Proteolytic Processing of the Open Reading Frame 1b-Encoded Part of Arterivirus Replicase Is Mediated by nsp4 Serine Protease and Is Essential for Virus Replication

The open reading frame (ORF) 1b-encoded part of the equine arteritis virus (EAV) replicase is expressed by ribosomal frameshifting during genome translation, which results in the production of an ORF1ab fusion protein (345 kDa). Four ORF1b-encoded processing products, nsp9 (p80), nsp10 (p50), nsp11 (p26), and nsp12 (p12), have previously been identified in EAV-infected cells (L. C. van Dinten, A. L. M. Wassenaar, A. E. Gorbalenya, W. J. M. Spaan, and E. J. Snijder, J. Virol. 70:6625–6633, 1996). In the present study, the generation of these four nonstructural proteins was shown to be mediated by the nsp4 serine protease, which is the main viral protease (E. J. Snijder, A. L. M. Wassenaar, L. C. van Dinten, W. J. M. Spaan, and A. E. Gorbalenya, J. Biol. Chem. 271:4864–4871, 1996). Mutagenesis of candidate cleavage sites revealed that Glu-2370/Ser, Gln-2837/Ser, and Glu-3056/Gly are the probable nsp9/10, nsp10/11, and nsp11/12 junctions, respectively. Mutations which abolished ORF1b protein processing were introduced into a recently developed infectious cDNA clone (L. C. van Dinten, J. A. den Boon, A. L. M. Wassenaar, W. J. M. Spaan, and E. J. Snijder, Proc. Natl. Acad. Sci. USA 94:991–997, 1997). An analysis of these mutants showed that the selective blockage of ORF1b processing affected different stages of EAV reproduction. In particular, the mutant with the nsp10/11 cleavage site mutation Gln-2837→Pro displayed an unusual phenotype, since it was still capable of RNA synthesis but was incapable of producing infectious virus.     

F Plasmid TraF and TraH Are Components of an Outer Membrane Complex Involved in Conjugation

F plasmid TraF and TraH are required for F pilus assembly and F plasmid transfer. Using flotation sucrose density gradients, we found that TraF and TraH (as well as TraU and TraW) localized to the outer membrane in the presence of the complete F transfer region, especially TraV, the putative anchor. Mutational analysis of TraH revealed two domains that are important for its function and possible interaction with TrbI, which in turn has a role in stabilizing TraH.The F plasmid (99,159 bp) of Escherichia coli is a model system for the study of the horizontal gene transfer among prokaryotes via conjugation (3, 10, 27). F encodes a 33.3-kb transfer region that is responsible for the formation of mating junctions between donor and recipient cells prior to DNA transfer and establishment in the recipient. The hallmark of F conjugation is the formation of extracellular filaments, F pili, that initiate contact between mating cells and retract, bringing the donor and recipient cells together (5, 19). Synthesis of the F pilus is not well understood, despite the morphological simplicity of this organelle (7, 15, 28). The F transfer region consists of nearly 40 tra genes, with 18 being involved in construction of the transferosome, which is involved in pilus synthesis, mating pair stabilization, and DNA transfer (9). Eight of the encoded Tra proteins (TraA, -B, -C, -E, -G [the N-terminal domain], -K, -L, and -V) correspond to widely conserved members of type IV secretion systems (T4SS), whereas another 9 (TraF, -G [C-terminal domain], -H, -N, -U, and -W and TrbB, -C, and -I) are involved in the F-specific T4SS (4, 18). Two other proteins (TraQ and -X) are specific to the F plasmid itself. The roles of the F-specific proteins that are involved in pilus assembly and DNA transfer are intriguing, since other conjugative T4SS appear to function efficiently without them (18). These tra proteins do not affect F pilin levels, and hence, they have been assigned functions in pilus assembly/retraction and mating pair stabilization, which are characteristics of F-like transfer systems (18). TraF, -H, and -W and TrbC are required for F pilus assembly (9), and mutations in traU reduce the number and the mean length of pili but do not abolish pilus outgrowth (24). TraU is required for DNA transfer and has been tentatively grouped with TraN and -G as proteins involved in mating pair stabilization (18). TrbI is thought to play a role in pilus retraction, since trbI mutants have unusually long pili (21). TrbB contains the thioredoxin-like domain with a C-X-X-C motif and appears to be a periplasmic disulfide bond isomerase (6). Previously, we hypothesized that TrbB and TraF, the latter of which also has the thioredoxin-like domain but lacks the C-X-X-C motif, might have chaperone-like activity. These proteins might help F T4SS proteins such as TraH, -U, and -N, which have 6, 10, and 22 conserved cysteines, respectively, achieve the correct conformation for assembly into the transferosome complex (6). Interestingly, yeast two-hybrid (Y2H) analysis demonstrated that TraF, -H, -U, and -W and TrbB and -I form an interaction group, with TraH directly linked to TraF, TraU, and TrbI (14). TraH is the only one of the three cysteine-rich proteins required for pilus assembly; it is the largest protein (458 amino acids [aa]; 50.2-kDa precursor, processed to 47.8 kDa) in the interaction group and contains a C-terminal coiled-coil domain that can contribute to its oligomerization and interaction with other T4SS proteins (18). Y2H analysis also showed that the C-terminal region of TraH is critical for its interaction with TraF (28 kDa, processed to 25.9 kDa) and TraU (36.8 kDa, processed to 34.3 kDa) and that a deletion within the N-terminal region of TraH enhanced its interaction with TrbI (14.1 kDa) (14).Mutations in traH affect pilus outgrowth but not pilus tip formation at the cell surface, since traH mutants are sensitive to the M13K07 transducing phage, which binds to the pilus tip (1). Membrane fractionation studies of cells containing subclones of the F transfer region originally suggested that TraH fractionates with the inner membrane (IM) (22). TraH contains three N-terminal hydrophobic domains of approximately 20 aa each, which supports this model. In contrast, Ham et al. predicted TraH to be a soluble periplasmic protein (12). Sucrose density gradient sedimentation studies suggested that FLAG-tagged TraH, in the presence of F lac traH80, is in the outer membrane (OM) (23). Since TraH is extracted from membrane preparations with guanidine-HCl or urea but not Triton X-100, Manwaring concluded that TraH is a peripherally associated outer membrane protein (23). By use of subclones of the F transfer region, TraF, -U, -W, and TrbB were localized to the periplasm, whereas TrbI was thought to be an inner membrane protein (21, 24, 29, 30). Using the F plasmid derivative pOX38-Tc (2), which carries the entire F transfer region, we reassessed the localization of TraH as well as TraF, TraU, and TraW (23.6 kDa, processed to 21.7 kDa).E. coli strains were grown at 37°C in Luria-Bertani (LB) broth (1% tryptone [Difco], 0.5% yeast extract [Difco], 1% NaCl [BDH]) with shaking to mid-exponential phase (optical density at 600 nm [OD₆₀₀] of ca. 0.5) with appropriate antibiotics at the following concentrations: 50 μg/ml ampicillin (Ap), 20 μg/ml chloramphenicol (Cm), 25 μg/ml kanamycin (Km), 200 μg/ml streptomycin (Sm), 100 μg/ml spectinomycin (Sp), and 10 μg/ml tetracycline (Tc). Sucrose density flotation studies of cell membrane fractions and immunoblot analysis were performed as previously described (17). Cell pellets corresponding to 0.1 OD₆₀₀ equivalents were used in all immunoblot assays. Samples were boiled in sodium dodecyl sulfate (SDS) sample buffer for 5 min and were analyzed by resolving SDS-15% polyacrylamide gel by using the Bio-Rad Minigel system. The positions of the inner and outer membrane fractions were determined using polyclonal antibodies to the C-terminal region of OmpA, the major outer membrane porin, and CpxA, the inner membrane sensor of the CpxAR two-component system (25). Anti-CpxA, anti-TraE, anti-TraF, anti-TraH, anti-TraU, anti-TraW, and anti-TrbB polyclonal antisera (raised in rabbits) were diluted 1:7,000, 1:5,000, 1:2,000, 1:1,000; 1:500, 1:20,000 and 1:10,000, respectively, in blocking solution and were incubated with the blots at room temperature for 1 h. Anti-OmpA antibodies were used at a 10⁻⁵ dilution in 5% bovine serum albumin (BSA; Roche) to avoid heavy background. Unfortunately, TrbI protein could not be overproduced and specific antibodies could not be raised.Log-phase cultures of E. coli MC4100 (Sm^r) (17) containing pOX38-Tc (2) were separated into periplasmic, cytoplasmic, and membrane fractions according to a previously described method (26). The fractions were tested for the presence of TraH, TraF, TraU, and TraW by SDS-PAGE, followed by immunoblot analysis. All four proteins were found associated with the membrane fraction and not the periplasmic fraction (Fig. ​(Fig.1A).1A). TrbB was found in the periplasmic fraction, in agreement with its proposed role in disulfide bond isomerization (6; data not shown).Open in a separate windowFIG. 1.(A) The F-specific proteins TraH, -F, -U, and -W were detected in the membrane fraction when expressed from MC4100/pOX38-Tc. Proteins were detected by immunoblotting using antisera specific for each protein as described in the text. (B) TraF localization was tested in pOX38-Km and pOX38 ΔtraV::cat. The cells were fractionated into cytoplasmic (C), periplasmic (P), and total membrane (M) fractions, and TraF was detected by immunoblotting with anti-TraF antibodies. TraV was complemented by pRS29 (pRS31 acted as a negative control). The following abbreviations are used: WT, wild type; ΔV, pOX38 ΔtraV::cat; ΔH, pOX38-Tc ΔtraH::cat; and ΔF, pOX38 ΔtraF::kan. The positions of the proteins are indicated by arrows on the right of each panel. The asterisk indicates a band that reacts nonspecifically with anti-F antiserum.Sucrose density flotation gradients of the membrane preparations of MC4100 (Sm^r) cells harboring pOX38-Tc (2), pOX38-Tc ΔtraF::kan (6) and pOX38-Tc ΔtraH::cat were performed to distinguish between OM and IM proteins according to reference 17. pOX38-Tc ΔtraH::cat was constructed according to the method described by Elton et al. (6) by inserting a chloramphenicol acetyltransferase cassette into traH. Gradients were fractionated, and a subset of the fractions (fractions 26 to 54, renamed 1 to 29) that contained the proteins of interest were subjected to SDS-PAGE and immunoblot analyses (Fig. ​(Fig.2).2). OmpA and CpxA were controls for the outer and inner membrane fractions and helped define the subset of fractions examined (Fig. ​(Fig.2,2, panels 1 and 2, respectively). The TraE pilus assembly protein of the F plasmid was used as an IM marker for the F transfer system (Fig. ​(Fig.2,2, panel 9) (9). TraH fractionated as an OM protein in MC4100/pOX38-Tc (Fig. ​(Fig.2,2, panel 3), as did TraF, TraU, and TraW (Fig. ​(Fig.2,2, panels 5, 7, and 8, respectively). TraH did not appear to be required for TraF localization, which was unaffected in a traH mutant (Fig. ​(Fig.2,2, panel 6). In addition, TraF did not appear to be required for TraH localization, although its absence caused a reduction in the levels of TraH (Fig. ​(Fig.2,2, panel 4; see below).Open in a separate windowFIG. 2.The cellular localizations of TraE, TraF, TraH, TraU, and TraW in subcellular fractions of E. coli MC4100/pOX38-Tc and its derivatives. Flotation sucrose density gradients were performed with subsequent immunodetection of tra proteins in a subset of gradient fractions (fractions 26 to 54, renumbered 1 to 29). The positions of the IM and OM fractions are shown above the gels, and the identities of the samples are indicated on the left. The panel numbers are indicated on the right.TraF, -H, -U, and -W appear to be periplasmic proteins that associate with the outer membrane when in the context of the complete transfer apparatus. TrbC, which is fused to TraW in the F-like R27 T4SS, might also be part of this complex (18). Therefore, an as yet unidentified transfer protein should act as an anchor in the outer membrane, directing these proteins to this location. Of the 18 transferosome proteins, only TraV and TraN are known to be located in the OM, with TraV being the only OM protein involved in pilus assembly. Preliminary localization studies using TraF as a test case and a traV insertion mutant, pOX38 ΔtraV::cat (this study, constructed as described above for pOX38-Tc ΔtraH::cat), demonstrated that the levels of TraF decreased dramatically. However, the remaining TraF was found in the periplasm (Fig. ​(Fig.1B).1B). Complementation of the traV mutation with pRS29, but not pRS31 (1), restored TraF localization to the outer membrane. Thus, TraV is probably the anchor protein for both the F-specific transferosome proteins (TraF, -H, -U, and -W) as well as the TraV, -K, and -B complex (13).MC4100 (Sm^r) cells bearing pOX38-Tc (2) or insertion mutant pOX38-Tc ΔtraH::cat, pOX38-Tc ΔtraF::kan (6), pOX38-Tc ΔtrbB::cat (6), pOX38-Tc ΔtraW::cat (this study), pOX38 traU347 (Km^r) (24), or pOX38-trbI472 (Km^r) (21) were used in subsequent experiments. pOX38-Tc ΔtraW::cat was constructed according to the method described by Elton et al. (6) by inserting a chloramphenicol acetyltransferase gene within traW. Mating efficiencies of these mutants were determined according to previously described methods using E. coli ED24 (Sp^r) as the recipient (20). Transconjugants were selected based on double resistance toward chloramphenicol or kanamycin and spectinomycin (Fig. ​(Fig.2).2). Observed mating efficiencies were in agreement with the data obtained previously, as were the results of complementation assays using subclones carrying the appropriate transfer gene (1, 6, 21, 24, 29, 30). These subclones were pK184TraH (Km^r) (this study), pFTraF and pFTrbB (Ap^r) (6), and pKI175 (Ap^r; traWU) (30) (Fig. ​(Fig.2).2). pK184TraH is based on the vector pK184 (Km^r) and contains the traH gene plus its ribosome binding site cloned into the EcoRI and HindIII sites in pK184 (16). Immunoblot analyses revealed that traF, traU, or trbB, but not traW, insertion mutants had slightly reduced levels of TraH in MC4100 cells whereas the trbI insertion mutant had undetectable levels of TraH (Fig. ​(Fig.3).3). Since TraH interacts directly with TrbI, TraF, and TraU in Y2H assays (14), the absence of these proteins would be expected to destabilize TraH. TraH is thought to interact indirectly with TraW via TraU (14); its levels were unaffected in a traW mutant. TraH was destabilized in a dsbA mutant and was undetectable by immunoblotting (data not shown) and decreased slightly in a trbB mutant, suggesting that disulfide bond formation (DsbA) and isomerization (TrbB) are important for TraH.Open in a separate windowFIG. 3.Immunoblot analysis of the levels of TraH in the absence of other members of Y2H interaction group by using pOX38-Tc and its derivatives containing insertion mutations in traH, traF, trbB, traW, traU, or trbI. A loading control is shown in the lower panel, and the mating efficiency (ME) expressed as a percentage of transconjugants relative to donor cells is given below the gels. n.d., not detected; n.a., not applicable. The last line of data are the complementation data (percent complementation mating efficiency [CME]) obtained by use of clones as described in the text. Previously, TraH was found to interact with TraF, TraU, and TrbI, and TraU interacts with TraW (14).The absence of TrbI appeared to have the most profound effect on the level of TraH, although there was only a 20-fold decrease in mating efficiency, suggesting that enough TraH was present to support mating (Fig. ​(Fig.3).3). Complementation assays performed with pOX38-trbI472 and pBAD24TrbI plasmids (this study) restored the levels of TraH, possibly by stabilizing it (Fig. ​(Fig.3).3). pBAD24TrbI is based on the vector pBAD24 (Ap^r) and contains the trbI gene cloned into the EcoRI site in pBAD24 (11). However, complementation with pBAD24TrbI did not restore mating efficiency to wild-type levels, confirming that the insertion mutation within pOX38-trbI472 has a weak polar effect on downstream genes in the tra operon (21). Alternatively, overexpression of TrbI from pBAD24TrbI affected mating efficiency.Y2H analysis revealed two regions within TraH that appeared to be important for TraH-TrbI interactions (14). The deletion of 50 N-terminal amino acids (aa 25 to 75) from the mature TraH gave a 40-fold increase in TraH-TrbI interaction in the Y2H assay (14). This region of TraH also contains the highly conserved residues N31, T44, G60, and R65 (numeration includes the 25-aa signal peptide) (Fig. ​(Fig.4A).4A). Site-directed mutagenesis was performed on plasmid pK184TraH by using the QuikChange kit (Stratagene). The mating abilities of MC4100/pOX38-Tc ΔtraH::cat/pK184TraH and derivatives with amino acid substitutions N31A, T44A, G60A, and R65A were determined according to previously described methods using ED24 (Sp^r) as the recipient (20). Transconjugants were selected based on double resistance toward tetracycline and spectinomycin. TraH levels within the donor cells were monitored by immunoblot analysis. The N31A and T44A substitutions did not affect mating efficiency and did not change the level of TraH within donor cells (Fig. ​(Fig.5).5). The G60A and R65A substitutions decreased mating efficiency to undetectable levels. TraH levels remained unchanged in both mutants (Fig. ​(Fig.5).5). MC4100/pOX38-Tc ΔtraH::cat cells with pK184TraHG60A or pK184TraHR65A were also resistant toward pilus-specific phage f1, suggesting that the pilus was not assembled.Open in a separate windowFIG. 4.Multiple sequence alignment of F-like TraH proteins. (A) Alignment of the N-terminal regions. (B) Alignment of the putative TraH-TrbI interaction region. The leader peptide is cleaved after A24 in F TraH, which is marked by an arrow. The degrees of identity are indicated by black and gray boxes above the sequences, with the tallest black boxes representing conservation over all 7 sequences. Positions with 5 or more different amino acids are marked with the shortest black boxes. The gray boxes in the residue number line indicate gaps in some of the sequences. The putative nucleotide triphosphate (NTP) binding site (aa 193 to 200) and the conserved sequence (aa 220 to 226) are underlined. The regions thought to interact with TrbI are bracketed. Asterisks refer to amino acids selected for mutational analysis. GenBank protein accession numbers for the sequences are as follows: for F, {"type":"entrez-protein","attrs":{"text":"BAA97968","term_id":"8918921","term_text":"BAA97968"}}BAA97968; for SXT, {"type":"entrez-protein","attrs":{"text":"AAL59676","term_id":"21885270","term_text":"AAL59676"}}AAL59676; for R391, {"type":"entrez-protein","attrs":{"text":"AAM08008","term_id":"20095142","term_text":"AAM08008"}}AAM08008; for pNL1, {"type":"entrez-protein","attrs":{"text":"NP_049152","term_id":"10956932","term_text":"NP_049152"}}NP_049152; for RTS1, {"type":"entrez-protein","attrs":{"text":"NP_640201","term_id":"21233903","term_text":"NP_640201"}}NP_640201; for pED208, {"type":"entrez-protein","attrs":{"text":"AAM90722","term_id":"22539465","term_text":"AAM90722"}}AAM90722; and for R27, {"type":"entrez-protein","attrs":{"text":"NP_058340","term_id":"10957316","term_text":"NP_058340"}}NP_058340. Sequence alignment was performed with DNAStar software (LazerGene), using the ClustalW algorithm. Highly conserved amino acids as well as a consensus sequence are given above the residue number line.Open in a separate windowFIG. 5.Immunoblot analysis of intracellular levels of TraH in MC4100/pOX38-Tc ΔtraH::cat complemented with different pK184TraH plasmids. C represents the vector control pK184, WT is the wild-type pK184TraH plasmid, and an asterisk refers to the nonspecific band used as the loading control. Mating efficiency (ME), expressed as a percentage of transconjugants relative to donor cells, is given below the gels. n.d., not detected; MW, molecular mass.Sequence analysis also showed the presence of conserved residues N(L/I/Y)X(W/Y)XX(F/L) (N₂₂₀IMWNAL₂₂₆ in F TraH) within the putative TrbI interaction domain (aa 193 to 226) (Fig. ​(Fig.4B)4B) (14). Substitution of N220 with alanine (N220A) did not change the levels of TraH protein in pOX38-Tc ΔtraH::cat/pK184TraHN220A but decreased the mating ability to undetectable levels. The W223A mutation in TraH decreased the level of TraH within donor cells and reduced the mating efficiency 1,000-fold compared to the wild-type level (Fig. ​(Fig.5).5). The N220A and W223A mutants were resistant to f1 phage and could not assemble functional pili. Thus, mutations in N220 and W223 could affect TraH-TrbI interaction, or they may act independently to block TraH function. If TrbI is in the IM as previously reported (21), then the TrbI:TraH pair could be part of a second envelope-spanning structure analogous to the TraV:TraK:TraB scaffold (8, 13).Primary sequence analysis also revealed the presence of a putative Walker A motif within aa 193 to 226 of TraH (G₁₉₃CTVGGKS₂₀₀) (9). Comparison of seven TraH orthologs revealed that this motif is not conserved among TraH-like sequences (Fig. ​(Fig.4B).4B). To confirm whether this sequence might be important in the F plasmid, a triple mutant (G193A/K199A/S200A) was constructed. It reduced mating efficiency 20-fold but did not change the levels of TraH within donor cells (data not shown). Single substitutions (G193A, K199A, or S200A) did not change the mating efficiency or the level of TraH (data not shown). Thus, TraH, a peripheral OM protein, is probably not an NTPase, nor does it bind nucleotides.Our data also revealed that several conserved amino acid residues are critical for TraH function and structure and that TraH stability is dependent on TrbI as well as DsbA and TrbB, which affect disulfide bond formation and isomerization, respectively. Thus, TrbI, in which mutations have only a minor effect on mating ability, plays a more important role than previously thought (21).     

Membrane Rafts Are Involved in Intracellular Miconazole Accumulation in Yeast Cells

Azoles inhibit ergosterol biosynthesis, resulting in ergosterol depletion and accumulation of toxic 14α-methylated sterols in membranes of susceptible yeast. We demonstrated previously that miconazole induces actin cytoskeleton stabilization in Saccharomyces cerevisiae prior to induction of reactive oxygen species, pointing to an ancillary mode of action. Using a genome-wide agar-based screening, we demonstrate in this study that S. cerevisiae mutants affected in sphingolipid and ergosterol biosynthesis, namely ipt1, sur1, skn1, and erg3 deletion mutants, are miconazole-resistant, suggesting an involvement of membrane rafts in its mode of action. This is supported by the antagonizing effect of membrane raft-disturbing compounds on miconazole antifungal activity as well as on miconazole-induced actin cytoskeleton stabilization and reactive oxygen species accumulation. These antagonizing effects point to a primary role for membrane rafts in miconazole antifungal activity. We further show that this primary role of membrane rafts in miconazole action consists of mediating intracellular accumulation of miconazole in yeast cells.     

Heat Shock Protein 72 Is Associated with the Hepatitis C Virus Replicase Complex and Enhances Viral RNA Replication

The NS5A protein of the hepatitis C virus (HCV) is an integral component of the viral replicase. It also modulates cellular signaling and perturbs host interferon responses. The multifunctional characteristics of NS5A are mostly attributed to its ability to interact with various cellular proteins. This study aimed to identify the novel cellular factors that interact with NS5A and decipher the significance of this interaction in viral replication. The NS5A-interacting proteins were purified by the tandem affinity purification (TAP) procedure from cells expressing NS5A and identified by mass spectrometry. The chaperone protein Hsp72 was identified herein. In vivo protein-protein interaction was verified by co-immunoprecipitation and an in situ proximity ligation assay. In addition to NS5A, Hsp72 was also associated with other members of the replicase complex, NS3 and NS5B, suggesting that it might be directly involved in the HCV replication complex. Hsp72 plays a positive regulatory role in HCV RNA replication by increasing levels of the replicase complex, which was attributed either to the increased stability of the viral proteins in the replicase complex or to the enhanced translational activity of the internal ribosome entry site of HCV. The fact that the host chaperone protein Hsp72 is involved in HCV RNA replication may represent a therapeutic target for controlling virus production.     

Replication of Adenovirus 12 Deoxyribonucleic Acid in Association with the Nuclear Membrane

Analysis of nuclei of adenovirus 12-infected cells revealed that viral DNA replicated in association with the nuclear membrane and that complete viral DNA was liberated from the nuclear membrane. Analysis of isolated nuclei in vitro showed that DNA polymerase activity increased in the nuclear membrane of adenovirus 12-infected cells without addition of primer DNA.     

Caveolae Are Highly Immobile Plasma Membrane Microdomains, Which Are not Involved in Constitutive Endocytic Trafficking

To investigate whether caveolae are involved in constitutive endocytic trafficking, we expressed N- and C- terminally green fluorescent protein (GFP)-tagged caveolin- 1 fusion proteins in HeLa, A431, and Madin-Darby canine kidney cells. The fusion proteins were shown by immunogold labeling to be sorted correctly to caveolae. By using confocal microscopy and photobleaching techniques, it was found that although intracellular structures labeled with GFP-tagged caveolin were dynamic, GFP-labeled caveolae were very immobile. However, after incubation with methyl- beta-cyclodextrin, distinct caveolae disappeared and the mobility of GFP-tagged caveolin in the plasma membrane increased. Treatment of cells with cytochalasin D caused lateral movement and aggregation of GFP-labeled caveolae. Therefore, both cholesterol and an intact actin cytoskeleton are required for the integrity of GFP-labeled caveolae. Moreover, stimulation with okadaic acid caused increased mobility and internalization of the labeled caveolae. Although the calculated mobile fraction (for t = infinity) of intracellular, GFP-tagged caveolin- associated structures was 70-90%, GFP-labeled caveolae in unstimulated cells had a mobile fraction of <20%, a value comparable to that previously reported for E-cadherin in junctional complexes. We therefore conclude that caveolae are not involved in constitutive endocytosis but represent a highly stable plasma membrane compartment anchored by the actin cytoskeleton.     

Basic Domains Target Protein Subunits of the RNase MRP Complex to the Nucleolus Independently of Complex Association

The RNase MRP and RNase P ribonucleoprotein particles both function as endoribonucleases, have a similar RNA component, and share several protein subunits. RNase MRP has been implicated in pre-rRNA processing and mitochondrial DNA replication, whereas RNase P functions in pre-tRNA processing. Both RNase MRP and RNase P accumulate in the nucleolus of eukaryotic cells. In this report we show that for three protein subunits of the RNase MRP complex (hPop1, hPop4, and Rpp38) basic domains are responsible for their nucleolar accumulation and that they are able to accumulate in the nucleolus independently of their association with the RNase MRP and RNase P complexes. We also show that certain mutants of hPop4 accumulate in the Cajal bodies, suggesting that hPop4 traverses through these bodies to the nucleolus. Furthermore, we characterized a deletion mutant of Rpp38 that preferentially associates with the RNase MRP complex, giving a first clue about the difference in protein composition of the human RNase MRP and RNase P complexes. On the basis of all available data on nucleolar localization sequences, we hypothesize that nucleolar accumulation of proteins containing basic domains proceeds by diffusion and retention rather than by an active transport process. The existence of nucleolar localization sequences is discussed.     

The Endoplasmic Reticulum Provides the Membrane Platform for Biogenesis of the Flavivirus Replication Complex

The cytoplasmic replication of positive-sense RNA viruses is associated with a dramatic rearrangement of host cellular membranes. These virus-induced changes result in the induction of vesicular structures that envelop the virus replication complex (RC). In this study, we have extended our previous observations on the intracellular location of West Nile virus strain Kunjin virus (WNV_KUN) to show that the virus-induced recruitment of host proteins and membrane appears to occur at a pre-Golgi step. To visualize the WNV_KUN replication complex, we performed three-dimensional (3D) modeling on tomograms from WNV_KUN replicon-transfected cells. These analyses have provided a 3D representation of the replication complex, revealing the open access of the replication complex with the cytoplasm and the fluidity of the complex to the rough endoplasmic reticulum. In addition, we provide data that indicate that a majority of the viral RNA species housed within the RC is in a double-stranded RNA (dsRNA) form.West Nile virus (WNV) belongs to the Flaviviridae, which is a large family of enveloped, positive-strand RNA viral pathogens that are responsible for causing severe disease and mortality in humans and animals each year. The Australian WNV strain Kunjin virus (WNV_KUN) is a relatively low-pathogenic virus that is closely related to the pathogenic WNV strain New York 99 (WNV_NY99), the causative agent of the 1999 epidemic of encephalitis in New York City (11).It has become increasingly known that the replication of most, if not all, positive-sense RNA viruses, whether they infect plants, insects, or humans, is associated with dramatic membrane alterations resulting in the formation of membranous microenvironments that facilitate efficient virus replication. In most cases the induced membrane structures house the actively replicating viral RNA and comprise 70- to 100-nm membrane “vesicles” (sometimes referred to as spherules). Although this distinct morphology is shared across virus families, the cellular origins of these membranes is diverse: the endoplasmic reticulum (ER), mitochondria, peroxisomes, and trans-Golgi membranes have been implicated in different viral systems (1, 8, 13, 23, 31, 38, 41, 45). This diversity implies that the processes involved in inducing the membrane vesicles/spherules are shared, rather than the composition of the membrane itself, although the exact purpose for utilizing membranes derived from different cellular compartments is still not completely resolved or understood.The replication of the flavivirus WNV_KUN is associated with the induction of morphologically distinct membrane structures that have defined roles during the WNV_KUN replication cycle. Three well-defined structures can be seen as large convoluted membranes (CM), paracrystalline arrays (PC), or membrane sacs containing small vesicles, termed vesicle packets (VP) (18, 20, 48). Based on localization studies with viral proteins of specific functions, we observed that components of the virus protease complex (namely, nonstructural protein 3 [NS3] with cofactor NS2B) localize specifically to the CM/PC, whereas viral double-stranded RNA (dsRNA) and the viral RNA-dependent RNA polymerase (RdRp) NS5 localized primarily to VP (20-22, 47, 48). Additionally, we observed that the CM and PC originate from and are modified membranes of the intermediate compartment (IC) and rough endoplasmic reticulum (RER), whereas the VP appear to be derived from trans-Golgi network (TGN) membranes (19). Recently, we have found that the WNV_KUN NS4A protein by itself has the intrinsic capacity to induce the CM and PC structures (35), a property also subsequently shown for Dengue virus (DENV) NS4A (29). Additionally, we have shown that upon WNV infection cellular cholesterol and cholesterol-synthesizing proteins are redistributed to the virus-induced membranes and that this redistribution severely disrupted the formation of cholesterol-rich microdomains (23). Furthermore, we have shown that the membranous structures induced during WNV replication provide partial protection of the WNV replication components from the interferon (IFN)-induced antiviral MxA protein, suggesting that the distinct compartmentalization of viral replication and components of the cellular antiviral response may be an evolutionary mechanism by which flaviviruses can protect themselves from host surveillance (6).In this study we focused on three-dimensional (3D) modeling to give insight into the 3D structure of the VP and provide evidence of how these complexes are organized and formed within the RER membrane. These results add valuable information to our understanding of how the WNV replication complex (RC) functions.     

The Resistance Protein Tm-1 Inhibits Formation of a Tomato Mosaic Virus Replication Protein-Host Membrane Protein Complex

The Tm-1 gene of tomato confers resistance to Tomato mosaic virus (ToMV). Tm-1 encodes a protein that binds ToMV replication proteins and inhibits the RNA-dependent RNA replication of ToMV. The replication proteins of resistance-breaking mutants of ToMV do not bind Tm-1, indicating that the binding is important for inhibition. In this study, we analyzed how Tm-1 inhibits ToMV RNA replication in a cell-free system using evacuolated tobacco protoplast extracts. In this system, ToMV RNA replication is catalyzed by replication proteins bound to membranes, and the RNA polymerase activity is unaffected by treatment with 0.5 M NaCl-containing buffer and remains associated with membranes. We show that in the presence of Tm-1, negative-strand RNA synthesis is inhibited; the replication proteins associate with membranes with binding that is sensitive to 0.5 M NaCl; the viral genomic RNA used as a translation template is not protected from nuclease digestion; and host membrane proteins TOM1, TOM2A, and ARL8 are not copurified with the membrane-bound 130K replication protein. Deletion of the polymerase read-through domain or of the 3′ untranslated region (UTR) of the genome did not prevent the formation of complexes between the 130K protein and the host membrane proteins, the 0.5 M NaCl-resistant binding of the replication proteins to membranes, and the protection of the genomic RNA from nucleases. These results indicate that Tm-1 binds ToMV replication proteins to inhibit key events in replication complex formation on membranes that precede negative-strand RNA synthesis.     

Intestinal Scavenger Receptors Are Involved in Vitamin K1 Absorption

Vitamin K₁ (phylloquinone) intestinal absorption is thought to be mediated by a carrier protein that still remains to be identified. Apical transport of vitamin K₁ was examined using Caco-2 TC-7 cell monolayers as a model of human intestinal epithelium and in transfected HEK cells. Phylloquinone uptake was then measured ex vivo using mouse intestinal explants. Finally, vitamin K₁ absorption was compared between wild-type mice and mice overexpressing scavenger receptor class B type I (SR-BI) in the intestine and mice deficient in cluster determinant 36 (CD36). Phylloquinone uptake by Caco-2 cells was saturable and was significantly impaired by co-incubation with α-tocopherol (and vice versa). Anti-human SR-BI antibodies and BLT1 (a chemical inhibitor of lipid transport via SR-BI) blocked up to 85% of vitamin K₁ uptake. BLT1 also decreased phylloquinone apical efflux by ∼80%. Transfection of HEK cells with SR-BI and CD36 significantly enhanced vitamin K₁ uptake, which was subsequently decreased by the addition of BLT1 or sulfo-N-succinimidyl oleate (CD36 inhibitor), respectively. Similar results were obtained in mouse intestinal explants. In vivo, the phylloquinone postprandial response was significantly higher, and the proximal intestine mucosa phylloquinone content 4 h after gavage was increased in mice overexpressing SR-BI compared with controls. Phylloquinone postprandial response was also significantly increased in CD36-deficient mice compared with wild-type mice, but their vitamin K₁ intestinal content remained unchanged. Overall, the present data demonstrate for the first time that intestinal scavenger receptors participate in the absorption of dietary phylloquinone.     

Control of Plasmid R1 Replication: Functions Involved in Replication, Copy Number Control, Incompatibility, and Switch-off of Replication

A small derivative of plasmid R1 was used to integratively suppress a chromosomal dnaA(Ts) mutation. The strain obtained grew normally at 42°C. The integratively suppressed strain was used as recipient for various plasmid R1 derivatives. Plasmid R1 and miniplasmid derivatives of R1 could be established in the strain that carried an integrated R1 replicon, but they were rapidly lost during growth. However, plasmids also carrying ColE1 replication functions were almost completely stably inherited. The integratively suppressed strain therefore allows the establishment of bacteria diploid with respect to plasmid R1 and forms a useful and sensitive system for studies of interaction between plasmid R1 replication functions. Several of the chimeric plasmids caused inhibition of growth at high temperatures. All plasmids that inhibited growth carried one particular PstI fragment from plasmid R1 (the PstI F fragment), and in all cases the growth inhibition could be ascribed to repression of initiation of chromosome replication at 42°C, i.e., they carry a trans-acting switch-off function. Furthermore, the analogous PstI fragments from different copy mutants of plasmid R1 were analyzed similarly, and one mutant was found to lack the switch-off function. The different chimeric plasmids were also tested for their incompatibility properties. All plasmids that carried the switch-off function (and no other plasmids) also carried R1 incompatibility gene(s). Since the PstI F fragment, which is present on all these plasmids, is very small (0.35 × 10⁶), it is suggested that the switch-off regulation of replication (by an inhibitor), incompatibility, and copy number control are governed by the same gene.     

Werner Syndrome Protein and the MRE11 Complex are Involved in a Common Pathway of Replication Fork Recovery

Werner syndrome (WS) is an autosomal recessive disease that predisposes individuals toa wide range of cancers. The gene mutated in WS, WRN, encodes a member of the RecQfamily of DNA helicases. The precise DNA metabolic processes in which WRN participatesremain to be elucidated. However, it has been proposed that WRN might play an importantrole in the maintenance of genetic stability during DNA replication, possibly cooperatingwith other proteins. Here, we show that, following DNA replication arrest, WRN associatesand colocalises with the MRE11 complex at PCNA sites. We also provide evidence thatboth WRN/MRE11 complex association and proper WRN relocalisation after HU treatmentrequire a functional MRE11 complex. We demonstrate that mutations altering thefunctionality of WRN or that of the MRE11 complex result in chromosomal breakage duringDNA replication and enhanced cell death following replication arrest. Finally, we show thatthe DNA breakage in replicating cells and apoptosis observed in WS are not enhanced byconcomitant knock down of MRE11 by RNAi, indicating that WRN and MRE11 complexact in a common pathway. These results suggest a functional relationship between WRNand the MRE11 complex in response to replication fork arrest, disclosing a common actionof WRN and the MRE11 complex in the pathway(s) preserving genome stability duringDNA replication.     

The Endosomal Pathway and the Golgi Complex Are Involved in the Infectious Bursal Disease Virus Life Cycle

Infectious bursal disease virus (IBDV), a double-stranded RNA virus belonging to the Birnaviridae family, causes immunosuppression in chickens. In this study, we defined the localization of IBDV replication complexes based on colocalization analysis of VP3, the major protein component of IBDV ribonucleoproteins (RNPs). Our results indicate that VP3 localizes to vesicular structures bearing features of early and late endocytic compartments located in the juxtanuclear region. Interfering with the endocytic pathway with a dominant negative version of Rab5 after the internalization step leads to a reduction in virus titer. Triple-immunostaining studies between VP3, the viral RNA-dependent RNA polymerase VP1, and viral double-stranded RNA (dsRNA) showed a well-defined colocalization, indicating that the three critical components of the RNPs colocalize in the same structure, likely representing replication complexes. Interestingly, recombinant expressed VP3 also localizes to endosomes. Employing Golgi markers, we found that VP3-containing vesicles were closely associated with this organelle. Depolymerization of microtubules with nocodazole caused a profound change in VP3 localization, showing a punctate distribution scattered throughout the cytoplasm. However, these VP3-positive structures remained associated with Golgi ministacks. Similarly, brefeldin A (BFA) treatment led to a punctate distribution of VP3, scattered throughout the cytoplasm of infected cells. In addition, analysis of intra- and extracellular viral infective particles after BFA treatment of avian cells suggested a role for the Golgi complex in viral assembly. These results constitute the first study elucidating the localization of IBDV replication complexes (i.e., in endocytic compartments) and establishing a role for the Golgi apparatus in the assembly step of a birnavirus.