Angewandte Chemie (International ed. in English), Jan 19, 2015
N-glycosylation is a fundamental modification of proteins and exists in the three domains of life... more N-glycosylation is a fundamental modification of proteins and exists in the three domains of life and in some viruses, including the chloroviruses, for which a new type of core N-glycan is herein described. This N-glycan core structure, common to all chloroviruses, is a pentasaccharide with a β-glucose linked to an asparagine residue which is not located in the typical sequon N-X-T/S. The glucose is linked to a terminal xylose unit and a hyperbranched fucose, which is in turn substituted with a terminal galactose and a second xylose residue. The third position of the fucose unit is always linked to a rhamnose, which is a semiconserved element because its absolute configuration is virus-dependent. Additional decorations occur on this core N-glycan and represent a molecular signature for each chlorovirus.
When mouse L-cells are infected with vesicular stomatitis virus, there is a decrease in the rate ... more When mouse L-cells are infected with vesicular stomatitis virus, there is a decrease in the rate of protein synthesis ranging from 20 to 85% of that in mock-infected cells. Vesicular stomatitis virus, irradiated with increasing doses of UV light, eventually loses this capacity to inhibit protein synthesis. The UV inactivation curve was biphasic, suggesting that transcription of two regions of the viral genome is necessary for the virus to become inactivated in this capacity. The first transcription product corresponded to about 373 nucleotides, and the second corresponded to about 42 nucleotides. Inhibition of transcription of the larger product by irradiating the virus with low doses of UV light left a residual inhibition of protein synthesis consisting of approximately 60 to 65% of the total inhibition. This residual inhibition could be obviated by irradiating the virus with a UV dose of greater than 20,000 ergs/mm(2) and was thus considered to represent the effect of the smaller transcription product. In the R1 mutant of C. P. Stanners et al. (Cell 11:273-281, 1977), inhibition of transcription of the larger product sufficed to restore protein synthesis to the mock-infected level, suggesting that the smaller transcription product is nonfunctional with respect to protein synthesis inhibition. It thus appears that the inhibition of protein synthesis by wild-type vesicular stomatitis virus involved at least two separate viral transcription products, and the inhibition by the R1 mutant involved only one. Extracts from cells infected with virus irradiated with low doses of UV light showed a protein synthesis capacity quite similar to that of their in vivo counterparts, indicating that these extracts closely reflect the in vivo effects of virus infection.
It was recently reported that 44% of healthy humans in a study cohort had DNA sequences similar t... more It was recently reported that 44% of healthy humans in a study cohort had DNA sequences similar to Chlorovirus ATCV-1 (family Phycodnaviridae) in oropharyngeal samples and had decreases in visual processing and visual motor speed compared with individuals in whom no virus was detected. Moreover, mice inoculated orally with ATCV-1 developed immune responses to ATCV-1 proteins and had decreases in certain cognitive domains. Because heightened IL-6, nitric oxide (NO), and ERK MAP-kinase activation from macrophages are linked to cognitive impairments, we evaluated cellular responses and viral plaque forming units in murine RAW264.7 and primary macrophages after exposure to ATCV-1 in vitro for up to 72 h after virus challenge. Approximately 8% of the ATCV-1 inoculum was associated with macrophages after 1 h and increased 2-3 fold over 72 h. Immunoblots using rabbit anti-ATCV-1 detected a 55 kDa protein consistent with viral capsid protein from 1 to 72 h and an increasing de novo synthesi...
Transactions of the American Microscopical Society, 1993
... State-of-the-art methods and detailed protocols are provided, and more advanced applications ... more ... State-of-the-art methods and detailed protocols are provided, and more advanced applications are highlighted for the benefit of the more experienced user. Emphasis is placed on practical applications, interpretation of results, and potential problems and pitfalls. ...
Expression of the hypersensitive response (HSR) to tobacco mosaic virus (TMV) infection in Nicoti... more Expression of the hypersensitive response (HSR) to tobacco mosaic virus (TMV) infection in Nicotiana tabacum L. cv. Xanthi-nc (genotype NN) is controlled by the single dominant "N" gene and is temperature sensitive. TMV-infected Xanthi-nc tobacco plants grown at the HSR-restrictive temperature of 31 degrees for 3 days postinoculation show necrosis approximately 8 hr after the temperature shift to the HSR-permissive temperature of 25 degrees . Both polyribosomal and total cytoplasmic poly(A)-containing RNAs were isolated at various times after the temperature shift from leaves of TMV-infected and mock-infected Xanthi-nc tobacco plants and from TMV-infected Turkish Samsun tobacco plants (genotype nn; systemic for TMV infection). The RNAs were translated in vitro and the products were analyzed by polyacrylamide gel electrophoresis. A minimum of four polypeptide translation products specific to both the polyribosomal and total cytoplasmic poly(A)-containing RNAs derived from hypersensitively responding Xanthi-nc were observed.
Antibodies raised against the 126K nonstructural protein (replicase) encoded by tobacco mosaic vi... more Antibodies raised against the 126K nonstructural protein (replicase) encoded by tobacco mosaic virus (TMV) RNA or the viral coat protein have been used to localize these proteins within virus-infected tobacco leaf cells by an immunogold labeling technique. A protocol is given for low-temperature fixation to facilitate immunogold labeling. In cells of TMV-infected leaf tissue, the 126K protein immunogold label was found almost exclusively in "viroplasms" in the cytoplasm and in pockets of virus particles at the viroplasmic periphery. When utilizing the coat protein antiserum, very little labeling was seen within the viroplasms, although virus particles throughout the cytoplasm were heavily labeled. Viroplasms contained electron-dense rope-like structures embedded in a ribosome-rich matrix. In their "mature" form, viroplasms are the well-known "X body" inclusions. The rope-like structures were up to 1.2 micron long and appear twisted, undergoing several revolutions throughout their length, but were not of a constant pitch. In transverse section, they appeared to be composed of several hollow, radially segmented cylinders 21 nm in diameter, with a 9-nm hole. Antibody labeling showed them to be composed, at least in part, of the 126K protein. Clusters of virus particles at the edge of or within the viroplasms were also labeled with the 126K antiserum, in contrast to virus particles in other areas of the cell, which were not. TMV-infected tobacco mesophyll protoplasts cultured for up to 27 hr did not contain the rope-like ribbons. Instead, isolated protoplasts contained amorphous cytoplasmic areas which were labeled with 126K antibody. Since the 126K protein is most probably a constituent of the TMV RNA-replicating enzyme (replicase), its intracellular location is considered to be indicative of the site of replication of TMV RNA. Therefore these results suggest that replication occurs at the edges of the viroplasms.
The anti-apoptotic nematode CED-9 gene and a 3' non-coding mRNA region of the human Bcl-2 gen... more The anti-apoptotic nematode CED-9 gene and a 3' non-coding mRNA region of the human Bcl-2 gene, referred to as 161-1, enhanced resistance to drought and recovery after re-watering in transgenic Nicotiana tabacum L. CV Glurk plants. The effect of drought on plant functions was investigated by withholding water from pots for 6, 8, 10 and 13 days. Results showed that
The 5' end of tobacco mosaic virus (TMV) genomic RNA is capped with 7-methylguanosine. A viru... more The 5' end of tobacco mosaic virus (TMV) genomic RNA is capped with 7-methylguanosine. A virus-coded polypeptide with guanylyltransferase activity has been investigated. This enzyme is responsible for forming the 5'----5' linkage of guanosine 5'-monophosphate to the 5'-diphosphate of an acceptor RNA, thereby forming the cap. A critical step in the mechanism for cap formation in the eukaryotic nucleus is for guanylyltransferase to bind covalently to guanosine 5'-monophosphate with the hydrolysis of pyrophosphate when guanosine 5'-triphosphate is the substrate. The TMV 126-kilodalton protein, which is most probably a component of the TMV replicase, was found to have this activity. The mechanism of this reaction has been characterized biochemically.
Recently a new tetrazolium was described for the use of monitoring cell viability in culture. Thi... more Recently a new tetrazolium was described for the use of monitoring cell viability in culture. This tetrazolium, commonly referred to as MTS [3-(4,5-dimethylthiazol-2-yl)- 5-(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt], has the unusual property that it can be reduced to a water-soluble formazan. beta-Nicotinamide adenine dinucleotide/reduced (NADH) and beta-nicotinamide adenine dinucleotide phosphate/reduced (NADPH) are examples of physiologically important reducing agents. In cell-free studies, MTS was reduce to the soluble formazan in the presence of NADH and NADPH, and reaction were compared to those with dithiothreitol (DTT) or 2-mercaptoethanol (2-ME). The efficiency of these reactions was enhanced 1000-fold by the presence of phenazine methosulfate. Selectivity in the electron transfer from NADPH was slightly greater than NADH, and NADPH or NADH was much greater than the thiols DTT or 2-ME. Generation of either NADH or NADPH in solution by malate dehyd...
Chloroviruses infect their hosts by specifically binding to and degrading the cell wall of their ... more Chloroviruses infect their hosts by specifically binding to and degrading the cell wall of their algal hosts at the site of attachment, using an intrinsic digesting enzyme(s). Chlorovirus PBCV-1 stored as a lysate survived longer than virus alone, suggesting virus attachment to cellular debris may be reversible. Ghost cells (algal cells extracted with methanol) were used as a model to study reversibility of PBCV-1 attachment because ghost cells are as susceptible to attachment and wall digestion as are live cells. Reversibility of attachment to ghost cells was examined by releasing attached virions with a cell wall degrading enzyme extract. The majority of the released virions retained infectivity even after re-incubating the released virions with ghost cells two times. Thus the chloroviruses appear to have a dynamic attachment strategy that may be beneficial in indigenous environments where cell wall debris can act as a refuge until appropriate host cells are available.
Angewandte Chemie (International ed. in English), Jan 19, 2015
N-glycosylation is a fundamental modification of proteins and exists in the three domains of life... more N-glycosylation is a fundamental modification of proteins and exists in the three domains of life and in some viruses, including the chloroviruses, for which a new type of core N-glycan is herein described. This N-glycan core structure, common to all chloroviruses, is a pentasaccharide with a β-glucose linked to an asparagine residue which is not located in the typical sequon N-X-T/S. The glucose is linked to a terminal xylose unit and a hyperbranched fucose, which is in turn substituted with a terminal galactose and a second xylose residue. The third position of the fucose unit is always linked to a rhamnose, which is a semiconserved element because its absolute configuration is virus-dependent. Additional decorations occur on this core N-glycan and represent a molecular signature for each chlorovirus.
When mouse L-cells are infected with vesicular stomatitis virus, there is a decrease in the rate ... more When mouse L-cells are infected with vesicular stomatitis virus, there is a decrease in the rate of protein synthesis ranging from 20 to 85% of that in mock-infected cells. Vesicular stomatitis virus, irradiated with increasing doses of UV light, eventually loses this capacity to inhibit protein synthesis. The UV inactivation curve was biphasic, suggesting that transcription of two regions of the viral genome is necessary for the virus to become inactivated in this capacity. The first transcription product corresponded to about 373 nucleotides, and the second corresponded to about 42 nucleotides. Inhibition of transcription of the larger product by irradiating the virus with low doses of UV light left a residual inhibition of protein synthesis consisting of approximately 60 to 65% of the total inhibition. This residual inhibition could be obviated by irradiating the virus with a UV dose of greater than 20,000 ergs/mm(2) and was thus considered to represent the effect of the smaller transcription product. In the R1 mutant of C. P. Stanners et al. (Cell 11:273-281, 1977), inhibition of transcription of the larger product sufficed to restore protein synthesis to the mock-infected level, suggesting that the smaller transcription product is nonfunctional with respect to protein synthesis inhibition. It thus appears that the inhibition of protein synthesis by wild-type vesicular stomatitis virus involved at least two separate viral transcription products, and the inhibition by the R1 mutant involved only one. Extracts from cells infected with virus irradiated with low doses of UV light showed a protein synthesis capacity quite similar to that of their in vivo counterparts, indicating that these extracts closely reflect the in vivo effects of virus infection.
It was recently reported that 44% of healthy humans in a study cohort had DNA sequences similar t... more It was recently reported that 44% of healthy humans in a study cohort had DNA sequences similar to Chlorovirus ATCV-1 (family Phycodnaviridae) in oropharyngeal samples and had decreases in visual processing and visual motor speed compared with individuals in whom no virus was detected. Moreover, mice inoculated orally with ATCV-1 developed immune responses to ATCV-1 proteins and had decreases in certain cognitive domains. Because heightened IL-6, nitric oxide (NO), and ERK MAP-kinase activation from macrophages are linked to cognitive impairments, we evaluated cellular responses and viral plaque forming units in murine RAW264.7 and primary macrophages after exposure to ATCV-1 in vitro for up to 72 h after virus challenge. Approximately 8% of the ATCV-1 inoculum was associated with macrophages after 1 h and increased 2-3 fold over 72 h. Immunoblots using rabbit anti-ATCV-1 detected a 55 kDa protein consistent with viral capsid protein from 1 to 72 h and an increasing de novo synthesi...
Transactions of the American Microscopical Society, 1993
... State-of-the-art methods and detailed protocols are provided, and more advanced applications ... more ... State-of-the-art methods and detailed protocols are provided, and more advanced applications are highlighted for the benefit of the more experienced user. Emphasis is placed on practical applications, interpretation of results, and potential problems and pitfalls. ...
Expression of the hypersensitive response (HSR) to tobacco mosaic virus (TMV) infection in Nicoti... more Expression of the hypersensitive response (HSR) to tobacco mosaic virus (TMV) infection in Nicotiana tabacum L. cv. Xanthi-nc (genotype NN) is controlled by the single dominant "N" gene and is temperature sensitive. TMV-infected Xanthi-nc tobacco plants grown at the HSR-restrictive temperature of 31 degrees for 3 days postinoculation show necrosis approximately 8 hr after the temperature shift to the HSR-permissive temperature of 25 degrees . Both polyribosomal and total cytoplasmic poly(A)-containing RNAs were isolated at various times after the temperature shift from leaves of TMV-infected and mock-infected Xanthi-nc tobacco plants and from TMV-infected Turkish Samsun tobacco plants (genotype nn; systemic for TMV infection). The RNAs were translated in vitro and the products were analyzed by polyacrylamide gel electrophoresis. A minimum of four polypeptide translation products specific to both the polyribosomal and total cytoplasmic poly(A)-containing RNAs derived from hypersensitively responding Xanthi-nc were observed.
Antibodies raised against the 126K nonstructural protein (replicase) encoded by tobacco mosaic vi... more Antibodies raised against the 126K nonstructural protein (replicase) encoded by tobacco mosaic virus (TMV) RNA or the viral coat protein have been used to localize these proteins within virus-infected tobacco leaf cells by an immunogold labeling technique. A protocol is given for low-temperature fixation to facilitate immunogold labeling. In cells of TMV-infected leaf tissue, the 126K protein immunogold label was found almost exclusively in "viroplasms" in the cytoplasm and in pockets of virus particles at the viroplasmic periphery. When utilizing the coat protein antiserum, very little labeling was seen within the viroplasms, although virus particles throughout the cytoplasm were heavily labeled. Viroplasms contained electron-dense rope-like structures embedded in a ribosome-rich matrix. In their "mature" form, viroplasms are the well-known "X body" inclusions. The rope-like structures were up to 1.2 micron long and appear twisted, undergoing several revolutions throughout their length, but were not of a constant pitch. In transverse section, they appeared to be composed of several hollow, radially segmented cylinders 21 nm in diameter, with a 9-nm hole. Antibody labeling showed them to be composed, at least in part, of the 126K protein. Clusters of virus particles at the edge of or within the viroplasms were also labeled with the 126K antiserum, in contrast to virus particles in other areas of the cell, which were not. TMV-infected tobacco mesophyll protoplasts cultured for up to 27 hr did not contain the rope-like ribbons. Instead, isolated protoplasts contained amorphous cytoplasmic areas which were labeled with 126K antibody. Since the 126K protein is most probably a constituent of the TMV RNA-replicating enzyme (replicase), its intracellular location is considered to be indicative of the site of replication of TMV RNA. Therefore these results suggest that replication occurs at the edges of the viroplasms.
The anti-apoptotic nematode CED-9 gene and a 3' non-coding mRNA region of the human Bcl-2 gen... more The anti-apoptotic nematode CED-9 gene and a 3' non-coding mRNA region of the human Bcl-2 gene, referred to as 161-1, enhanced resistance to drought and recovery after re-watering in transgenic Nicotiana tabacum L. CV Glurk plants. The effect of drought on plant functions was investigated by withholding water from pots for 6, 8, 10 and 13 days. Results showed that
The 5' end of tobacco mosaic virus (TMV) genomic RNA is capped with 7-methylguanosine. A viru... more The 5' end of tobacco mosaic virus (TMV) genomic RNA is capped with 7-methylguanosine. A virus-coded polypeptide with guanylyltransferase activity has been investigated. This enzyme is responsible for forming the 5'----5' linkage of guanosine 5'-monophosphate to the 5'-diphosphate of an acceptor RNA, thereby forming the cap. A critical step in the mechanism for cap formation in the eukaryotic nucleus is for guanylyltransferase to bind covalently to guanosine 5'-monophosphate with the hydrolysis of pyrophosphate when guanosine 5'-triphosphate is the substrate. The TMV 126-kilodalton protein, which is most probably a component of the TMV replicase, was found to have this activity. The mechanism of this reaction has been characterized biochemically.
Recently a new tetrazolium was described for the use of monitoring cell viability in culture. Thi... more Recently a new tetrazolium was described for the use of monitoring cell viability in culture. This tetrazolium, commonly referred to as MTS [3-(4,5-dimethylthiazol-2-yl)- 5-(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt], has the unusual property that it can be reduced to a water-soluble formazan. beta-Nicotinamide adenine dinucleotide/reduced (NADH) and beta-nicotinamide adenine dinucleotide phosphate/reduced (NADPH) are examples of physiologically important reducing agents. In cell-free studies, MTS was reduce to the soluble formazan in the presence of NADH and NADPH, and reaction were compared to those with dithiothreitol (DTT) or 2-mercaptoethanol (2-ME). The efficiency of these reactions was enhanced 1000-fold by the presence of phenazine methosulfate. Selectivity in the electron transfer from NADPH was slightly greater than NADH, and NADPH or NADH was much greater than the thiols DTT or 2-ME. Generation of either NADH or NADPH in solution by malate dehyd...
Chloroviruses infect their hosts by specifically binding to and degrading the cell wall of their ... more Chloroviruses infect their hosts by specifically binding to and degrading the cell wall of their algal hosts at the site of attachment, using an intrinsic digesting enzyme(s). Chlorovirus PBCV-1 stored as a lysate survived longer than virus alone, suggesting virus attachment to cellular debris may be reversible. Ghost cells (algal cells extracted with methanol) were used as a model to study reversibility of PBCV-1 attachment because ghost cells are as susceptible to attachment and wall digestion as are live cells. Reversibility of attachment to ghost cells was examined by releasing attached virions with a cell wall degrading enzyme extract. The majority of the released virions retained infectivity even after re-incubating the released virions with ghost cells two times. Thus the chloroviruses appear to have a dynamic attachment strategy that may be beneficial in indigenous environments where cell wall debris can act as a refuge until appropriate host cells are available.
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