K.G. Murti

Cloning and characterization of a second human CTP:phosphocholine cytidylyltransferase.

CTP:phosphocholine cytidylyltransferase (CCT) is a key regulator of phosphatidylcholine biosynthesis, and only a single isoform of this enzyme, CCTα, is known. We identified and sequenced a human cDNA that encoded a distinct CCT isoform, called CCTβ, that is derived from a gene different from that encoding CCTα. CCTβ transcripts were detected in human adult and fetal tissues, and very high transcript levels were found in placenta and testis. CCTβ and CCTα proteins share highly related, but not identical, catalytic domains followed by three amphipathic helical repeats. Like CCTα, CCTβ required the presence of lipid regulators for maximum catalytic activity. The amino terminus of CCTβ bears no resemblance to the amino terminus of CCTα, and CCTβ protein was localized to the cytoplasm as detected by indirect immunofluorescent microscopy. Whereas CCTα activity is regulated by reversible phosphorylation, CCTβ lacks most of the corresponding carboxyl-terminal domain and contained only 3 potential phosphorylation sites of the 16 identified in CCTα. Transfection of COS-7 cells with a CCTβ expression construct led to the overexpression of CCT activity, the accumulation of cellular CDP-choline, and enhanced radiolabeling of phosphatidylcholine. CCTβ protein was posttranslationally modified in COS-7 cells, resulting in slower migration during polyacrylamide gel electrophoresis. Expression of CCTβ/CCTα chimeric proteins showed that the amino-terminal portion of CCTβ was required for posttranslational modification. These data demonstrate that a second, distinct CCT enzyme is expressed in human tissues and provides another mechanism by which cells regulate phosphatidylcholine production.

Distribution of hemagglutinin and neuraminidase on influenza virions as revealed by immunoelectron microscopy

Monoclonal antibodies specific for the hemagglutinin (HA) or the neuraminidase (NA) of influenza viruses were used in immunoelectron microscopic studies to determine the distribution of the two surface spikes on the virion. Indirect immunogold staining revealed that the HA is uniformly distributed on the virion while the NA occurs in discrete areas. Crosslinking and low temperature studies argue against redistribution of the HA and NA after antibody attachment and indicate that the NA on influenza virus occurs in patches.

A Novel CRM1-mediated Nuclear Export Signal Governs Nuclear Accumulation of Glyceraldehyde-3-phosphate Dehydrogenase following Genotoxic Stress

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional protein with glycolytic and non-glycolytic functions, including pro-apoptotic activity. GAPDH accumulates in the nucleus after cells are treated with genotoxic drugs, and it is present in a protein complex that binds DNA modified by thioguanine incorporation. We identified a novel CRM1-dependent nuclear export signal (NES) comprising 13 amino acids (KKVVKQASEGPLK) in the C-terminal domain of GAPDH, truncation or mutation of which abrogated CRM1 binding and caused nuclear accumulation of GAPDH. Alanine scanning of the sequence encompassing the putative NES demonstrated at least two regions important for nuclear export. Site mutagenesis of Lys259 did not affect oligomerization but impaired nuclear efflux of GAPDH, indicating that this amino acid residue is essential for proper functioning of this NES. This novel NES does not contain multiple leucine residues unlike other CRM1-interacting NES, is conserved in GAPDH from multiple species, and has sequence similarities to the export signal found in feline immunodeficiency virus Rev protein. Similar sequences (KKVV*7-13PLK) were found in two other human proteins, U5 small nuclear ribonucleoprotein, and transcription factor BT3.

Assembly of influenza ribonucleoprotein in vitro using recombinant nucleoprotein

The influenza A virus nucleoprotein previously expressed in Escherichia coli after fusion to 32 heterologous amino acids has now been purified and tested for its ability to form complexes with RNA in vitro. By using a simple filter binding assay, we show that ribonucleoprotein (RNP) complexes form readily with single-stranded RNA of viral or nonviral origin but not with double-stranded RNA. The RNP complexes formed were similar to authentic influenza virus RNPs in appearance under the electron microscope, in buoyant density in gradients of cesium chloride, and in sensitivities to pancreatic ribonuclease, to chaotropic reagents, and to high salt. We conclude that nucleoprotein synthesized in E. coli has all the properties required for correct assembly into ribonucleoprotein.

A functional role for intermediate filaments in the formation of frog virus 3 assembly sites.

During the course of frog virus 3 (FV3) infection in baby hamster kidney 21 (BHK) cells, vimentin-type intermediate filaments reorganize to surround the viruss cytoplasmic assembly sites. To determine whether the association between vimentin filaments and viral assembly sites has a functional role in the virus life-cycle, we treated cells with the antimicrotubule drugs taxol or colchicine, or injected them with monoclonal antivimentin antibodies prior to FV3 infection. Each of these reagents caused the collapse of the normally extended BHK intermediate filament system. In the case of taxol-treated or antivimentin-injected cells, the collapsed vimentin filaments were unable to reorganize around the newly forming viral assembly sites. The viral assembly sites that did form were aberrant and there was a significant reduction in the number of mature virions present. Colchicine, which also caused the collapse of vimentin filament organization, did not block the reorganization of vimentin filaments in response to viral infection and viral assembly sites appeared normal. These results suggest that intermediate filaments play an important role in maintaining the structural and functional integrity of FV3 assembly sites.

Interaction of frog virus 3 with the cytomatrix: III. Role of microfilaments in virus release

The role of microfilaments in the release of frog virus 3 (FV3) from the plasma membrane was studied. Scanning electron microscopic study of FV3-infected baby hamster kidney (BHK) cells showed that late in infection (15 hr), numerous microvillus-like projections containing virions and microfilaments occur on the cell surface. Two microfilament-disrupting drugs, cytochalasin B and cytochalasin D, inhibited both the formation of microvillus-like projections and virus release. In the drug-treated cells, the virions accumulated in large numbers beneath the plasma membrane (transmission electron microscopy), suggesting that both drugs affected the release of the virus at the level of plasma membrane rather than the traverse of the virus to the plasma membrane. Two-dimensional gel analysis of actin from FV3-infected and uninfected cells revealed the following. There was no difference in the synthesis of actin in infected versus uninfected cells. However, the actin of infected cells is more acidic than its counterpart in uninfected cells. Temporally, the change in actin preceded the formation of microvilli-like projections involved in virus release. The change in actin is virus induced and is linked to virus maturation since a ts mutant of FV3 (ts9467), which is deficient in virus production at the restrictive temperature (30 degrees), did not modify actin. The mutant, at the permissive temperature (25 degrees), produced virions and altered the actin. Together, the above results attribute an active role for microfilaments in virus release.

Nuclear distribution of Oct-4 transcription factor in transcriptionally active and inactive mouse oocytes and its relation to RNA polymerase II and splicing factors

The intranuclear distribution of the transcription factor Oct‐4, which is specifically expressed in totipotent mice stem and germ line cells, was studied in mouse oocytes using immunogold labeling/electron microscopy and immunofluorescence/confocal laser scanning microcopy. The localization of Oct‐4 was studied in transcriptionally active (uni/bilaminar follicles) and inactive (antral follicles) oocytes. Additionally, the Oct‐4 distribution was examined relative to that of the unphosphorylated form of RNA polymerase II (Pol II) and splicing factor (SC 35) in the intranuclear entities such as perichromatin fibrils (PFs), perichromatin granules (PGs), interchromatin granule clusters (IGCs), Cajal bodies (CBs), and nucleolus‐like bodies (NLBs). It was shown that: (i) Oct‐4 is localized in PFs, IGCs, and in the dense fibrillar component (DFC) of the nucleolus at the transcriptionally active stage of the oocyte nucleus; (ii) Oct‐4 present in PFs and IGCs colocalizes with Pol II and SC 35 at the transcriptionally active stage; (iii) Oct‐4 accumulates in NLBs, CBs, and PGs at the inert stage of the oocyte. The results confirm the previous suggestion that PFs represent the major nucleoplasmic structural domain involved in active pre‐mRNA transcription/processing. The colocalization of Oct‐4 with Pol II in both IGCs and PFs in active oocytes (uni/bilaminar follicles) suggests that Oct‐4 is intimately associated with the Pol II holoenzyme before and during transcription. The colocalization of Oct‐4, Pol II, and SC 35 with coilin‐containing structures such as NLBs and CBs at the inert stage (antral follicles) suggests that the latter may represent storage sites for the transcription/splicing machinery during the decline of transcription. J. Cell. Biochem. 89: 720–732, 2003.

The role of DNA methylation in virus replication: inhibition of frog virus 3 replication by 5-azacytidine

Frog virus 3 (FV3) DNA is the most highly methylated DNA of any known DNA virus; about 20% of the cytosine residues in FV3 DNA are methylated (D. Willis and A. Granoff, 1980, Virology 107, 250-257). To understand the role of DNA methylation in virus replication, we have examined the effect of 5-azacytidine, a drug that inhibits DNA methylation. 5-Azacytidine (10 microM) reduced the production of infectious FV3 by 100-fold or more and inhibited methylation of viral DNA by about 80%. Inhibition of DNA methylation did not affect viral gene expression since there was no detectable inhibition of virus-specific RNA or protein synthesis in 5-azacytidine-treated cells. In contrast, the size of the replicating DNA measured under completely denaturing conditions, was much smaller than that found during infection in the absence of drug. These results suggest that the undermethylated DNA was susceptible to endodeoxyribonuclease(s). Additionally, electron microscopic examination of FV3-infected, 5-azacytidine-treated cells revealed that preformed capsids remained empty or were only partially filled with viral DNA. Based on these data, it is suggested that methylation of DNA protects it from endonucleolytic cleavage and that the integrity of genomic DNA is required for its proper packaging into virions.

Intracellular metabolism of Sendai virus nucleocapsids

Abstract We examined the intracellular pathways that Sendai virus polypeptides P and NP take into nucleocapsids by use of pulse-chase labeling and cell fractionation. Although most molecules of the major polypeptide NP entered nucleocapsids by way of a large soluble pool with slow exit kinetics, a fraction of NP molecules appeared to bypass this pool, entering nucleocapsids directly after synthesis. In contrast, all molecules of the minor polypeptide P passed directly into nucleocapsids, never appearing in a soluble pool. These data indicate that Sendai virus nucleocapsid assembly occurs in two steps: condensation of NP with viral genomic RNA followed by addition of P (and probably L) to the structure. The data also explain, in part, why newly synthesized P is rapidly incorporated into virions (Portner, A., and Kingsbury, D. W. (1976). Virology 73 , 79–88). We identified a population of nucleocapsids that had previously been unrecognized. This fraction, representing about half of all intracellular nucleocapsids, sedimented at low centrifugal forces, as if attached to a large cellular organelle such as the cytoplasmic cytoskeleton. The labeling data suggested that these “bound” nucleocapsids, rather than the “free” nucleocapsids, were the source of supply for virion assembly.

Composition of the helical internal components of influenza virus as revealed by immunogold labeling/electron microscopy

The composition of the large helical internal components of influenza virus was investigated by immunogold labeling/electron microscopy with antibodies to the nucleoprotein (NP), matrix protein (M), and polymerase complex (PB1, PB2, and PA) of the virus. The morphologically intact helices, obtained by air-drying of the virions on the electron microscope grid, showed little or no labeling with any of the above antibodies. However, partial to full degradation of the helix by proteinase K (2 ng/ml) prior to immunogold labeling made the helices accessible to all three antibodies. The results are consistent with a model that the helix represents a polymer of M protein enclosing or containing the influenza ribonucleoprotein(s).