K. Gopal Murti

The human homologue of yeast CRM1 is in a dynamic subcomplex with CAN/Nup214 and a novel nuclear pore component Nup88

The oncogenic nucleoporin CAN/Nup214 is essential in vertebrate cells. Its depletion results in defective nuclear protein import, inhibition of messenger RNA export and cell cycle arrest. We recently found that CAN associates with proteins of 88 and 112 kDa, which we have now cloned and characterized. The 88 kDa protein is a novel nuclear pore complex (NPC) component, which we have named Nup88. Depletion of CAN from the NPC results in concomitant loss of Nup88, indicating that the localization of Nup88 to the NPC is dependent on CAN binding. The 112 kDa protein is the human homologue of yeast CRM1, a protein known to be required for maintenance of correct chromosome structure. This human CRM1 (hCRM1) localized to the NPC as well as to the nucleoplasm. Nuclear overexpression of the FG‐repeat region of CAN, containing its hCRM1‐interaction domain, resulted in depletion of hCRM1 from the NPC. In CAN−/− mouse embryos lacking CAN, hCRM1 remained in the nuclear envelope, suggesting that this protein can also bind to other repeat‐containing nucleoporins. Lastly, hCRM1 shares a domain of significant homology with importin‐β, a cytoplasmic transport factor that interacts with nucleoporin repeat regions. We propose that hCRM1 is a soluble nuclear transport factor that interacts with the NPC.

Influenza virus neuraminidase contributes to secondary bacterial pneumonia.

Secondary bacterial pneumonia is a common cause of death during influenza epidemics. We hypothesized that virus-specific factors could contribute to differences in annual excess mortality. Recombinant influenza viruses with neuraminidases from representative strains from the past 50 years were created and characterized. The specific level of their neuraminidase activity correlated with their ability to support secondary bacterial pneumonia. Recombinant viruses with neuraminidases from 1957 and 1997 influenza strains had the highest level of activity, whereas a virus with the neuraminidase from a 1968 strain had the lowest level of activity. The high level of activity of the neuraminidase from the 1957 strain, compared with that of other neuraminidases, more strongly supported the adherence of Streptococcus pneumoniae and the development of secondary bacterial pneumonia in a mouse model. These data lend support to our hypothesis that the influenza virus neuraminidase contributes to secondary bacterial pneumonia and subsequent excess mortality.

Mutations in the PPPY Motif of Vesicular Stomatitis Virus Matrix Protein Reduce Virus Budding by Inhibiting a Late Step in Virion Release

ABSTRACT The N terminus of the matrix (M) protein of vesicular stomatitis virus (VSV) and of other rhabdoviruses contains a highly conserved PPPY sequence (or PY motif) similar to the late (L) domains in the Gag proteins of some retroviruses. These L domains in retroviral Gag proteins are required for efficient release of virus particles. In this report, we show that mutations in the PPPY sequence of the VSV M protein reduce virus yield by blocking a late stage in virus budding. We also observed a delay in the ability of mutant viruses to cause inhibition of host gene expression compared to wild-type (WT) VSV. The effect of PY mutations on virus budding appears to be due to a block at a stage just prior to virion release, since electron microscopic examination of PPPA mutant-infected cells showed a large number of assembled virions at the plasma membrane trapped in the process of budding. Deletion of the glycoprotein (G) in addition to these mutations further reduced the virus yield to less than 1% of WT levels, and very few particles were assembled at the cell surface. This observation suggested that G protein aids in the initial stage of budding, presumably during the formation of the bud site. Overall, our results confirm that the PPPY sequence of the VSV M protein possesses L domain activity analogous to that of the retroviral Gag proteins.

Predominant Nuclear Localization of Mammalian Target of Rapamycin in Normal and Malignant Cells in Culture

Mammalian target of rapamycin (mTOR) controls initiation of translation through regulation of ribosomal p70S6 kinase (S6K1) and eukaryotic translation initiation factor-4E (eIF4E) binding protein (4E-BP). mTOR is considered to be located predominantly in cytosolic or membrane fractions and may shuttle between the cytoplasm and nucleus. In most previous studies a single cell line, E1A-immortalized human embryonic kidney cells (HEK293), has been used. Here we show that in human malignant cell lines, human fibroblasts, and murine myoblasts mTOR is predominantly nuclear. In contrast, mTOR is largely excluded from the nucleus in HEK293 cells. Hybrids between HEK293 and Rh30 rhabdomyosarcoma cells generated cells co-expressing markers unique to HEK293 (E1A) and Rh30 (MyoD). mTOR distribution was mainly nuclear with detectable levels in the cytoplasm. mTOR isolated from Rh30 nuclei phosphorylated recombinant GST-4E-BP1 (Thr-46) in vitro and thus has kinase activity. We next investigated the cellular distribution of mTOR substrates 4E-BP, S6K1, and eIF4E. 4E-BP was exclusively detected in cytoplasmic fractions in all cell lines. S6K1 was localized in the cytoplasm in colon carcinoma, HEK293 cells, and IMR90 fibroblasts. S6K1 was readily detected in all cellular fractions derived from rhabdomyosarcoma cells. eIF4E was detected in all fractions derived from rhabdomyosarcoma cells but was not detectable in nuclear fractions from colon carcinoma HEK293 or IMR90 cells.

Localization of RNA polymerases on influenza viral ribonucleoproteins by immunogold labeling

Monospecific antisera for the influenza polymerase proteins and high resolution immunoelectron microscopy have been used to investigate the topographical distribution of the polymerase molecules on influenza ribonucleoproteins (RNPs). Antibodies to PB1, PB2, and PA identify a single polymerase binding site located at, or very close to, the end of each RNP. Double labeling experiments confirm that all three polymerases are at the same end of each RNP and that they are in close association.

Protein kinase C associates with intermediate filaments and stress fibers

The subcellular distribution of protein kinase C (PKC) was determined by immunofluorescence using anti-PKC monoclonal antibodies (MAbs). The antibodies used were: (1) 1.9 MAb that is directed against an epitope in the catalytic domain of PKC, (2) 1.3 MAb that recognizes an isozyme of PKC (Mochly-Rosen, D., and Koshland, D. E., 1987, J. Biol. Chem. 262, 2291-2297; Mochly-Rosen, D., et al. 1987 Proc. Natl. Acad. Sci. USA 84, 4660-4664) and (3) MC-2a MAb that is directed against the beta-isozyme of PKC (Usuda, N., et al. 1991, J. Cell Biol. 112, 1241-1247). The cells used in this study were baby hamster kidney cells, vimentin+ and vimentin- clones of SW13 (a human adrenal carcinoma cell line), CEM (a human T cell line), U937 (a histiocytic myeloid cell line), and HL60 (a promyelocytic leukemia cell line). The 1.9 MAb was found to recognize a variety of subcellular components, viz., nucleus (nucleoplasm and nucleolus), cytoplasm, vimentin-type intermediate filaments (IF), stress fibers, and cell membrane. Among these components the beta-isozyme-specific MAbs (1.3 and MC-2a) recognized only the IF network, stress fibers, and edges of the cell membrane. Experiments with vimentin+ and vimentin- mutants of SW13 cells, double indirect immunofluorescence studies with anti-vimentin and anti-PKC antibodies, and drug studies confirmed that the IF network is the predominant cytoskeletal network labeled with all anti-PKC MAbs. Immunoblotting studies with the MC-2a MAb revealed that the observed staining of the IF network was not due to a cross-reaction of the MAb with IF proteins and that the MAb specifically recognizes PKC. These studies, while identifying the diverse cell components to which PKC binds, have demonstrated, for the first time, that PKC associates with the IF network in a variety of cell types. Additionally, the studies have confirmed the studies by others concerning the association of PKC with stress fibers.

Antibodies against Sendai virus L protein: distribution of the protein in nucleocapsids revealed by immunoelectron microscopy

Antibodies against the L protein of Sendai virus were made by immunizing rabbits with a synthetic peptide representing a carboxyl-terminal region of the protein predicted from the base sequence of its gene. These antibodies were used to localize the L protein in viral nucleocapsids by electron microscopy. Immunogold labeling revealed that L protein molecules were distributed in clusters along nucleocapsids, suggesting that L molecules act cooperatively in viral RNA synthesis. Immunogold double-labeling showed that all L clusters were associated with clusters of P molecules. We believe that this morphological association reflects the functional cooperation of the L and P proteins in viral RNA synthesis.

Localization of P, NP, and M proteins on Sendai virus nucleocapsid using immunogold labeling

The distribution of NP, P, and M proteins on Sendai virus nucleocapsids purified from cells and virions were studied by immunogold staining using monoclonal antibodies. NP molecules were found uniformly along the entire length of both cytosol and virion derived nucleocapsids. This observation is in accord with the earlier proposals that NP molecules maintained the structural integrity of the nucleocapsid. The distribution of P in nucleocapsids derived from the cytosol differed from the distribution in those originating from virions. In nucleocapsids derived from the cytosol, P molecules occurred in 4 to 10 discreet clusters at varying locations along the length of the nucleocapsid. In contrast, on nucleocapsids derived from virions, P molecules were uniformly distributed over the entire length of the nucleocapsid. These observations suggest that the distribution of P depends on the functional state of the nucleocapsid. The occurrence of P clusters at different locations on intracellular nucleocapsids indicates that P is a mobile molecule; this suggestion is consistent with Ps role in viral RNA synthesis. The distribution of the matrix (M) protein also depended on where the nucleocapsids were derived from. Large quantities of M protein were found along the entire length of nucleocapsids derived from the cytosol, while in virion nucleocapsids, many fewer molecules of M were observed. The large amounts of M on the nucleocapsids originating from the cytosol supports the hypothesis that M protein mediates the recognition between the nucleocapsid and the envelope glycoproteins.

Loss of the N-Linked Glycan at Residue 173 of Human Parainfluenza Virus Type 1 Hemagglutinin-Neuraminidase Exposes a Second Receptor-Binding Site

ABSTRACT BCX 2798 (4-azido-5-isobutyrylamino-2,3-didehydro-2,3,4,5-tetradeoxy-d-glycero-d-galacto-2-nonulopyranosic acid) effectively inhibited the activities of the hemagglutinin-neuraminidase (HN) of human parainfluenza viruses (hPIV) in vitro and protected mice from lethal infection with a recombinant Sendai virus whose HN was replaced with that of hPIV-1 (rSeV[hPIV-1HN]) (I. V. Alymova, G. Taylor, T. Takimoto, T. H. Lin., P. Chand, Y. S. Babu, C. Li, X. Xiong, and A. Portner, Antimicrob. Agents Chemother. 48:1495-1502, 2004). The ability of BCX 2798 to select drug-resistant variants in vivo was examined. A variant with an Asn-to-Ser mutation at residue 173 (N173S) in HN was recovered from mice after a second passage of rSeV(hPIV-1HN) in the presence of BCX 2798 (10 mg/kg of body weight daily). The N173S mutant remained sensitive to BCX 2798 in neuraminidase inhibition assays but was more than 10,000-fold less sensitive to the compound in hemagglutination inhibition tests than rSeV(hPIV-1HN). Its susceptibility to BCX 2798 in plaque reduction assays was reduced fivefold and did not differ from that of rSeV(hPIV-1HN) in mice. The N173S mutant failed to be efficiently eluted from erythrocytes and released from cells. It demonstrated reduced growth in cell culture and superior growth in mice. The results for gel electrophoresis analysis were consistent with the loss of the N-linked glycan at residue 173 in the mutant. Sequence and structural comparisons revealed that residue 173 on hPIV-1 HN is located close to the region of the second receptor-binding site identified in Newcastle disease virus HN. Our study suggests that the N-linked glycan at residue 173 masks a second receptor-binding site on hPIV-1 HN.

Electron microscopic evidence for the association of M 2 protein with the influenza virion

SummaryImmunogold electron microscopy revealed that site-specific antibodies elicited by a synthetic peptide representing the N-terminal sequence (residues 2–10) of influenza virus M 2 protein were capable of binding to the surface of virions. Antibody binding was observed with two human influenza virus strains but not with an avian virus strain which has amino acid substitutions in the appropriate sequence of M 2. These results provide direct evidence for the presence of M 2 in the influenza virion.