Novel NG36/G9a gene products encoded within the human and mouse MHC class III regions.

Previous annotation of the Class III region of the human Major Histocompatibility Complex (MHC) depicts NG36 as an independent gene, which lies immediately centromeric to the G9a gene. However, data presented in this report show that in human and mouse cells the NG36 and G9a genes are predominantly expressed within a single approximately 3.9-kbp transcript. Thus, the human NG36/G9a gene contains 28 exons (4 exons from the NG36 gene and 24 exons from the G9a gene), spans 17.938 kb, and encodes a 1210-amino acid polypeptide. In addition, a splice variant (NG36G9a-SPI), which lacks exon 10, was found to be coexpressed together with the full-length NG36/G9a transcript in both human and mouse cells. To aid functional characterization of the novel NG36/G9a gene-product, T7-epitope-tagged versions of the complete NG36/G9a protein or the G9a region alone (amino acids 210 to 1210) was transiently expressed in mammalian cells. Surprisingly, the sub-cellular distribution of the NG36/G9a-T7 and G9a-T7 proteins was found to be quite distinct. Whereas the G9a-T7 protein was observed in both the cytoplasm and the nucleus, the NG36/G9a-T7 protein was extensively concentrated within the nucleus. Also, the G9a-T7 protein frequently appeared marginalized at the nuclear periphery, while the NG36/G9a-T7 protein was generally found throughout the nucleoplasm. As such, it would appear that the NG36 domain plays a key role in controlling the sub-cellular distribution of the NG36/G9a protein.

The vaccinia virus A27L protein is needed for the microtubule-dependent transport of intracellular mature virus particles.

The vaccinia virus (VV) A27L gene encodes a 14 kDa protein that is required for the formation of intracellular enveloped virus (IEV) and, consequently, normal sized plaques. Data presented here show that A27L plays an additional role in VV assembly. When cells were infected with the VV WR32-7/Ind 14K, under conditions that repress A27L expression, transport of intracellular mature virus (IMV) from virus factories was inhibited and some IMV was found in aberrant association with virus crescents. In contrast, other VV mutants (vDeltaB5R and vDeltaF13L) that are defective in IEV formation produce IMV particles that are transported out of virus factories. This indicated a specific role for A27L in IMV transport. Induction of A27L expression at 10 h post-infection promoted the dispersal of clustered IMV particles, but only when microtubules were intact. Formation of IEV particles was also impaired when cells were infected with WR32-7/14K, a VV strain expressing a mutated form of the A27L protein; however, this mutation did not inhibit intracellular transport of IMV particles. Collectively, these data define two novel aspects of VV morphogenesis. Firstly, A27L is required for both IMV transport and the process of envelopment that leads to IEV formation. Secondly, movement of IMV particles between the virus factory and the site of IEV formation is microtubule-dependent.

The effects of targeting the vaccinia virus B5R protein to the endoplasmic reticulum on virus morphogenesis and dissemination.

The consequence of redirecting the vaccinia virus (VV) B5R protein to the endoplasmic reticulum (ER) has been investigated by the addition of an ER retrieval signal KKSL (K(2)X(2)) to the B5R C-terminus. This mutant B5R gene and a version of the gene with the inactive ER retrieval sequence KKSLAL (K(2)X(4)) were inserted into the thymidine kinase locus of a VV mutant lacking the B5R gene, vDeltaB5R. Similar levels of B5R protein were made by each virus, but the B5R-K(2)X(2) protein remained sensitive to endoglycosidase H and colocalised with protein disulphide isomerase in the ER. In contrast, the B5R-K(2)X(4) protein colocalised with 1, 4-galactosyltransferase in the trans-Golgi network. Electron microscopy revealed that even when the B5R protein was redirected to the ER, intracellular mature virus particles were wrapped by cellular membranes to form intracellular enveloped virus particles, although more incompletely wrapped particles were evident compared with wild type. These intracellular enveloped virus particles were, however, unable to efficiently induce the polymerisation of actin and the plaque size formed by vB5R-K(2)X(2) was small. Nevertheless, the amount and specific infectivity of EEV produced by vB5R-K(2)X(2) were similar to those of wild type, despite the dramatic reduction in the amount of B5R protein present in vB5R-K(2)X(2) EEV.

Cell motility and cell morphology: how some viruses take control.

Viruses replicate inside host cells, where they use host biochemical and structural components to facilitate the production of new virus particles. As a consequence of co-evolution with their hosts, viruses have acquired host genes and genetic mutations that confer dominance over normal cell function. Research on virus-cell interactions has focused on the identification of mechanisms of virus dominance in order to develop therapeutic strategies for preventing productive infection. Although such research remains an essential part of molecular virology, viruses are also important genetic tools that can be used to analyse cell function. Because virus genomes contain genetic information, some of which was derived from host cells, it is possible that the analyses of virus-host interactions might lead to the identification of functionally dominant virus genes and novel eukaryotic counterparts. In this article, we have described how transforming and non-transforming viruses can control cell motility (cell migration or membrane projection), and explained how the analysis of virus cytopathic effects (CPEs) led to the identification of a novel family of cellular genes that regulate diverse aspects of cell motility.

Vaccinia virus induces Ca2+-independent cell-matrix adhesion during the motile phase of infection.

Vaccinia virus (VV) induces two forms of cell motility: cell migration, which is dependent on the expression of early genes, and the formation of cellular projections, which requires the expression of late genes. The need for viral gene expression prior to cell motility suggests that VV proteins may affect how infected cells interact with the extracellular matrix. To address this, we have analyzed changes in cell-matrix adhesion after infection of BS-C-1 cells with VV. Whereas uninfected cells round up and detach from the culture flask in the presence of EGTA, infected cells remain attached to the culture flask with a stellate morphology. Ca2+-independent cell-matrix adhesion was evident by 10 h postinfection, after the onset of cell motility but before the formation of virus-induced cellular projections. Progression to Ca2+-independent adhesion required the expression of late viral genes but not the formation of intracellular enveloped virus particles or intracellular actin tails. Analyses of specific matrix proteins identified vitronectin and fibronectin as optimal ligands for Ca2+-independent adhesion and the formation of cellular projections. Adhesion to fibronectin was mediated via RGD motifs alone and was not inhibited by 500 micrograms of heparin/ml. Kistrin, a disintegrin which binds preferentially to the alphav beta3 (vitronectin/fibronectin) receptor inhibited the formation of cellular projections without disrupting preformed matrix interactions. Finally, we show that Ca2+-independent cell-matrix adhesion is a dynamic process which mediates changes in the morphology of VV-infected cells and uninfected cells which exhibit a transformed phenotype.

Roles of vaccinia virus EEV-specific proteins in intracellular actin tail formation and low pH-induced cell-cell fusion.

During vaccinia virus (VV) morphogenesis intracellular mature virus (IMV) is wrapped by two additional membranes to form intracellular enveloped virus (IEV). IEV particles can nucleate the formation of actin tails which aid movement of IEVs to the cell surface where the outer IEV membrane fuses with the plasma membrane forming cell-associated enveloped virus (CEV) which remains attached to the cell, or extracellular enveloped virus (EEV) which is shed from the cell. In this report, we have used a collection of VV mutants lacking individual EEV-specific proteins to compare the roles of these proteins in the formation of IEV and IEV-associated actin tails and fusion of infected cells after a low pH shock. Data presented here show that p45-50 (A36R) is not required for IEV formation or for acid-induced cell-cell fusion, but is required for formation of IEV-associated actin tails. In contrast, gp86 (A56R), the virus haemagglutinin, is not required for formation of either IEV or IEV-associated actin tails. Data presented also confirm that p37 (gene F13L), gp42 (B5R) and gp22-24 (A34R) are needed for formation of IEV-associated actin tails and for cell-cell fusion after low pH shock. The phenotypes of these mutants were not affected by the host cell type as similar results were obtained in a range of different cells. Lastly, comparisons of the phenotypes of VV strains Western Reserve, deltaA34R and deltaA36R demonstrate that actin tails are not required for low pH-induced cell-cell fusion.

The extracellular domain of vaccinia virus protein B5R affects plaque phenotype, extracellular enveloped virus release, and intracellular actin tail formation.

Vaccinia virus produces two morphologically distinct forms of infectious virus, termed intracellular mature virus (IMV) and extracellular enveloped virus (EEV). EEV is important for virus dissemination within a host and has different surface proteins which bind to cell receptors different from those used by IMV. Six genes are known to encode EEV-specific proteins. One of these, B5R, encodes a 42-kDa glycoprotein with amino acid similarity to members of the complement control protein superfamily and contains four copies of a 50- to 70-amino-acid repeat called the short consensus repeat (SCR). Deletion of B5R causes a small-plaque phenotype, a 10-fold reduction in EEV formation, and virus attenuation in vivo. In this study, we inserted mutated versions of the B5R gene lacking different combinations of the SCRs into a virus deletion mutant lacking the B5R gene. The resultant viruses each formed small plaques only slightly larger than those of the deletion mutant; however, the virus containing only SCR 1 formed plaques slightly larger than those of viruses with SCRs 1 and 2 or SCRs 1, 2, and 3. All of these viruses produced approximately 50-fold more infectious EEV than wild-type virus and formed comet-shaped plaques under liquid overlay. Despite producing more EEV, the mutant viruses were unable to induce the polymerization of actin on intracellular virus particles. The implications of these results for our understanding of EEV formation, release, and infectivity are discussed.

Virus-induced cell motility.

Many viruses induce profound changes in cell metabolism and function. Here we show that vaccinia virus induces two distinct forms of cell movement. Virus-induced cell migration was demonstrated by an in vitro wound healing assay in which infected cells migrated independently into the wound area while uninfected cells remained relatively static. Time-lapse microscopy showed that the maximal rate of migration occurred between 9 and 12 h postinfection. Virus-induced cell migration was inhibited by preinactivation of viral particles with trioxsalen and UV light or by the addition of cycloheximide but not by addition of cytosine arabinoside or rifampin. The expression of early viral genes is therefore necessary and sufficient to induce cell migration. Following migration, infected cells developed projections up to 160 microm in length which had growth-cone-like structures and were frequently branched. Time-lapse video microscopy showed that these projections were formed by extension and condensation of lamellipodia from the cell body. Formation of extensions was dependent on late gene expression but not the production of intracellular enveloped (IEV) particles. The requirements for virus-induced cell migration and for the formation of extensions therefore differ from each other and are distinct from the polymerization of actin tails on IEV particles. These data show that poxviruses encode genes which control different aspects of cell motility and thus represent a useful model system to study and dissect cell movement.

Transmembrane domain of influenza virus neuraminidase, a type II protein, possesses an apical sorting signal in polarized MDCK cells.

The influenza virus neuraminidase (NA), a type II transmembrane protein, is directly transported to the apical plasma membrane in polarized MDCK cells. By using deletion mutants and chimeric constructs of influenza virus NA with the human transferrin receptor, a type II basolateral transmembrane protein, we investigated the location of the apical sorting signal of influenza virus NA. When these mutant and chimeric proteins were expressed in stably transfected polarized MDCK cells, the transmembrane domain of NA, and not the cytoplasmic tail, provided a determinant for apical targeting in polarized MDCK cells and this transmembrane signal was sufficient for sorting and transport of the ectodomain of a reporter protein (transferrin receptor) directly to the apical plasma membrane of polarized MDCK cells. In addition, by using differential detergent extraction, we demonstrated that influenza virus NA and the chimeras which were transported to the apical plasma membrane also became insoluble in Triton X-100 but soluble in octylglucoside after extraction from MDCK cells during exocytic transport. These data indicate that the transmembrane domain of NA provides the determinant(s) both for apical transport and for association with Triton X-100-insoluble lipids.

Overexpression of the vaccinia virus A38L integral membrane protein promotes Ca2+ influx into infected cells.

The vaccinia virus Western Reserve A38L protein is a hydrophobic integral membrane glycoprotein with amino acid similarity to mammalian integrin-associated protein. The protein has an N-terminal immunoglobulin superfamily domain, followed by five membrane-spanning domains and a short cytoplasmic tail. Deletion of the protein reduces virus plaque size but does not affect virus virulence (J. E. Parkinson, C. M. Sanderson, and G. L. Smith, Virology, in press). In this study, we have used a recombinant vaccinia virus in which the A38L gene may be inducibly overexpressed by addition of isopropyl-beta-D-thiogalactopyranoside (IPTG), to demonstrate that overexpression of the vaccinia virus A38L gene produces drastic changes in the morphology, permeability, and adhesion of infected cells. In particular, A38L overexpression caused swelling of cells, marginalization of nuclear chromatin, and vacuolization of the endoplasmic reticulum, features characteristic of cell necrosis. By 18 h postinfection, cells become permeable and lytic as defined by the free entry of propidium iodide and loss of the cytoplasmic enzyme lactate dehydrogenase. Chelation of extracellular Ca2+ 22 h postinfection inhibited further release of lactate dehydrogenase, showing that Ca2+ influx was required for A38L-induced lysis. Direct measurement of 45Ca2+ influx showed that the rate of Ca2+ uptake was directly related to the period of A38L induction. The A38L protein, therefore, promotes the formation of pores within the plasma membrane of cells, and these pores facilitate Ca2+ entry and induce necrosis. Addition of rifampin inhibited virus assembly but not the ability of A38L to induce necrosis, indicating that pore formation is independent of viral morphogenesis. Finally, overexpression of the A38L protein resulted in a reduced plaque size and a threefold decrease in production of infective particles in vitro. The A38L protein represents the first example of a virus protein which directly or indirectly promotes the influx of extracellular Ca2+.

The vaccinia virus A38L gene product is a 33-kDa integral membrane glycoprotein.

The vaccinia virus gene A38L encodes a highly hydrophobic protein with amino acid similarity to mammalian integrin-associated protein (IAP). In this report we have identified the A38L protein of strain Western Reserve (WR), defined its membrane topology, and analyzed its role in virus production and virulence. An antiserum raised against an A38L peptide identified the A38L gene product as a 33-kDa protein which is expressed at low levels during virus infection. A serum from a rabbit previously infected with WR virus recognized the A38L protein, thus confirming that the A38L gene is expressed in vivo. Using a coupled in vitro-translation/membrane-translocation system the 33-kDa protein was shown to be a membrane-associated and glycosylated form of a 29-kDa polypeptide precursor. The membrane topology of the A38L protein was defined by its glycosylation and protease sensitivity when associated with microsomal membranes. The N-terminal immunoglobulin-like variable domain was protected from exogenous protease and was therefore in the lumen of the vesicle, whereas the C-terminus was sensitive and therefore cytoplasmic. A38L deletion and revertant viruses were constructed and were used to study the involvement of A38L in virus assembly, release, and virulence. Deletion of the A38L gene caused a slight reduction in virus plaque size but did not affect the production of intracellular mature virus or extracellular enveloped virus particles in tissue culture cells nor the virulence of the virus in the murine intranasal model. The A38L protein therefore possesses similar sequence and membrane topology to the mammalian IAP protein but is not required for virus particle production or virulence.

Interaction of Sendai viral F, HN, and M proteins with host cytoskeletal and lipid components in Sendai virus-infected BHK cells.

We have studied the interaction of Sendai viral fusion (F), hemagglutinin/neuraminidase (H/N), and matrix (M) proteins with host cytoskeletal and lipid components in Sendai virus-infected BHK cells using two nonionic detergents Triton X-100 (TX-100) and octyl glucoside (OG). Our results show that while M protein acquired resistance to both TX-100 and OG extraction, F and HN exhibited insolubility only to TX-100 but not to OG. Furthermore, in the presence of high salt (1 M NaCl), M, but not F or HN, became TX-100 soluble. Both type I (F) and type II (HN) viral glycoproteins acquired TX-100 insolubility at a late stage during exocytic transport as they acquired endo H resistance. In addition, TX-100 insoluble F and HN exhibited a lighter density compared to TX-100 resistant M by flotation analysis. Using recombinant vaccinia viruses that express Sendai virus HN, F, or M protein individually, we observed that each viral protein (F, HN, or M) was independently capable of acquiring TX-100 insolubility in the absence of other viral components. These results would indicate that while Sendai viral F and HN became bound to TX-100 insoluble lipids, M protein bound ionically to TX-100 insoluble cytoskeletal components and not to TX-100 insoluble lipids.

Sendai virus M protein binds independently to either the F or the HN glycoprotein in vivo.

We have analyzed the mechanism by which M protein interacts with components of the viral envelope during Sendai virus assembly. Using recombinant vaccinia viruses to selectively express combinations of Sendai virus F, HN, and M proteins, we have successfully reconstituted M protein-glycoprotein interaction in vivo and determined the molecular interactions which are necessary and sufficient to promote M protein-membrane binding. Our results showed that M protein accumulates on cellular membranes via a direct interaction with both F and HN proteins. Specifically, our data demonstrated that a small fraction (8 to 16%) of M protein becomes membrane associated in the absence of Sendai virus glycoproteins, while > 75% becomes membrane bound in the presence of both F and HN proteins. Selective expression of M protein together with either F or HN protein showed that each viral glycoprotein is individually sufficient to promote efficient (56 to 73%) M protein-membrane binding. Finally, we observed that M protein associates with cellular membranes in a time-dependent manner, implying a need for either maturation or transport before binding to glycoproteins.

Sendai virus assembly: M protein binds to viral glycoproteins in transit through the secretory pathway.

We have examined the relative ability of Sendai virus M (matrix) protein to associate with membranes containing viral glycoproteins at three distinct stages of the exocytic pathway prior to cell surface appearance. By the use of selective low-temperature incubations or the ionophore monensin, the transport of newly synthesized viral glycoproteins was restricted to either the pre-Golgi intermediate compartment (by incubation at 15 degrees C), the medial Golgi (in the presence of monensin), or the trans-Golgi network (by incubation at 20 degrees C). All three of these treatments resulted in a marked accumulation of the M protein on perinuclear Golgi-like membranes which in each case directly reflected the distribution of the viral F protein. Subsequent redistribution of the F protein to the plasma membrane by removal of the low-temperature (20 degrees C) block resulted in a concomitant redistribution of the M protein, thus implying association of the two components during intracellular transit. The extent of M protein-glycoprotein association was further examined by cell fractionation studies performed under each of the three restrictive conditions. Following equilibrium sedimentation of membranes derived from monensin-treated cells, approximately 40% of the recovered M protein was found to cofractionate with membranes containing the viral glycoproteins. Also, by flotation analyses, a comparable subpopulation of M protein was found to be membrane associated whether viral glycoproteins were restricted to the trans-Golgi network, the medial Golgi, or the pre-Golgi intermediate compartment. Additionally, transient expression of M protein alone from cloned cDNA showed that neither membrane association nor Golgi localization occurs in the absence of Sendai virus glycoproteins.

Purification and functional characterization of membranes derived from the rough endoplasmic reticulum of Saccharomyces cerevisiae.

Isolation and biochemical analysis of the components involved in protein translocation into the rough endoplasmic reticulum (ER) requires starting material highly enriched in membranes derived from this organelle. We have chosen to study the yeast Saccharomyces cerevisiae in order to profit from the ease of genetic manipulation. To date, however, no efficient scheme has been devised that allows the purification of functional rough ER-derived membranes from yeast, largely because proteins have yet to be identified that are rough ER-specific. In the experiments described here, we expressed the human rough ER marker ribophorin I to facilitate the analysis of subcellular fractionation. We found that the endoplasmic reticulum of yeast could be separated into two distinct domains by fractionation on continuous sucrose gradients. This procedure revealed a bimodal distribution of ER markers. The yeast homologue of the heavy chain-binding protein, BiP (encoded by the KAR2 gene), and the product of the SEC62 gene were present in two fractions having equilibrium densities of 1.146 and 1.192 g/ml, respectively. In contrast, our analysis showed that preprotein translocation activity and retention of the rough ER-specific protein ribophorin I were specific only to the membrane fraction with an equilibrium density of 1.192 g/ml. To prepare fractions highly enriched in translocation competent rough ER-derived membranes for analysis, we developed a density shift fractionation scheme that optimizes the purity of membranes containing human ribophorin I. Membranes obtained by this method were found to possess the majority of the appropriate functional markers, including ATP-independent preprotein binding, ribosome binding, and post-translational translocation. Mitochondria, the major contaminant of the 1.192 g/ml fraction, were significantly depleted in density-shifted membrane populations.

Protein retention in yeast rough endoplasmic reticulum: expression and assembly of human ribophorin I.

The RER retains a specific subset of ER proteins, many of which have been shown to participate in the translocation of nascent secretory and membrane proteins. The mechanism of retention of RER specific membrane proteins is unknown. To study this phenomenon in yeast, where no RER-specific membrane proteins have yet been identified, we expressed the human RER-specific protein, ribophorin I. In all mammalian cell types examined, ribophorin I has been shown to be restricted to the membrane of the RER. Here we ascertain that yeast cells correctly target, assemble, and retain ribophorin I in their RER. Floatation experiments demonstrated that human ribophorin I, expressed in yeast, was membrane associated. Carbonate (pH = 11) washing and Triton X-114 cloud-point precipitations of yeast microsomes indicated that ribophorin I was integrated into the membrane bilayer. Both chromatography on Con A and digestion with endoglycosidase H were used to prove that ribophorin I was glycosylated once, consistent with its expression in mammalian cells. Proteolysis of microsomal membranes and subsequent immunoblotting showed ribophorin I to have assumed the correct transmembrane topology. Sucrose gradient centrifugation studies found ribophorin I to be included only in fractions containing rough membranes and excluded from smooth ones that, on the basis of the distribution of BiP, included smooth ER. Ribosome removal from rough membranes and subsequent isopycnic centrifugation resulted in a shift in the buoyant density of the ribophorin I-containing membranes. Furthermore, the rough and density-shifted fractions were the exclusive location of protein translocation activity. Based on these studies we conclude that sequestration of membrane proteins to rough domains of ER probably occurs in a like manner in yeast and mammalian cells.