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K G Murti

Publications and source records attributed to K G Murti.

At least 73 records · Page 4Linked to original sources
PubMed1986

Mouse monoclonal antibodies induced by anti-allotype antibody display internal images of the rabbit VHa1 allotype: direct visualization by immunoelectron microscopy.

We have previously shown that injection of adult rabbits with anti-immunoglobulin (Ig) antibody (Ab) induces the expression of genetically unexpected Ig markers, i.e., a1 allotypic determinants. We now show that these rabbit Ig markers can also be induced in mice by a similar treatment; in the latter case the a1 determinants are located in the antigen-combining site and thus represent "internal images". Two monoclonal antibodies (mAb) were developed from mice treated with anti-allotype Ab. These mAb were reactive with all homologous and heterologous anti-a1 Ab but not normal Ig; efficiently inhibited the binding of rabbit a1 Ig to anti-a1 Ab; and elicited the production of anti-allotype Ab when injected into normal mice. To determine whether the a1-like determinants on these mAb were located in the antigen-combining site, immunoelectron microscopy was utilized to directly visualize Ab complexes. Complexes composed of intact Ab and anti-a1 Fab fragments yielded uniform binding patterns which were identical to those produced by anti-idiotypic reactions. In each case, an identical tip-to-tip binding configuration was observed with a single Fab fragment attached at an approximate 180 degree angle to the V region terminus of each Ab arm. In contrast, rabbit a1 Ig bound as many as two anti-a1 fragments per Ig arm; these fragments were attached laterally and at right angles to the intact molecule. These mAb thus provide the first direct evidence that Ab2 beta determinants are located in, or near, the antigen-combining site.

Animals↗
PubMed1986

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.

Antibodies, Monoclonal↗
PubMed1986

Interaction of frog virus 3 with the cytomatrix. IV. Phosphorylation of vimentin precedes the reorganization of intermediate filaments around the virus assembly sites.

Frog virus 3 (FV3) assembles in morphologically distinct assembly sites in the cytoplasm of infected cells. As the assembly sites form, the intermediate filaments (IF) aggregate, delimit the assembly sites, and remain so throughout infection. To determine the molecular basis of reorganization of IF, we analysed the vimentin of uninfected and FV3-infected cells by two-dimensional gel electrophoresis. The results showed that (i) the vimentin was more acidic in FV3-infected cells than in uninfected cells, (ii) the acidification of vimentin in FV3-infected cells was possibly due to a fourfold increase in phosphorylation, and (iii) the phosphorylation of vimentin preceded the reorganization of IF around virus assembly sites. A temperature-sensitive mutant of FV3 (ts9467), which at the non-permissive temperature neither reorganized IF nor formed assembly sites, failed to increase the phosphorylation of vimentin. Together, the above results suggest that changes in phosphorylation may modulate IF organization and that changes in IF organization are required for FV3 assembly site formation.

Animals↗
PubMed1985

An 18-amino acid deletion in an influenza neuraminidase.

The genes coding for the neuraminidase (NA) enzyme in antigenic variants of influenza virus X-7(F1) (which contains the NA of A/RI/5+/57) are shorter than other N2 neuraminidase genes. When the parent X-7(F1) virus was cloned at limiting dilution prior to selection of variants with monoclonal antibodies, a minor component with a shorter neuraminidase gene was cloned out, and this deletion was retained in all the variants. Sequence analysis has shown that there is a single deletion, 54 nucleotides long, which occurs in the coding region of the NA. The 18 deleted amino acids are from the stalk region of the protein, which is a thin structure containing the four polypeptide chains of the tetrameric enzyme which separates the enzymatically and antigenically active "head" from the hydrophobic sequence embedded in the viral membrane. The deletion can be seen as a shortened stalk when rosettes of detergent-released NA are examined by electron microscopy, which confirms that the NA stalk is a highly extended structure. Although the more abundant hemagglutinin (HA) would probably extend beyond the shortened NA molecule on the surface of the virus, the NA head is still accessible to all of the available monoclonal antibodies to the different antigenic sites on the molecule. Enzymatic activity toward a small substrate molecule, N-acetyl neuraminyl lactose, is the same for both the parental X-7(F1) virus and the strain having the deleted NA; however, the latter virus is slower to cleave a large substrate, fetuin, and elutes from red cells at a reduced rate.

Amino Acid Sequence↗
PubMed1985

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.

Actin Cytoskeleton↗
PubMed1984

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.

Animals↗
PubMed1984

Interaction of frog virus 3 with the cytomatrix. II. Structure and composition of the virus assembly site.

We have described the structure of virus assembly sites in frog virus 3-infected tissue culture cells based on an examination of sectioned and whole cells by conventional and high voltage (1 000 kV) electron microscopy (HVEM), respectively. We have also attempted to identify the cellular and viral components within the assembly sites using immunofluorescence and a combination of DNase digestion and EM autoradiography. Immunofluorescence studies showed that the sites do not contain tubulin, vimentin, actin, or myosin, confirming EM and HVEM studies which showed the absence of these cytoskeletal filaments from these sites. Enzymatic digestion and autoradiographic studies identified viral DNA in clumps of electron-dense material which are suspended within the matrix of the assembly site. Examination of whole, virus-infected cells by HVEM revealed that the matrix of the assembly site appears as a compaction of the cytoplasmic matrix and appears to be continuous with it. The fine strands of cytomatrix, equated with the microtrabeculae of other cells, are covered with or contain granules measuring 6-8 nm; similar granules also populate the virus assembly sites. Disruption of purified FV 3 with chloroform yielded capsomeres measuring 6-8 nm. Based on all these observations, we postulate that the virus assembly sites are regions of the cytomatrix specialized for virus assembly and that the viral components are transported along the cytomatrix to the assembly sites.

Actins↗
PubMed1983

Interaction of frog virus-3 with the cytoskeleton. I. Altered organization of microtubules, intermediate filaments, and microfilaments.

The progressive cytoskeletal alterations of frog virus 3-infected baby hamster kidney (BHK) and fathead minnow (FHM) cells were studied by immunofluorescence and electron microscopy. The virus assembly sites, which contain viral genomes and viral proteins, were detected in the cytoplasm at 4 h (FHM) or 6 h (BHK) and mature virions appeared 2 h later. When infected cells were treated with Triton X-100, the assembly sites were found in association with the cytoskeleton. In infected cells, the number of microtubules progressively decreased but a few microtubules traversing in the vicinity of the assembly sites remained intact. Early in infection, the intermediate filaments retracted from the cell periphery, delimited the forming assembly sites, and remained there throughout infection. We suggest that intermediate filaments are involved in the formation of assembly sites. In addition, the filaments either by themselves or in conjunction with microtubules may anchor the assembly sites near the nucleus. The microfilament bundles (stress fibers) disappeared with the formation of assembly sites, and late in infection many projections containing microfilaments and virus particles appeared at the cell surface. The observation suggests a role for microfilaments in virus release. Taken together, these results provide the first example of a virus-infected cell in which all three cytoskeletal filaments show profound organizational changes and suggest an active participation of the host cytoskeleton in viral functions.

Animals↗
PubMed1983

Replication forms of the gene-sized DNA molecules of hypotrichous ciliates.

Using a method for obtaining DNA from 10 to 40 macronuclei for electron microscopy, we analyzed the structure of gene-sized, linear DNA molecules from S-phase macronuclei of two hypotrichous ciliates, Euplotes eurystomus and Styx sp. Three types of putative replicating intermediates were observed: (i) molecules with a bubble close to one end, (ii) molecules with single forks, and (iii) molecules with two forks. We conclude that: (i) each macronuclear DNA molecule replicates as an independent unit, (ii) the molecules contain an origin of replication close to one or both ends, and (iii) the mode of replication is bidirectional.

Animals↗
PubMed1982

The genome of frog virus 3, an animal DNA virus, is circularly permuted and terminally redundant.

We examined the structure of the frog virus 3 (FV 3) genome by using electron microscopic and biochemical techniques. The linear FV 3 DNA molecules (Mr approximately 100 x 10(6) formed circles when partially degraded with bacteriophage lambda 5'-exonuclease and annealed, but not when the annealing was done without prior exonuclease digestion. The results suggest that the DNA molecules contain direct terminal repeats. The repeated region composed about 4% of the genome. Complete denaturation of native FV 3 DNA molecules followed by renaturation produced duplex circles each bearing two single-stranded tails at different points along the circumference. The tails presumably represent the terminal repeats. The formation of duplex circles suggests that the FV 3 genome is circularly permuted. This is further borne out by (i) failure to identify a specific restriction endonuclease fragment containing the label when the molecular ends were radiolabeled by using the polynucleotide kinase procedure, and (ii) similarity in the restriction patterns of virion DNA and large concatemeric replicating viral DNA as revealed by endonucleolytic cleavage of both DNAs with HindIII. From the above data, we conclude that the FV3 genome is both circularly permuted and terminally redundant--unique features for an animal virus.

Animals↗
PubMed1981

Secondary structural features in the 70S RNAs of Moloney murine leukemia and Rous sarcoma viruses as observed by electron microscopy.

The secondary structural features in the 70S RNAs of the Prague strain of avian Rous sarcoma virus, subgroup A (PR-RSV-A), and Moloney murine leukemia virus (M-MuLV) were compared by electron microscopy. The PR-RSV-A genome contained two subunits joined by a linkage structure as in the genomes of M-MuLV and other mammalian retroviruses. In both viral genomes, a highly reproducible hairpin occurred at about 70 nucleotides from the 5' end of each subunit and contained 320 +/- 8 nucleotides. The stable point of linkage between the subunits in both viral genomes involved fewer than 50 nucleotides and occurred at 466 +/- 9 nucleotides from the 5' end. This places the linkage about 350 nucleotides further toward the 3' end of the subunit than the binding site of primer tRNA. Another structural feature common to both genomes was a loop in each subunit. In M-MuLV, the loop contained 3.9 +/- 0.10 kilobases (kb) and occurred at a distance of 2.2 +/- 0.05 kb from the 5' end. In PR-RSV-A, the loop was smaller (2.3 +/- 0.10 kb) and further (3.3 +/- 0.10 kb) from the 5' end. When M-MuLV RNA was heated to 70, 85, or 90 degrees C and cooled, the hairpin consistently reformed at the 5' end. No other structures typical of the native molecules reappeared. In RNA samples heated to 70 degrees C, a new loop reproducibly occurred near the 5' end of each subunit, but this loop was not found in samples heated to higher temperatures. Based on all of these findings, we conclude that the genome of PR-RSV-A shares several features with M-MuLV and other mammalian retroviruses and that the primer tRNA molecules are not involved in the linkage of the two subunits in either genome. We also conclude that the dimer linkage and the loops in subunits are typical of the native molecules and that their formation requires a special environment.

Avian Sarcoma Viruses↗