Bovine viral diarrhea virus NS4B protein is an integral membrane protein associated with Golgi markers and rearranged host membranes

<p>Abstract</p> <p>Background</p> <p>Very little is known about BVDV NS4B, a protein of approximately 38 kDa. However, a missense mutation in NS4B has been implicated in changing BVDV from a cytopathic to noncytopathic virus, suggesting that NS4B might play a role in BVDV pathogenesis. Though this is one possible function, it is also likely that NS4B plays a role in BVDV genome replication. For example, BVDV NS4B interacts with NS3 and NS5A, implying that NS4B is part of a complex, which contains BVDV replicase proteins. Other possible BVDV NS4B functions can be inferred by analogy to hepatitis C virus (HCV) NS4B protein. For instance, HCV NS4B remodels host membranes to form the so-called membranous web, the site for HCV genome replication. Finally, HCV NS4B is membrane-associated, implying that HCV NS4B may anchor the virus replication complex to the membranous web structure. Unlike its HCV counterpart, we know little about the subcellular distribution of BVDV NS4B protein. Further, it is not clear whether NS4B is localized to host membrane alterations associated with BVDV infection.</p> <p>Results</p> <p>We show first that release of infectious BVDV correlates with the kinetics of BVDV genome replication in infected cells. Secondly, we found that NS4B subcellular distribution changes over the course of BVDV infection. Further, BVDV NS4B is an integral membrane protein, which colocalizes mainly with the Golgi compartment when expressed alone or in the context of BVDV infection. Additionally, BVDV induces host membrane rearrangement and these membranes contain BVDV NS4B protein. Finally, NS4B colocalizes with replicase proteins NS5A and NS5B proteins, raising the possibility that NS4B is a component of the BVDV replication complex. Interestingly, NS4B was found to colocalize with mitochondria suggesting that this organelle might play a role in BVDV genome replication or cytopathogenicity.</p> <p>Conclusion</p> <p>These results show that BVDV NS4B is an integral membrane protein associated with the Golgi apparatus and virus-induced membranes, the putative site for BVDV genome replication. On the basis of NS4B Colocalization with NS5A and NS5B, we conclude that NS4B protein is an integral component of the BVDV replication complex.</p

Formation and function of hepatitis C virus replication complexes require residues in the carboxy-terminal domain of NS4B protein

AbstractDuring replication, hepatitis C virus (HCV) NS4B protein rearranges intracellular membranes to form foci, or the web, the putative site for HCV replication. To understand the role of the C-terminal domain (CTD) in NS4B function, mutations were introduced into NS4B alone or in the context of HCV polyprotein. First, we show that the CTD is required for NS4B-induced web structure, but it is not sufficient to form the web nor is it required for NS4B membrane association. Interestingly, all the mutations introduced into the CTD impeded HCV genome replication, but only two resulted in a disruption of NS4B foci. Further, we found that NS4B interacts with NS3 and NS5A, and that mutations causing NS4B mislocalization have a similar effect on these proteins. Finally, we show that the redistribution of Rab5 to NS4B foci requires an intact CTD, suggesting that Rab5 facilitates NS4B foci formation through interaction with the CTD

Conserved Cysteine-Rich Domain of Paramyxovirus Simian Virus 5 V Protein Plays an Important Role in Blocking Apoptosis

The paramyxovirus family includes many well-known human and animal pathogens as well as emerging viruses such as Hendra virus and Nipah virus. The V protein of simian virus 5 (SV5), a prototype of the paramyxoviruses, contains a cysteine-rich C-terminal domain which is conserved among all paramyxovirus V proteins. The V protein can block both interferon (IFN) signaling by causing degradation of STAT1 and IFN production by blocking IRF-3 nuclear import. Previously, it was reported that recombinant SV5 lacking the C terminus of the V protein (rSV5VΔC) induces a severe cytopathic effect (CPE) in tissue culture whereas wild-type (wt) SV5 infection does not induce CPE. In this study, the nature of the CPE and the mechanism of the induction of CPE were investigated. Through the use of DNA fragmentation, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling, and propidium iodide staining assays, it was shown that rSV5VΔC induced apoptosis. Expression of wt V protein prevented apoptosis induced by rSV5VΔC, suggesting that the V protein has an antiapoptotic function. Interestingly, rSV5VΔC induced apoptosis in U3A cells (a STAT1-deficient cell line) and in the presence of neutralizing antibody against IFN, suggesting that the induction of apoptosis by rSV5VΔC was independent of IFN and IFN-signaling pathways. Apoptosis induced by rSV5VΔC was blocked by a general caspase inhibitor, Z-VAD-FMK, but not by specific inhibitors against caspases 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 13, suggesting that rSV5VΔC-induced apoptosis can occur in a caspase 12-dependent manner. Endoplasmic reticulum stress can lead to activation of caspase 12; compared to the results seen with mock and wt SV5 infection, rSV5VΔC infection induced ER stress, as demonstrated by increased expression levels of known ER stress indicators GRP 78, GRP 94, and GADD153. These data suggest that rSV5VΔC can trigger cell death by inducing ER stress

Multiple poliovirus-induced organelles suggested by comparison of spatiotemporal dynamics of membranous structures and phosphoinositides

<div><p>At the culmination of poliovirus (PV) multiplication, membranes are observed that contain phosphatidylinositol-4-phosphate (PI4P) and appear as vesicular clusters in cross section. Induction and remodeling of PI4P and membranes prior to or concurrent with genome replication has not been well studied. Here, we exploit two PV mutants, termed EG and GG, which exhibit aberrant proteolytic processing of the P3 precursor that substantially delays the onset of genome replication and/or impairs virus assembly, to illuminate the pathway of formation of PV-induced membranous structures. For WT PV, changes to the PI4P pool were observed as early as 30 min post-infection. PI4P remodeling occurred even in the presence of guanidine hydrochloride, a replication inhibitor, and was accompanied by formation of membrane tubules throughout the cytoplasm. Vesicular clusters appeared in the perinuclear region of the cell at 3 h post-infection, a time too slow for these structures to be responsible for genome replication. Delays in the onset of genome replication observed for EG and GG PVs were similar to the delays in virus-induced remodeling of PI4P pools, consistent with PI4P serving as a marker of the genome-replication organelle. GG PV was unable to convert virus-induced tubules into vesicular clusters, perhaps explaining the nearly 5-log reduction in infectious virus produced by this mutant. Our results are consistent with PV inducing temporally distinct membranous structures (organelles) for genome replication (tubules) and virus assembly (vesicular clusters). We suggest that the pace of formation, spatiotemporal dynamics, and the efficiency of the replication-to-assembly-organelle conversion may be set by both the rate of P3 polyprotein processing and the capacity for P3 processing to yield 3AB and/or 3CD proteins.</p></div

EG PV exhibits delayed induction and redistribution of PI4P.

<p><b>(A)</b> Immunostaining of PI4P in mock-infected HeLa cells. <b>(B)</b> Time-course of PI4P-staining in HeLa cells infected with WT or EG PV. HeLa cells were infected with WT or EG virus at an MOI of 10, fixed at indicated times post-infection, and immunostained for PI4P. <b>(C)</b> Impact of GuHCl on PI4P induction by WT and EG PVs. HeLa cells were either incubated with PBS or infected with WT or EG virus (MOI 10) in presence of 3 mM GuHCl and immunostained. In all cases, PI4P was stained using anti-PI4P antibody (red) and nuclei were stained with DAPI (blue).</p

WT PV induces tubules in the presence of a replication inhibitor.

<p>(<b>A</b>) GuHCl has no impact on cell ultrastructure. HeLa cells were grown for 10 h at 37 ^oC in the presence of 3 mM GuHCl, and the cell ultrastructure was visualized by TEM. Bar = 1 μm. N denotes nucleus. (<b>B</b>) Ultrastructural changes are observed in the absence of replication. HeLa cells were infected with WT PV at an MOI of 10 in the presence of 3 mM GuHCl at 37 ^oC. Ten hours post-infection, cell ultrastructure was visualized by TEM. Bar = 1 μm. Representative images are shown in panels i, ii, and iii; the lower panels are enlargements of the boxed fields in the panels above. Some of the tubular-reticular structures are marked by the dotted line and/or arrowheads in the various panels to highlight the structures to which we refer but not to be exhaustive in our labeling. N denotes nucleus.</p

Kinetics of genome replication precedes the kinetics of vesicular cluster formation for EG PV.

<p>(<b>A</b>) Kinetics of RNA synthesis (○) and virus production (●) by EG PV. HeLa cells were infected with EG PV at an MOI of 10, placed at 37°C, and at the indicated times post-infection, total RNA was isolated and subjected to either Northern blotting or assayed for virus production by standard plaque assay. (<b>B</b>) Image of a representative blot visualized by phosphorimaging. <b>(C)</b> Kinetics of formation of virus-induced vesicular clusters by transmission electron microscope (TEM). Vesicular clusters begin to form at 4 h post-infection and continue throughout the time course are indicated by white dotted circles. HeLa cells were infected with EG PV at MOI of 10, placed at 37°C, and at the indicated times post-infection, infected cells were fixed and visualized by TEM, bar = 1 μm. UN denotes uninfected control; N denotes nucleus.</p

GG PV induces and redistributes PI4P in spite of impaired formation of vesicular clusters.

<p>(<b>A</b>) Kinetics of RNA synthesis by WT (●) and GG (Δ) subgenomic replicon RNA. HeLa cells were co-transfected with two different replicon RNAs, luciferase replicon (2 μg) and EGFP replicon (4 μg), placed at 34°C and at the indicated times post-transfection, luciferase activity was measured. (<b>B</b>) Cell sorting to isolate PV replicon-positive cells. WT replicon RNA-transfected cells were 61% positive in pre-sort cells (top-left) and 98% positive in post-sort cells (bottom-left). GG replicon RNA transfected cells were 18% positive in pre-sort cells (top-right) and 94% positive in post-sort cells (bottom-right). (<b>C</b>) WT and GG PV-induced membranes visualized by TEM. Vesicular clusters that form with the WT replicon are indicated by a white dotted circle. Vesicular clusters are not observed with the GG replicon. The right most panel is an enlargement of the area indicated by the black box in the middle panel for the GG replicon. The tubular/reticular network that forms with the GG replicon is indicated by white arrows. HeLa cells were transfected with either WT or GG replicon RNA, placed at 34°C for 5 h or 14 h, respectively, at which time cells were fixed and visualized by TEM. Bar = 1 μm. N denotes nucleus. <b>(D)</b> Kinetics of PI4P induction and redistribution by the GG PV subgenomic replicon. HeLa cells were transfected with replicon RNA expressing EGFP and samples were fixed at the indicated time post-transfection and subjected to IFM using anti-PI4P antibody (red) and nuclei were stained with DAPI (blue).</p