Biomedical subjects

K G Murti

Publications and source records attributed to K G Murti.

At least 19 recordsLinked to original sources

PubMed2002

Topological organization of DNA molecules in the macronucleus of hypotrichous ciliated protozoa.

The DNA in the macronucleus of a hypotrichous ciliate occurs as millions of short molecules packed into dense chromatin bodies 0.1-2 microm in diameter. We have studied by electron microscopy the organization of DNA molecules in these chromatin bodies of macronuclei lysed in water at pH 9. Proteinase K treatment of lysed macronuclei progressively releases from chromatin bodies many rosettes of DNA molecules bound at one or both ends to a central core of protein. With longer treatment with proteinase K, rosettes disappear, leaving individual free DNA molecules. We propose that, in the native state, both ends of DNA molecules are bound through telomere-binding protein to a central core to form rosettes. Many rosettes, with collapsed DNA loops, aggregate to form a chromatin body. Chromatin bodies are believed to dissociate into individual collapsed rosettes to form the granules in the forward zone of the replication band. In the rear zone of the band, the rosettes dissociate, presumably as a result of release of telomere-binding protein, which is preliminary to the replication of the DNA molecules.

Animals↗

PubMed2001

A novel protein complex distinct from mismatch repair binds thioguanylated DNA.

To elucidate molecular mechanism(s) of cellular response to mercaptopurine, a widely used antileukemic agent, we assessed mercaptopurine (MP) sensitivity in mismatch repair (MMR) proficient and MMR deficient human acute lymphoblastic leukemia (ALL) cells. Sensitivity to thiopurine cytotoxicity was not dependent on MMR (i.e., MutSalpha) competence among six cell lines tested. Using electrophoretic mobility shift assay analysis, we found that the incubation of nuclear extracts from ALL cells with synthetic 34-mer DNA duplexes containing deoxythioguanosine (G(S)) within either G(S).T or G(S).C pairs, resulted in formation of a DNA-protein complex distinct from the DNA-MutSalpha complex and unaffected by ATP. Isolation and sequence analysis of proteins involved in this DNA-protein complex identified glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a component. Western blot analysis of nuclear extracts from a panel of human lymphoblastic leukemia cell lines revealed markedly different basal levels of GAPDH in nuclei, which was significantly related to thiopurine sensitivity (p = 0.001). Confocal analysis revealed markedly different intracellular distribution of GAPDH between nucleus and cytosol in six human ALL cell lines. Redistribution of GAPDH from cytosol to nucleus was evident after MP treatment. These findings indicate that a new DNA-protein complex containing GAPDH and distinct from known MMR protein-DNA complexes binds directly to thioguanylated DNA, suggesting that this may act as a sensor of structural alterations in DNA and serve as an interface between these DNA modifications and apoptosis.

Antimetabolites, Antineoplastic↗

PubMed2001

Role of matrix and fusion proteins in budding of Sendai virus.

Paramyxoviruses are assembled at the surface of infected cells, where virions are formed by the process of budding. We investigated the roles of three Sendai virus (SV) membrane proteins in the production of virus-like particles. Expression of matrix (M) proteins from cDNA induced the budding and release of virus-like particles that contained M, as was previously observed with human parainfluenza virus type 1 (hPIV1). Expression of SV fusion (F) glycoprotein from cDNA caused the release of virus-like particles bearing surface F, although their release was less efficient than that of particles bearing M protein. Cells that expressed only hemagglutinin-neuraminidase (HN) released no HN-containing vesicles. Coexpression of M and F proteins enhanced the release of F protein by a factor greater than 4. The virus-like particles containing F and M were found in different density gradient fractions of the media of cells that coexpressed M and F, a finding that suggests that the two proteins formed separate vesicles and did not interact directly. Vesicles released by M or F proteins also contained cellular actin; therefore, actin may be involved in the budding process induced by viral M or F proteins. Deletion of C-terminal residues of M protein, which has a sequence similar to that of an actin-binding domain, significantly reduced release of the particles into medium. Site-directed mutagenesis of the cytoplasmic tail of F revealed two regions that affect the efficiency of budding: one domain comprising five consecutive amino acids conserved in SV and hPIV1 and one domain that is similar to the actin-binding domain required for budding induced by M protein. Our results indicate that both M and F proteins are able to drive the budding of SV and propose the possible role of actin in the budding process.

Actins↗

PubMed2001

Nucleocapsid incorporation into parainfluenza virus is regulated by specific interaction with matrix protein.

The paramyxovirus nucleoproteins (NPs) encapsidate the genomic RNA into nucleocapsids, which are then incorporated into virus particles. We determined the protein-protein interaction between NP molecules and the molecular mechanism required for incorporating nucleocapsids into virions in two closely related viruses, human parainfluenza virus type 1 (hPIV1) and Sendai virus (SV). Expression of NP from cDNA resulted in in vivo nucleocapsid formation. Electron micrographs showed no significant difference in the morphological appearance of viral nucleocapsids obtained from lysates of transfected cells expressing SV or hPIVI NP cDNA. Coexpression of NP cDNAs from both viruses resulted in the formation of nucleocapsid composed of a mixture of NP molecules; thus, the NPs of both viruses contained regions that allowed the formation of mixed nucleocapsid. Mixed nucleocapsids were also detected in cells infected with SV and transfected with hPIV1 NP cDNA. However, when NP of SV was donated by infected virus and hPIV1 NP was from transfected cDNA, nucleocapsids composed of NPs solely from SV or solely from hPIVI were also detected. Although almost equal amounts of NP of the two viruses were found in the cytoplasm of cells infected with SV and transfected with hPIV1 NP cDNA, 90% of the NPs in the nucleocapsids of the progeny SV virions were from SV. Thus, nucleocapsids containing heterologous hPIV1 NPs were excluded during the assembly of progeny SV virions. Coexpression of hPIV1 NP and hPIV1 matrix protein (M) in SV-infected cells increased the uptake of nucleocapsids containing hPIV1 NP; thus, M appears to be responsible for the specific incorporation of the nucleocapsid into virions. Using SV-hPIV1 chimera NP cDNAs, we found that the C-terminal domain of the NP protein (amino acids 420 to 466) is responsible for the interaction with M.

Animals↗

PubMed2001

[Immunoelectron study of RNA polymerase II distribution in human oocyte nuclei].

The intranuclear distribution of two (unphosphorylated and hyperphosphorylated) forms of RNA polymerase II (Pol II) was studied in human oocytes from antral follicles using immunogold labeling/electron microscopy. The distribution of Pol II was as well as to the distribution of two splicing factors (snRNPs and SC-35) in the intranuclear entities, namely, interchromatin granule clusters (IGCs), nucleolus-like bodies (NLBs), and perichromatin fibrils (PFs). The results have shown that 1) antibodies directed against two forms of Pol II have a similar pattern of intranuclear distribution 2) both Pol II and splicing factors progressively accumulate in IGCs with a decrease in the transcriptional activity of the oocyte nucleus, 3) both Pol II and splicing factors are located on PFs, and 4) Pol II is present in the NLBs at all transcriptional states of the oocyte nucleus. The accumulation of Pol II and splicing factors in IGCs, concomitant with a decrease in the transcriptional activity, suggests a coordinated mechanism for the movement of both Pol II and splicing factors from the sites of action to the sites of storage.

Adult↗

PubMed2000

The amino terminus targets the mixed lineage leukemia (MLL) protein to the nucleolus, nuclear matrix and mitotic chromosomal scaffolds.

The mixed-lineage leukemia gene (MLL) is associated with more than 25 chromosomal translocations involving band 11q23 in diverse subtypes of human acute leukemia. Conditional expression of a 50 kDa amino terminal fragment spanning the AT hook motifs of MLL (MLL3AT) causes cell cycle arrest, upregulation of p21Cip1 and p27KiP1 and partial monocytic differentiation of the monoblastic U937 cell line, suggesting a major role for MLL3AT in MLL-AF9-induced myelomonocytic differentiation. In this study, we analyzed the subcellular localization of conditionally expressed MLL3AT in both U937 and HeLa cell lines. Immunofluorescence staining, confocal laser scanning microscopy and immunoelectron microscopy indicated that MLL3AT, like endogenous MLL, localized in the nucleoplasm in a punctate pattern of distribution, including regions attached to the nuclear envelope and the periphery of the nucleolus. We found that MLL3AT and endogenous MLL were present in interphase nuclear matrices and colocalized with topoisomerase II to mitotic chromosomal scaffolds. Nucleoplasm and nucleolar localization was observed even for MLL-AF9 and MLL-AF4 conditionally expressed chimeric proteins, suggesting a common target conferred by the amino terminus of MLL to many if not all the chimeric MLL proteins. The nuclear matrix/scaffold association suggests a role for the amino terminus of MLL in the modulation of chromatin structure, leading to epigenetic effects on the maintenance of gene expression.

Active Transport, Cell Nucleus↗

PubMed2000

Mutations in the PPPY motif of vesicular stomatitis virus matrix protein reduce virus budding by inhibiting a late step in virion release.

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.

Amino Acid Motifs↗

PubMed2000

Nuclear distribution of RNA polymerase II in human oocytes from antral follicles: dynamics relative to the transcriptional state and association with splicing factors.

The intranuclear distribution of two (unphosphorylated and hyperphosphorylated) forms of RNA polymerase II (Pol II) was studied in human oocytes from antral follicles using immunogold labeling/electron microscopy. The distribution of Pol II was analyzed relative to the transcriptional state of the oocyte as well as to the distribution of two splicing factors (snRNPs and SC-35) in the intranuclear entities, namely, interchromatin granule clusters (IGCs), nucleolus-like bodies (NLBs), and perichromatin fibrils (PFs). The results showed that (1) antibodies directed against two forms of Pol II have similar pattern of intranuclear distribution, (2) both Pol II and splicing factors progressively accumulate in IGCs with decrease in the transcriptional activity of the oocyte nucleus, (3) both Pol II and splicing factors localize to PFs, and (4) Pol II is present in the NLBs at all transcriptional states of the oocyte nucleus. These studies confirm earlier proposals that PFs represent a nuclear domain in which RNA transcription/processing are spatially coupled. The accumulation of Pol II and splicing factors in IGCs concomitant with a decrease in the transcriptional activity suggests a coordinated mechanism for the movement of both Pol II and splicing factors from the sites of action to the sites of storage.

Adult↗

PubMed1999

Telomeres of polytene chromosomes in a ciliated protozoan terminate in duplex DNA loops.

The end of a telomeric DNA sequence isolated from a polytene chromosome of a hypotrichous ciliate folds back and hybridizes with downstream telomeric sequence to form a t loop that is stable in the absence of protein and DNA cross-linking. The single-stranded, telomeric DNA sequence at the end of a macronuclear molecule does not form a t loop but, instead, is complexed with a heterodimeric, telomere-binding protein. Thus, two mechanisms for capping the ends of DNA molecules are used in the same cell.

Animals↗

PubMed1999

The polyamine oxidase inhibitor MDL-72,527 selectively induces apoptosis of transformed hematopoietic cells through lysosomotropic effects.

Polyamine oxidase functions in the polyamine catabolic pathway, converting N1-acetyl-spermidine and -spermine into putrescine (Put) and spermidine (Spd), respectively, thereby facilitating homeostasis of intracellular polyamine pools. Inhibition of polyamine oxidase in hematopoietic cells by a specific inhibitor, N,N'-bis(2,3-butadienyl)-1,4-butanediamine (MDL-72,527), reduces the levels of Put and Spd and induces the accumulation of N1-acetylated Spd. Although previously thought to be relatively nontoxic, we now report that this inhibitor overrides survival factors to induce cell death of several immortal and malignant murine and human hematopoietic cells, but not of primary myeloid progenitors. Cells treated with MDL-72,527 displayed biochemical changes typical of apoptosis, and cell death was associated with the down-regulation of the antiapoptotic protein Bcl-X(L). However, enforced overexpression of Bcl-X(L), or treatment with the universal caspase inhibitor zVAD-fmk, failed to block MDL-72,527-induced apoptosis in these hematopoietic cells. Despite decreases in Put and Spd pools, MDL-72,527-induced apoptosis was not blocked by cotreatment with exogenous Put or Spd, nor was it influenced by overexpression or inhibition of the polyamine biosynthetic enzyme ornithine decarboxylase. Significantly, MDL-72,527-induced apoptosis was associated with the rapid formation of numerous lysosomally derived vacuoles. Malignant leukemia cells were variably sensitive to the lysosomotropic effects of MDL-72,527, yet pretreatment with the ornithine decarboxylase inhibitor L-alpha-difluoromethylornithine sensitized all of these leukemia cells to the deleterious effects of the inhibitor by stimulating its intracellular accumulation. The lysosomotropic nature of select polyamine analogues may, thus, provide a novel chemotherapeutic strategy to selectively induce apoptosis of malignant hematopoietic cells.

Animals↗

PubMed1999

Hyperdiploid acute lymphoblastic leukemia with 51 to 65 chromosomes: a distinct biological entity with a marked propensity to undergo apoptosis.

To determine the cellular basis for the excellent clinical outcome of hyperdiploid acute lymphoblastic leukemia (ALL), defined by a modal chromosome number of 51 to 65, we assessed the growth potential of leukemic cells from 129 children with newly diagnosed ALL. Flow cytometric analysis was used to compare leukemic cell recoveries at the beginning and at the end of 7-day cultures on allogeneic bone marrow-derived stromal layers. The median percentage of cell recovery after culture was 91% (range, <1% to 550%). Among the 25 hyperdiploid cases, only two had cell recoveries above the median value, compared with 63 of 104 cases with different ploidies (P <.001); 21 had recoveries within the first quartile, in contrast to only 12 of the 104 other cases. Cell recoveries in the 16 cases with duplications of chromosomes 4 and 10, a feature previously associated with a superior outcome, were all within the first quartile. Flow cytometric studies indicated that rapid induction of apoptosis was the underlying cause of low cell recoveries in cases with hyperdiploidy. The demise of hyperdiploid cells on stroma was not due to failure to adhere with stromal elements (as shown by electron microscopy) or to deficiencies of interleukin-1 (IL-1), IL-2, IL-3, IL-4, IL-6, IL-7, IL-11, stem-cell factor, interferon- (IFN-), tumor necrosis factor- (TNF-), or to combinations of these cytokines. Inactivation of IL-4, IFN- and TNF-, which if secreted by stromal layers could be toxic to ALL cells, failed to improve the survival of hyperdiploid blasts. We conclude that leukemic cells bearing 51 to 65 chromosomes have a marked propensity to undergo apoptosis. The stringent survival requirements of these cells, together with their potentially higher sensitivity to antileukemic drugs, may well account for the high cure rates achieved in patients with this form of ALL.

Adolescent↗

PubMed1999

Human parainfluenza virus type 1 matrix and nucleoprotein genes transiently expressed in mammalian cells induce the release of virus-like particles containing nucleocapsid-like structures.

The matrix (M) protein plays an essential role in the assembly and budding of some enveloped RNA viruses. We expressed the human parainfluenza virus type 1 (hPIV-1) M and/or NP genes into 293T cells using the mammalian expression vector pCAGGS. Biochemical and electron microscopic analyses of transfected cells showed that the M protein alone can induce the budding of virus-like particles (vesicles) from the plasma membrane and that the NP protein can assemble into intracellular nucleocapsid-like (NC-like) structures. Furthermore, the coexpression of both the M and NP genes resulted in the production of vesicles enclosing NC-like structures, suggesting that the hPIV-1 M protein has the intrinsic ability to induce membrane vesiculation and to incorporate NC-like structures into these budding vesicles.

Animals↗

PubMed1998

Characterization of avian H5N1 influenza viruses from poultry in Hong Kong.

The transmission of avian H5N1 influenza viruses to 18 humans in Hong Kong in 1997 with six deaths established that avian influenza viruses can transmit to and cause lethal infection in humans. This report characterizes the antigenic and biological properties of the H5N1 influenza viruses isolated from chickens, ducks, and geese from farms and poultry markets in Hong Kong during 1997 and compares them with those of virus isolated from the index human case. Each of the H5N1 viruses from Hong Kong poultry markets that were tested were lethal in chickens, possessed polybasic amino acids at the carboxy-terminus of HA1, and by definition were highly pathogenic in poultry. The available nonpathogenic H5 influenza viruses and the pathogenic H5N1 virus from Hong Kong were analyzed with monoclonal antibodies prepared to A/chicken/Pennsylvania/1370/83 (H5N2). The analysis revealed limited antigenic drift in 15 years and established that monoclonal antibodies are useful reagents for identification and antigenic analysis of avian strains that may transmit to humans in the future. One of the monoclonal antibodies permitted separation of the H5N1 influenza viruses from poultry into two groups that correlated with the presence or absence of a carbohydrate at residue 158 adjacent to the receptor binding site on HA. The H5N1 viruses examined replicated in geese, pigs, rats, and mice, but to only a very limited extent in ducks. It is noteworthy that all infected geese shed virus and that the H5N1 viruses caused disease signs and death in a portion (3 of 16) of the geese, with evidence of systemic spread to the brain. The tropism for geese is unusual and may provide insight into the origin of these viruses. In mice, the H5N1 virus caused lethal pneumonia and spread systemically to the brain. Mice would thus provide an ideal model system for studying immune responses and pathogenesis. Transmission experiments in chickens revealed that the H5N1 viruses are spread by fecal-oral transmission rather than by aerosol, and that the viruses are inactivated by drying of feces at ambient temperature. However, infectivity is maintained for at least 4 days in wet feces at 25 degreesC. There were differences in the morphology of the H5N1 viruses isolated from birds and humans. The perpetuation of H5N1 influenza viruses in the poultry markets in Hong Kong and the transmission of these viruses to humans emphasize the importance of these markets in the epidemiology of influenza. The poultry markets are of critical importance in the perpetuation and transmission of influenza viruses to other avian species and to mammals, including humans.

Animals↗

PubMed1998

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

Adult↗

PubMed1998

Dynamics of distribution of splicing components relative to the transcriptional state of human oocytes from antral follicles.

The distribution of two splicing components (snRNP and SC-35) and coilin were studied by immunogold/electron microscopy in human oocytes from antral follicles at different levels of transcriptional activity (i.e., active, intermediate, and inactive). The results showed a decrease of snRNPs and SC-35 in the karyoplasm as the oocytes progress from a transcriptionally active to the inactive state. The main areas of accumulation of both these splicing components in all stages of oocytes appeared to be the interchromatin granule clusters (IGCs). Within the IGCs, the two splicing components seemed to be spatially segregated, with the snRNPs predominantly bound to the fibrillar region, whereas the SC-35 factors are being enriched in the granular zone. The p80 coilin was found only in the nucleolus-like body (NLB), which is present in all three stages of oocytes; no coiled bodies were evident. These data are consistent with the notion that splicing occurs in the karyoplasm and that the splicing components are mobilized from a storage site (IGCs) to the site of action.

Adult↗

PubMed1998

[Splicing factors in oocyte nuclei from human antral follicles].

Three groups of oocytes in the human antral follicules were previously distinguished on the basis of nuclear structures arrangement and 3H-uridine incorporation in the oocyte nuclei revealed by ultrastructural and autoradiographic research (Parfenov et al., 1984, 1989). These groups can be regarded as consecutive states of oocyte development, i. e. active, intermediate and inactive ones. The latter is characterized by compactization of nuclear structures arranged within a limited nuclear volume. The present study concerns the distribution of splicing factors (snRNP and SC35) and p80 coilin in the nuclei of oocytes being at either of the three states. Along with transcription decreasing in oocyte nuclei, reduction of snRNP and SC35 amounts in the karyoplasm was detected. Simultaneously, accumulation of these splicing factors occurred in clusters of interchromatin granules (CIG). snRNP and SC35 are spatially segregated in CIG. snRNP are located within the fibrillar zones of CIG, while SC35 corresponds to the granular component of CIG. CIG are the only structures containing splicing factors in the nuclei of oocytes from the human antral follicules. These nuclei lack typical coiled bodies (CB). Considerable amounts of the marker protein of CB--p80 coilin are revealed in the nucleolus-like bodies (NLB) of human oocyte nuclei. Contrary to the data obtained on the oocytes from the antral follicules of other mammals (Kopecny et al., 1996a, 1996b) NLB in human oocytes do not contain snRNPs and SC35. The present study allows to make the following conclusions: a) splicing factors recruted to the sites of transcription in karyoplasm of oocytes are assembled in CIG when inactivation of transcription takes place; b) CIG in preovulated human oocytes play substantial role in the storage and preservation of splicing factors.

Adult↗

PubMed1997

A system for functional analysis of Ebola virus glycoprotein.

Ebola virus causes hemorrhagic fever in humans and nonhuman primates, resulting in mortality rates of up to 90%. Studies of this virus have been hampered by its extraordinary pathogenicity, which requires biosafety level 4 containment. To circumvent this problem, we developed a novel complementation system for functional analysis of Ebola virus glycoproteins. It relies on a recombinant vesicular stomatitis virus (VSV) that contains the green fluorescent protein gene instead of the receptor-binding G protein gene (VSVDeltaG*). Herein we show that Ebola Reston virus glycoprotein (ResGP) is efficiently incorporated into VSV particles. This recombinant VSV with integrated ResGP (VSVDeltaG*-ResGP) infected primate cells more efficiently than any of the other mammalian or avian cells examined, in a manner consistent with the host range tropism of Ebola virus, whereas VSVDeltaG* complemented with VSV G protein (VSVDeltaG*-G) efficiently infected the majority of the cells tested. We also tested the utility of this system for investigating the cellular receptors for Ebola virus. Chemical modification of cells to alter their surface proteins markedly reduced their susceptibility to VSVDeltaG*-ResGP but not to VSVDeltaG*-G. These findings suggest that cell surface glycoproteins with N-linked oligosaccharide chains contribute to the entry of Ebola viruses, presumably acting as a specific receptor and/or cofactor for virus entry. Thus, our VSV system should be useful for investigating the functions of glycoproteins from highly pathogenic viruses or those incapable of being cultured in vitro.

Animals↗

PubMed1997

A single sequence change destabilizes the influenza virus neuraminidase tetramer.

A single change (E119G) in the influenza A virus N9 neuraminidase (NA) results in resistance of the enzyme to the NA inhibitor 4-Guanidino-Neu5Ac2en (4-GuDANA). This change causes a salt link between Glu119, which sits in a pocket in the bottom of the active site of the enzyme, and the 4-guanidinium moiety of the inhibitor to be lost. NA "heads" of the resistant enzyme produced only a few small crystals under conditions in which the wild-type enzyme readily formed large crystals. These small crystals were of sufficient quality to yield X-ray crystallographic data which confirmed the E119G change and demonstrated the presence of electron density representing either a strong structural-water molecule or an anionic species in place of the glutamate carboxylate. NA heads of the resistant enzyme also have greatly reduced NA activity per milligram of total protein. We have now found that the mutant NA heads consist predominantly of monomers with a few dimers and tetramers, as determined by electron microscopic analysis of the protein. The low level of enzymatic activity as well as the small number of crystals obtained were probably from the few tetramers remaining intact in the preparation. The purified wild-type and 4-GuDANA-resistant enzymes were treated with the homobifunctional NHS-ester cross linker, DTSSP. SDS-PAGE analysis of the treated enzymes clearly revealed cross-linked dimers of the wild-type enzyme. In contrast, only a small proportion of the 4-GuDANA-resistant neuraminidase was cross-linked. An examination of the known X-ray crystallographic structure of the wild-type NA reveals a salt bridge between Glu119 and Arg156 of the same monomer. Arg156 is a conserved amino acid that is situated at the interface between monomers, and a salt link between this amino acid and Glu119 may contribute to the stability of enzyme tetramers. It is suggested that the E119G alteration in the 4-GuDANA-resistant NA leads to the abrogation of this interaction and thus to the instability of the NA tetramers.

Animals↗