes contain double lipid bilayers that surround apparently cytosolic material. Immunolabeling experiments showed that poliovirus proteins 2C and 3D were localized to the same membranes as the cellular markers tested. The morphological and biochemical data are consistent with the hypothesis that autophagy or a similar host process is involved in the formation of the poliovirus-induced membranes

Katanin localization requires triplet microtubules in Chlamydomonas reinhardtii

Centrioles and basal bodies are essential for a variety of cellular processes that include the recruitment of proteins to these structures for both centrosomal and ciliary function. This recruitment is compromised when centriole/basal body assembly is defective. Mutations that cause basal body assembly defects confer supersensitivity to Taxol. These include bld2, bld10, bld12, uni3, vfl1, vfl2, and vfl3. Flagellar motility mutants do not confer sensitivity with the exception of mutations in the p60 (pf19) and p80 (pf15) subunits of the microtubule severing protein katanin. We have identified additional pf15 and bld2 (ε-tubulin) alleles in screens for Taxol sensitivity. Null pf15 and bld2 alleles are viable and are not essential genes in Chlamydomonas. Analysis of double mutant strains with the pf15-3 and bld2-6 null alleles suggests that basal bodies in Chlamydomonas may recruit additional proteins beyond katanin that affect spindle microtubule stability. The bld2-5 allele is a hypomorphic allele and its phenotype is modulated by nutritional cues. Basal bodies in bld2-5 cells are missing proximal ends. The basal body mutants show aberrant localization of an epitope-tagged p80 subunit of katanin. Unlike IFT proteins, katanin p80 does not localize to the transition fibers of the basal bodies based on an analysis of the uni1 mutant as well as the lack of colocalization of katanin p80 with IFT74. We suggest that the triplet microtubules are likely to play a key role in katanin p80 recruitment to the basal body of Chlamydomonas rather than the transition fibers that are needed for IFT localization

Nonstructural Protein Precursor NS4A/B from Hepatitis C Virus Alters Function and Ultrastructure of Host Secretory Apparatus

The nonstructural proteins of hepatitis C virus (HCV) have been shown previously to localize to the endoplasmic reticulum (ER) when expressed singly or in the context of other HCV proteins. To determine whether the expression of HCV nonstructural proteins alters ER function, we tested the effect of expression of NS2/3/4A, NS4A, NS4B, NS4A/B, NS4B/5A, NS5A, and NS5B from genotype 1b HCV on anterograde traffic from the ER to the Golgi apparatus. Only the nominal precursor protein NS4A/B affected the rate of ER-to-Golgi traffic, slowing the rate of Golgi-specific modification of the vesicular stomatitis virus G protein expressed by transfection by approximately threefold. This inhibition of ER-to-Golgi traffic was not observed upon expression of the processed proteins NS4A and NS4B, singly or in combination. To determine whether secretion of other cargo proteins was inhibited by NS4A/B expression, we monitored the appearance of newly synthesized proteins on the cell surface in the presence and absence of NS4A/B expression; levels of all were reduced in the presence of NS4A/B. This reduction is also seen in cells that contain genome length HCV replicons: the rate of appearance of major histocompatibility complex class I (MHC-I) on the cell surface was reduced by three- to fivefold compared to that for a cured cell line. The inhibition of protein secretion caused by NS4A/B does not correlate with the ultrastructural changes leading to the formation a “membranous web” (D. Egger et al., J. Virol. 76:5974-5984, 2002), which can be caused by expression of NS4B alone. Inhibition of global ER-to-Golgi traffic could, by reducing cytokine secretion, MHC-I presentation, and transport of labile membrane proteins to the cell surface, have significant effects on the host immune response to HCV infection

Numbers of flagella in <i>bld2-5</i>, <i>bld2-6</i> and intragenic revertant strains.

a<p><i>BLD2TG</i> indicates the ε-tubulin transgene described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053940#pone.0053940-Dutcher3" target="_blank">[24]</a>.</p

Basal body mutant strains show supersensitivity to Taxol.

<p>(A) Serial dilution of mutant, rescued, and intragenic revertant strains on control medium and (B) 8 µM Taxol-containing medium. Phase images of cells on media with different Taxol concentrations. (C, G) Wild-type, (D, H) <i>pf15-1</i>, (E, I) <i>bld2-6</i> and (F, J) <i>bld2-6, pf15-1</i> double mutant on 10 µM (C–F) or 6 µM Taxol (G–J) containing medium. The <i>bld2-6, pf15-1</i> double mutant is unable to grow on 6 µM Taxol containing medium compared to the single mutant strains. Scale bar in Panel C equals 10 µm. Panels C–J are at the same magnification.</p

The <i>bld2-5</i> and <i>bld2-6</i> strains misplace the cleavage furrow.

<p>A. The ratio of the areas of wild-type sister cells is approximately equal to one (black bars), whereas the ratio of the areas of <i>bld2-5</i> (gray bars) and <i>bld2-6</i> (white bars) sister cells is equal to or greater than one, which suggests a defect in proper placement of the cleavage furrow <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053940#pone.0053940-Preble1" target="_blank">[34]</a>. These results are statistically significant compared by a permutation test <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053940#pone.0053940-Good1" target="_blank">[37]</a>.</p

Katanin localizes to the spindle poles in mitotic cells.

<p>Synchronized cultures were fixed and stained for α-tubulin (red, left panel), HA (green, middle panel), and DNA (blue). Cell cycle stages were determined based on the DNA and tubulin staining patterns. The prometaphase, metaphase, and top row of anaphase are deconvoluted maximum projections of the z-stack. The bottom three rows for anaphase are maximum projections of the z-stack without deconvolution. Scale bar equals 5 µm.</p