Sundararajan Venkatesan - Academia.edu (original) (raw)

Papers by Sundararajan Venkatesan

Research paper thumbnail of Nucleotide Sequence oftheGeneEncoding Respiratory Syncytial VirusMatrix Protein

Theaminoacid sequenceofthematrix protein ofthehumanrespiratory syncytial virus (RSvirus) was dedu... more Theaminoacid sequenceofthematrix protein ofthehumanrespiratory syncytial virus (RSvirus) was deduced fromthesequenceofacDNAinsert inarecombinant plasmid harboring analmost full-length copy ofthis gene.Itspecifically hybridized toa single 1,050-base mRNA frominfected cells. Therecombinant containing 944basepairs ofRSviral matrix protein gene sequencelacked five nucleotides corresponding to the5'endofthemRNA. Thenucleotide sequenceofthe5'endofthemRNA was determined bythe dideoxy sequencing methodandfound tobe5'NGGGC,wherein theCresidue isone nucleotide upstream ofthecloned viral sequence.Theinitiator ATG codonforthematrix protein isembeddedinanAATATGG sequencesimilar tothecanonical PXXATGG sequencepresent aroundfunctional eucaryotic translation initiation codons. Thereisno conserved sequenceupstreamofthepolyadenylate tail, unlike vesicular stomatitis virusandSendaivirus, inwhichfournucleotides upstreamofthepolyadenylate tail are conserved inall genes.Thereisno equivalent o...

Research paper thumbnail of Modification of the 5' end of mRNA. Association of RNA triphosphatase with the RNA guanylyltransferase-RNA (guanine-7-)methyltransferase complex from vaccinia virus

Journal of Biological Chemistry, 1980

Modification of the 5' End of mRNA ASSOCIATION OF RNA TRIPHOSPHATASE WITH THE RNA GUANYLYLTRANSFE... more Modification of the 5' End of mRNA ASSOCIATION OF RNA TRIPHOSPHATASE WITH THE RNA GUANYLYLTRANSFERASE. RNA (GUANINE-7-)METHYLTRANSFERASE COMPLEX FROM VACCINIA VIRUS*

Research paper thumbnail of A specific sequence with a bulged guanosine residue(s) in a stem-bulge-stem structure of Rev-responsive element RNA is required for trans activation by human immunodeficiency virus type 1 Rev

Journal of Virology, 1992

We demonstrate that both the in vitro RNA binding and in vivo trans activation functions of human... more We demonstrate that both the in vitro RNA binding and in vivo trans activation functions of human immunodeficiency virus type 1 Rev regulatory protein Rev require the presence of a 9-nucleotide 5'-CACUAUGGG-3' RNA motif on its cognate target, the Rev-responsive element RNA. For optimal Rev recognition, this sequence must be presented as a stem-bulge-stem structure and must contain at least two G's, one of which must be unpaired, and include some or all of the CACUAU sequence upstream of the three G's. Distal mutations which result in the base pairing of the G's eliminate the Rev response. The first G is crucial, but changes at the other G's are tolerated if at least one G is unpaired. The secondary structure or the three-dimensional orientation of the B1 and B2 stem-loops of the Rev-responsive element are not relevant as long as the 5'-CACUAUGGG-3' sequence is preserved, with at least one bulged G residue.

Research paper thumbnail of Proteins associated with human parainfluenza virus type 3

Journal of Virology, 1985

The polypeptides associated with human parainfluenza virus type 3 were identified. Five proteins ... more The polypeptides associated with human parainfluenza virus type 3 were identified. Five proteins were present in detergent- and salt-resistant viral cores. Of these, three proteins designated NP0, NP1, and NP2 of 68,000, 58,000, and 52,000 daltons, respectively, were stably associated with 50S RNA in CsCl gradient-purified nucleocapsids. The amounts of NP1 and NP2 were variable, and these proteins were shown to be structurally related to the major nucleocapsid protein (NP0) by partial Staphylococcus aureus V8 protease mapping. The other core proteins included a 240K protein designated L (candidate for the viral polymerase) and an 84K protein designated as the phosphoprotein (P) on the basis of a predominant incorporation of Pi. The viral envelope had four prominent proteins (72, 53, 40, and 12K) under reducing conditions of electrophoresis. The 72 and 53K proteins were specifically labeled with [3H]glucosamine and [3H]mannose. When sulfhydryl reagents were removed, a new 62K protein...

Research paper thumbnail of Molecular Functional Studies of HIV-1 REV and NEF Proteins

Advances in Molecular Biology and Targeted Treatment for AIDS, 1991

Human immunodeficiency virus type 1 (HIV-1), the etiological agent of AIDS, preferentially infect... more Human immunodeficiency virus type 1 (HIV-1), the etiological agent of AIDS, preferentially infects the CD4+ helper subset of human T lymphocytes. Clinically, HIV-1 infection is characterized by a chronic phase lasting several years with a paucity of infected lymphocytes in the circulation. Notwithstanding, HIV-1 infection of primary human T lymphocytes or CD4+ cell lines in vitro leads to massive acute infection and cell death (20). This dichotomy between the natural history of virus infection and its behavior in tissue culture implies the existence of viral and cellular determinants of viral latency and reactivation.

Research paper thumbnail of Human immunodeficiency virus type 1 (HIV-1) provirus expression and LTR transcription are repressed in NEF-expressing cell lines

Virology, 1991

Human immunodeficiency virus type 1 (HIV-l) NEF protein has been demonstrated to be a negative re... more Human immunodeficiency virus type 1 (HIV-l) NEF protein has been demonstrated to be a negative regulator of HIV-1 replication and HIV-1 LTR transcription under transient expression conditions. The difficulty of several laboratories to reproduce these findings led us to reexamine the role of NEF in HIV-1 provirus expression and HIV-1 LTR transcription. Basal transcription from the HIV-l LTR in the presence of a NEF expression vector was compared to that in the presence of a mutated NEF vector. NEF expression led to a greater than lo-fold repression of LTR transcription under these conditions. HeLa and Jurkat cell lines carrying the nef gene linked to the CMV promoter or the HIV-l LTR were isolated by coselection for neomycin resistance. Single cell isolates were further selected for the expression of nef transcripts. With the exception of the anti-sense nef cell lines, all the nef cell lines expressed the 27-kDa NEF protein, detectable by immunoprecipitation. NEF+ HeLa cell lines were at least 5-fold less efficient than NEF-HeLa cell lines in transient proviral expression. Provirus expression was also repressed in the NEF+ Jurkat cell lines. TAT-activated LTR transcription from an HIV-1 LTR-linked CAT expression vector was repressed 1 O-fold in the NEF+ HeLa and NEF+ Jurkat cell lines. When infected with HIV-l, NEF expressing T lymphoid cell lines showed moderate delays in onset and peak of reverse transcriptase production. However, none of these cell lines completely arrested virus replication. Our data confirm a negative regulatory effect of NEF on both virus production and LTR driven CAT expression in the cell lines tested. It is possible that cell specific factors may influence NEF activity.

Research paper thumbnail of Biochemical and biological comparison of HIV-1 NEF and ras gene products

Virology, 1991

Human immunodeficiency virus type 1 (HIV-l) NEf protein has been reported to share certain bioche... more Human immunodeficiency virus type 1 (HIV-l) NEf protein has been reported to share certain biochemical and structural properties with known oncoproteins like src or ras. To determine whether this is a general property of NEF from various HIV isolates, three different NEF proteins were expressed in Escherichia co/i using a thermoinducible expression system previously exploited to overproduce functionally active p21 ras proteins. ras and NEF proteins expressed in this manner were evaluated in parallel to compare their biochemical and biological properties. In contrast to ras, our NEF protein preparations had no detectable GTP binding but showed autophosphorylation activity when incubated in the presence of either GTP or ATP. This putative autokinase activity was higher in NEF proteins containing threonine at position 15 than in those carrying alanine at that position. Two different NEF genes also failed to induce oncogenic transformation of permanently transfected NIH 3T3 cells under conditions that led to oncogenic transformation using activated ras genes. Also, unlike ras, the NEf gene products failed to induce meiotic maturation when injected into fully grown Xenopus OOCytSS.

Research paper thumbnail of Recognition of dileucine-based sorting signals from HIV-1 Nef and LIMP-II by the AP-1 γ–σ1 and AP-3 δ–σ3 hemicomplexes

Journal of Cell Biology, 2003

The sorting of transmembrane proteins to endosomes and lysosomes is mediated by signals present i... more The sorting of transmembrane proteins to endosomes and lysosomes is mediated by signals present in the cytosolic tails of the proteins. A subset of these signals conform to the [DE]XXXL[LI] consensus motif and mediate sorting via interactions with heterotetrameric adaptor protein (AP) complexes. However, the identity of the AP subunits that recognize these signals remains controversial. We have used a yeast three-hybrid assay to demonstrate that [DE]XXXL[LI]-type signals from the human immunodeficiency virus negative factor protein and the lysosomal integral membrane protein II interact with combinations of the γ and σ1 subunits of AP-1 and the δ and σ3 subunits of AP-3, but not the analogous combinations of AP-2 and AP-4 subunits. The sequence requirements for these interactions are similar to those for binding to the whole AP complexes in vitro and for function of the signals in vivo. These observations reveal a novel mode of recognition of sorting signals involving the γ/δ and σ ...

Research paper thumbnail of TAR RNA-binding protein is an inhibitor of the interferon-induced protein kinase PKR

Proceedings of the National Academy of Sciences, 1994

A cDNA encoding a double-stranded-RNA (dsRNA)-binding protein was isolated by screening a HeLa ce... more A cDNA encoding a double-stranded-RNA (dsRNA)-binding protein was isolated by screening a HeLa cell cDNA expression library for proteins that bind the HIV-1 Rev-responsive-element RNA. The cDNA encoded a protein that was identical to TRBP, the previously reported cellular protein that binds the transactivation response element (TAR) RNA of human immunodeficiency virus type 1. TRBP inhibited phosphorylation of the interferon-induced ribosome-associated protein kinase PKR and of the eukaryotic translation initiation factor eIF-2 alpha in a transient-expression system in which the translation of a reporter gene was inhibited by the localized activation of PKR. TRBP expression in HeLa cells complemented the growth and protein-synthesis defect of a vaccinia virus mutant lacking the expression of the dsRNA-binding protein E3L. These results implicate TRBP as a cellular regulatory protein that binds RNAs containing specific secondary structure(s) to mediate the inhibition of PKR activation...

Research paper thumbnail of Rev-induced modulation of Nef protein underlies temporal regulation of human immunodeficiency virus replication

Proceedings of the National Academy of Sciences, 1989

The replication of human immunodeficiency virus type 1 (HIV-1) requires the concerted action of t... more The replication of human immunodeficiency virus type 1 (HIV-1) requires the concerted action of two virus-encoded transactivator proteins, Tat and Rev, and is in turn moderated by the viral transcriptional repressor Nef. We show here that the phenotype of a Rev- HIV-1 provirus was nonreplicating and was distinguished by accumulation of Nef protein and reduced Tat function. Provirus defective in both the rev and nef genes (Rev-Nef-) was also nonreplicating but had normal Tat function. Trans-complementation of the Rev- mutant with Rev caused a decrease of both the steady-state level and the rate of synthesis of Nef. This was accompanied by enhanced synthesis of viral structural proteins. Rev induced even greater levels of virus production from the Rev-Nef- double mutant. In contrast, exogenous Rev did not augment virus production from wild-type provirus. Virus production from Rev- and Rev-Nef- mutants induced by Rev was repressed by exogenous Nef. The repression induced by Nef could n...

Research paper thumbnail of Eukaryotic mRNA capping enzyme-guanylate covalent intermediate

Proceedings of the National Academy of Sciences, 1982

Incubation of HeLa cell mRNA guanylyltransferase (GTP:mRNA guanylyltransferase, EC 2.7.7.50) with... more Incubation of HeLa cell mRNA guanylyltransferase (GTP:mRNA guanylyltransferase, EC 2.7.7.50) with [alpha-32P]GTP and a divalent cation in the absence of an RNA acceptor results in the formation of a covalent enzyme-guanylate complex. The complex, after purification by phosphocellulose chromatography, can transfer its bound GMP moiety to pyrophosphate, regenerating GTP, or to the 5'-diphosphate end of poly(A), forming a cap structure G(5')pppA(pA)n. The GMP-polypeptide has a molecular weight of 65,000 and is stable to heating in the presence of sodium dodecyl sulfate. On the basis of the alkali-stable and acid-labile nature of the bond and its susceptibility to nucleophilic attack by hydroxylamine at low pH, the GMP-polypeptide linkage appears to be a phosphoamine bond. After digestion with trypsin, a single GMP-peptide was resolved by two dimensional electrophoresis and chromatography.

Research paper thumbnail of Resistance to human respiratory syncytial virus (RSV) infection induced by immunization of cotton rats with a recombinant vaccinia virus expressing the RSV G glycoprotein

Proceedings of the National Academy of Sciences, 1986

A cDNA copy of the G glycoprotein gene of human respiratory syncytial virus (RSV) was placed unde... more A cDNA copy of the G glycoprotein gene of human respiratory syncytial virus (RSV) was placed under control of a vaccinia virus promoter and inserted into the thymidine kinase locus of the vaccinia virus genome. The recombinant vaccinia virus retained infectivity and expressed a 93-kDa protein that migrated with the authentic RSV G glycoprotein upon polyacrylamide gel electrophoresis. Glycosylation of the expressed protein and transport to the cell surface were demonstrated in the absence of other RSV proteins. Cotton rats that were inoculated intradermally with the infectious recombinant virus produced serum antibody to the G glycoprotein that neutralized RSV in vitro. Furthermore, the vaccinated animals were resistant to lower respiratory tract infection upon intranasal inoculation with RSV and had reduced titers of RSV in the nose.

Research paper thumbnail of Preparation and properties of a poly A resin and its use in the isolation of naturally occurring poly U sequences

Nucleic Acids Research, 1976

A method for the covalent attachment of poly A, as well as other nucleic acids and nucleosides, t... more A method for the covalent attachment of poly A, as well as other nucleic acids and nucleosides, to a methylene dianiline derivative of starch is described. The properties of this poly A resin and its use for the recovery of poly U sequences from both nuclear and cytoplasmic extracts of HeLa cells is described.

Research paper thumbnail of Amino acid sequence of human respiratory syncytial virus nucleocapsid protein

Nucleic Acids Research, 1983

Amino add sequence of the human respiratory syncytial (RS) virus nudeocapsid (NC) protein, deduce... more Amino add sequence of the human respiratory syncytial (RS) virus nudeocapsid (NC) protein, deduced from the DNA sequence of a recombinant plasmid, is presented. The cDNA plasmid (pRSBll) has 1412 bp of RS viral NC sequence and lacks six nucleotides of the 5'end of mRNA. There is a single long open reading frame encoding 467 amino acids. This 51540 dal protein is rich in basic amino acids and has no homologies with other known viral capsid proteins.

Research paper thumbnail of Respiratory syncytial virus envelope glycoprotein (G) has a novel structure

Nucleic Acids Research, 1985

ASTno acid sequence of human respiratory syncytiai virus envelope glycoprotein (G) was deduced fr... more ASTno acid sequence of human respiratory syncytiai virus envelope glycoprotein (G) was deduced from the DNA sequence of a recombinant plasmid and confirmed by limited amino add microsequendng of purified 90K G protein. The calculated molecular mass of the protein encoded by the only long open reading frame of 298 amino acids was 32,588 daltons and was somewhat smaller than the 36K polypeptide translated J[n vitro from mRNA selected by this plasmid. Inspection of the sequence revealed a single hydrophobic domain of 23 amino acids capable of membrane Insertion at 41 residues from the N-term1nus. There was no N-terminal signal sequence and the hydrophHic N-term1nal 20 residues probably represent the cytoplasmic tall of the protein. The N-terminally oriented membrane Insertion was somewhat analogous to paramyxovims hemagglutinin-neuraminidase (HN) and Influenza neuraminidase (NA). The protein was moderately hydrophilic and rich 1n hydroxy-amino adds. It was both N-and O-glycosylated with the latter contributing significantly to the net molecular mass 90K.

Research paper thumbnail of Cholesterol is obligatory for polarization and chemotaxis but not for endocytosis and associated signaling from chemoattractant receptors in human neutrophils

Journal of Biomedical Science, 2008

Plasma membrane cholesterol is critical for neutrophil chemotaxis, although how cholesterol affec... more Plasma membrane cholesterol is critical for neutrophil chemotaxis, although how cholesterol affects chemotactic signaling pathway has not been clearly delineated. Here we demonstrate that cholesterol was absolutely required for polarized redistribution of key chemotactic mediators in human neutrophils in response to all chemoattractants tested (fMet-Leu-Phe, and the chemokines CXCL1, CXCL8 and CXCL12). In particular, PI3K and phosphatidylinositol-3,4,5 triphosphate (PIP 3) failed to accumulate at the front and phosphatase and tensin homolog (PTEN) at the back of chemoattractant-stimulated neutrophils after cholesterol depletion. Cholesterol depletion did not affect early chemoattractant signaling events such as G-protein activation, intracellular calcium flux or G-protein-independent endocytosis-linked signaling, including the activation of mitogen-activated protein kinase (MAPK), Hck and Fgr transduced by b-arrestin. During cell polarization, F-actin assemblies redistributed the cholesterol-rich microdomains and cytoskeletonanchored proteins, including CD16 and CD44 from the leading edge. These data suggest that spatial polarization of chemotactic mediators is orchestrated by protein:protein interactions that organize cholesterol-rich domains of the plasma membrane. Keywords Neutrophils Á Chemotaxis Á Cholesterol Á Lipid rafts Á Chemoattractants Á G-proteins Á Endocytosis Á Arrestin Á Degranulation Á Protein kinases Á F-actin Abbreviations APC Allophycocyanin DRMs Detergent-resistant membranes 123

Research paper thumbnail of Real-time Kinetics of HIV-1 Rev-Rev Response Element Interactions

Journal of Biological Chemistry, 1999

The kinetics of interaction between the human immunodeficiency virus-1 Rev protein and its RNA ta... more The kinetics of interaction between the human immunodeficiency virus-1 Rev protein and its RNA target, Rev response element (RRE) RNA was determined in vitro using a biosensor technique. Our results showed that the primary Rev binding site is a core stem-loop RNA molecule of 30 nucleotides that bound Rev at a 1:1 ratio, whereas the 244-nucleotide full-length RRE bound four Rev monomers. At high Rev concentrations, additional binding of Rev to RRE was observed with ratios of more than 10:1. Because RRE mutants that lacked the core binding site and were inactive in vivo bound Rev nonspecifically at these concentrations, the real stoichiometric ratio of Rev-RRE is probably closer to 4:1. Binding affinity of Rev for RRE was approximately 10 ؊10 M, whereas the affinity for the core RNA was about 10 ؊11 M, the difference being due to the contribution of low affinity binding sites on the RRE. Mathematical analysis suggested cooperativity of Rev binding, probably mediated by the Rev oligomerization domains. C-terminal deletions of Rev had no effect on RRE binding, but truncation of the N terminus by as few as 11 residues significantly reduced binding specificity. This method was also useful to rapidly evaluate the potential of aminoglycoside antibiotics, to inhibit the Rev-RRE interaction.

Research paper thumbnail of Reduced Cell Surface Expression of CCR5 in CCR5Δ32 Heterozygotes Is Mediated by Gene Dosage, Rather Than by Receptor Sequestration

Journal of Biological Chemistry, 2001

Research paper thumbnail of Structure and Replication of Vaccinia Virus Telomeres

Cold Spring Harbor Symposia on Quantitative Biology, 1983

Page 1. Structure and Replication of Vaccinia Virus Telomeres BM BAROUDY, S. VENKATESAN, AND B. M... more Page 1. Structure and Replication of Vaccinia Virus Telomeres BM BAROUDY, S. VENKATESAN, AND B. MOSS Laboratory of Biology of Viruses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20205 ...

Research paper thumbnail of Incompletely base-paired flip-flop terminal loops link the two DNA strands of the vaccinia virus genome into one uninterrupted polynucleotide chain

Cell, 1982

The nature of the ends of the vaccinia virus genome was determined by nucleotide sequencing. Our ... more The nature of the ends of the vaccinia virus genome was determined by nucleotide sequencing. Our finding of terminal hairpins indicated that the linear double-stranded DNA molecule consists of a single continuous polynucleotide chain. The 104 nucleo tide apex of the hairpin contains predominantly A and T residues and is incompletely base-paired. These loops exist in two forms, which when inverted with respect to each other are complementary in sequence. Both forms of the 104 nucleotide loop are present in nearly equimolar amounts at each end of the genome. A set of 13 tandem 70 bp repeats begins 87 bp from the proximal segment of the terminal loop, followed by a unique sequence of 325 bp, and then by a second set of 18 tandem 70 bp repeats. The sequence of the 70 bp repeats reveals a 13 bp internal redundancy. Self-priming and de novo start replication models, which involve a site-specific nick in one DNA strand proximal to the 104 nucleotlde loop, account for the observed sequence inversions and incomplete base-pairing. Similar mechanisms may be involved in replication of the ends of the eucaryotic chromosome.

Research paper thumbnail of Nucleotide Sequence oftheGeneEncoding Respiratory Syncytial VirusMatrix Protein

Theaminoacid sequenceofthematrix protein ofthehumanrespiratory syncytial virus (RSvirus) was dedu... more Theaminoacid sequenceofthematrix protein ofthehumanrespiratory syncytial virus (RSvirus) was deduced fromthesequenceofacDNAinsert inarecombinant plasmid harboring analmost full-length copy ofthis gene.Itspecifically hybridized toa single 1,050-base mRNA frominfected cells. Therecombinant containing 944basepairs ofRSviral matrix protein gene sequencelacked five nucleotides corresponding to the5'endofthemRNA. Thenucleotide sequenceofthe5'endofthemRNA was determined bythe dideoxy sequencing methodandfound tobe5'NGGGC,wherein theCresidue isone nucleotide upstream ofthecloned viral sequence.Theinitiator ATG codonforthematrix protein isembeddedinanAATATGG sequencesimilar tothecanonical PXXATGG sequencepresent aroundfunctional eucaryotic translation initiation codons. Thereisno conserved sequenceupstreamofthepolyadenylate tail, unlike vesicular stomatitis virusandSendaivirus, inwhichfournucleotides upstreamofthepolyadenylate tail are conserved inall genes.Thereisno equivalent o...

Research paper thumbnail of Modification of the 5' end of mRNA. Association of RNA triphosphatase with the RNA guanylyltransferase-RNA (guanine-7-)methyltransferase complex from vaccinia virus

Journal of Biological Chemistry, 1980

Modification of the 5' End of mRNA ASSOCIATION OF RNA TRIPHOSPHATASE WITH THE RNA GUANYLYLTRANSFE... more Modification of the 5' End of mRNA ASSOCIATION OF RNA TRIPHOSPHATASE WITH THE RNA GUANYLYLTRANSFERASE. RNA (GUANINE-7-)METHYLTRANSFERASE COMPLEX FROM VACCINIA VIRUS*

Research paper thumbnail of A specific sequence with a bulged guanosine residue(s) in a stem-bulge-stem structure of Rev-responsive element RNA is required for trans activation by human immunodeficiency virus type 1 Rev

Journal of Virology, 1992

We demonstrate that both the in vitro RNA binding and in vivo trans activation functions of human... more We demonstrate that both the in vitro RNA binding and in vivo trans activation functions of human immunodeficiency virus type 1 Rev regulatory protein Rev require the presence of a 9-nucleotide 5'-CACUAUGGG-3' RNA motif on its cognate target, the Rev-responsive element RNA. For optimal Rev recognition, this sequence must be presented as a stem-bulge-stem structure and must contain at least two G's, one of which must be unpaired, and include some or all of the CACUAU sequence upstream of the three G's. Distal mutations which result in the base pairing of the G's eliminate the Rev response. The first G is crucial, but changes at the other G's are tolerated if at least one G is unpaired. The secondary structure or the three-dimensional orientation of the B1 and B2 stem-loops of the Rev-responsive element are not relevant as long as the 5'-CACUAUGGG-3' sequence is preserved, with at least one bulged G residue.

Research paper thumbnail of Proteins associated with human parainfluenza virus type 3

Journal of Virology, 1985

The polypeptides associated with human parainfluenza virus type 3 were identified. Five proteins ... more The polypeptides associated with human parainfluenza virus type 3 were identified. Five proteins were present in detergent- and salt-resistant viral cores. Of these, three proteins designated NP0, NP1, and NP2 of 68,000, 58,000, and 52,000 daltons, respectively, were stably associated with 50S RNA in CsCl gradient-purified nucleocapsids. The amounts of NP1 and NP2 were variable, and these proteins were shown to be structurally related to the major nucleocapsid protein (NP0) by partial Staphylococcus aureus V8 protease mapping. The other core proteins included a 240K protein designated L (candidate for the viral polymerase) and an 84K protein designated as the phosphoprotein (P) on the basis of a predominant incorporation of Pi. The viral envelope had four prominent proteins (72, 53, 40, and 12K) under reducing conditions of electrophoresis. The 72 and 53K proteins were specifically labeled with [3H]glucosamine and [3H]mannose. When sulfhydryl reagents were removed, a new 62K protein...

Research paper thumbnail of Molecular Functional Studies of HIV-1 REV and NEF Proteins

Advances in Molecular Biology and Targeted Treatment for AIDS, 1991

Human immunodeficiency virus type 1 (HIV-1), the etiological agent of AIDS, preferentially infect... more Human immunodeficiency virus type 1 (HIV-1), the etiological agent of AIDS, preferentially infects the CD4+ helper subset of human T lymphocytes. Clinically, HIV-1 infection is characterized by a chronic phase lasting several years with a paucity of infected lymphocytes in the circulation. Notwithstanding, HIV-1 infection of primary human T lymphocytes or CD4+ cell lines in vitro leads to massive acute infection and cell death (20). This dichotomy between the natural history of virus infection and its behavior in tissue culture implies the existence of viral and cellular determinants of viral latency and reactivation.

Research paper thumbnail of Human immunodeficiency virus type 1 (HIV-1) provirus expression and LTR transcription are repressed in NEF-expressing cell lines

Virology, 1991

Human immunodeficiency virus type 1 (HIV-l) NEF protein has been demonstrated to be a negative re... more Human immunodeficiency virus type 1 (HIV-l) NEF protein has been demonstrated to be a negative regulator of HIV-1 replication and HIV-1 LTR transcription under transient expression conditions. The difficulty of several laboratories to reproduce these findings led us to reexamine the role of NEF in HIV-1 provirus expression and HIV-1 LTR transcription. Basal transcription from the HIV-l LTR in the presence of a NEF expression vector was compared to that in the presence of a mutated NEF vector. NEF expression led to a greater than lo-fold repression of LTR transcription under these conditions. HeLa and Jurkat cell lines carrying the nef gene linked to the CMV promoter or the HIV-l LTR were isolated by coselection for neomycin resistance. Single cell isolates were further selected for the expression of nef transcripts. With the exception of the anti-sense nef cell lines, all the nef cell lines expressed the 27-kDa NEF protein, detectable by immunoprecipitation. NEF+ HeLa cell lines were at least 5-fold less efficient than NEF-HeLa cell lines in transient proviral expression. Provirus expression was also repressed in the NEF+ Jurkat cell lines. TAT-activated LTR transcription from an HIV-1 LTR-linked CAT expression vector was repressed 1 O-fold in the NEF+ HeLa and NEF+ Jurkat cell lines. When infected with HIV-l, NEF expressing T lymphoid cell lines showed moderate delays in onset and peak of reverse transcriptase production. However, none of these cell lines completely arrested virus replication. Our data confirm a negative regulatory effect of NEF on both virus production and LTR driven CAT expression in the cell lines tested. It is possible that cell specific factors may influence NEF activity.

Research paper thumbnail of Biochemical and biological comparison of HIV-1 NEF and ras gene products

Virology, 1991

Human immunodeficiency virus type 1 (HIV-l) NEf protein has been reported to share certain bioche... more Human immunodeficiency virus type 1 (HIV-l) NEf protein has been reported to share certain biochemical and structural properties with known oncoproteins like src or ras. To determine whether this is a general property of NEF from various HIV isolates, three different NEF proteins were expressed in Escherichia co/i using a thermoinducible expression system previously exploited to overproduce functionally active p21 ras proteins. ras and NEF proteins expressed in this manner were evaluated in parallel to compare their biochemical and biological properties. In contrast to ras, our NEF protein preparations had no detectable GTP binding but showed autophosphorylation activity when incubated in the presence of either GTP or ATP. This putative autokinase activity was higher in NEF proteins containing threonine at position 15 than in those carrying alanine at that position. Two different NEF genes also failed to induce oncogenic transformation of permanently transfected NIH 3T3 cells under conditions that led to oncogenic transformation using activated ras genes. Also, unlike ras, the NEf gene products failed to induce meiotic maturation when injected into fully grown Xenopus OOCytSS.

Research paper thumbnail of Recognition of dileucine-based sorting signals from HIV-1 Nef and LIMP-II by the AP-1 γ–σ1 and AP-3 δ–σ3 hemicomplexes

Journal of Cell Biology, 2003

The sorting of transmembrane proteins to endosomes and lysosomes is mediated by signals present i... more The sorting of transmembrane proteins to endosomes and lysosomes is mediated by signals present in the cytosolic tails of the proteins. A subset of these signals conform to the [DE]XXXL[LI] consensus motif and mediate sorting via interactions with heterotetrameric adaptor protein (AP) complexes. However, the identity of the AP subunits that recognize these signals remains controversial. We have used a yeast three-hybrid assay to demonstrate that [DE]XXXL[LI]-type signals from the human immunodeficiency virus negative factor protein and the lysosomal integral membrane protein II interact with combinations of the γ and σ1 subunits of AP-1 and the δ and σ3 subunits of AP-3, but not the analogous combinations of AP-2 and AP-4 subunits. The sequence requirements for these interactions are similar to those for binding to the whole AP complexes in vitro and for function of the signals in vivo. These observations reveal a novel mode of recognition of sorting signals involving the γ/δ and σ ...

Research paper thumbnail of TAR RNA-binding protein is an inhibitor of the interferon-induced protein kinase PKR

Proceedings of the National Academy of Sciences, 1994

A cDNA encoding a double-stranded-RNA (dsRNA)-binding protein was isolated by screening a HeLa ce... more A cDNA encoding a double-stranded-RNA (dsRNA)-binding protein was isolated by screening a HeLa cell cDNA expression library for proteins that bind the HIV-1 Rev-responsive-element RNA. The cDNA encoded a protein that was identical to TRBP, the previously reported cellular protein that binds the transactivation response element (TAR) RNA of human immunodeficiency virus type 1. TRBP inhibited phosphorylation of the interferon-induced ribosome-associated protein kinase PKR and of the eukaryotic translation initiation factor eIF-2 alpha in a transient-expression system in which the translation of a reporter gene was inhibited by the localized activation of PKR. TRBP expression in HeLa cells complemented the growth and protein-synthesis defect of a vaccinia virus mutant lacking the expression of the dsRNA-binding protein E3L. These results implicate TRBP as a cellular regulatory protein that binds RNAs containing specific secondary structure(s) to mediate the inhibition of PKR activation...

Research paper thumbnail of Rev-induced modulation of Nef protein underlies temporal regulation of human immunodeficiency virus replication

Proceedings of the National Academy of Sciences, 1989

The replication of human immunodeficiency virus type 1 (HIV-1) requires the concerted action of t... more The replication of human immunodeficiency virus type 1 (HIV-1) requires the concerted action of two virus-encoded transactivator proteins, Tat and Rev, and is in turn moderated by the viral transcriptional repressor Nef. We show here that the phenotype of a Rev- HIV-1 provirus was nonreplicating and was distinguished by accumulation of Nef protein and reduced Tat function. Provirus defective in both the rev and nef genes (Rev-Nef-) was also nonreplicating but had normal Tat function. Trans-complementation of the Rev- mutant with Rev caused a decrease of both the steady-state level and the rate of synthesis of Nef. This was accompanied by enhanced synthesis of viral structural proteins. Rev induced even greater levels of virus production from the Rev-Nef- double mutant. In contrast, exogenous Rev did not augment virus production from wild-type provirus. Virus production from Rev- and Rev-Nef- mutants induced by Rev was repressed by exogenous Nef. The repression induced by Nef could n...

Research paper thumbnail of Eukaryotic mRNA capping enzyme-guanylate covalent intermediate

Proceedings of the National Academy of Sciences, 1982

Incubation of HeLa cell mRNA guanylyltransferase (GTP:mRNA guanylyltransferase, EC 2.7.7.50) with... more Incubation of HeLa cell mRNA guanylyltransferase (GTP:mRNA guanylyltransferase, EC 2.7.7.50) with [alpha-32P]GTP and a divalent cation in the absence of an RNA acceptor results in the formation of a covalent enzyme-guanylate complex. The complex, after purification by phosphocellulose chromatography, can transfer its bound GMP moiety to pyrophosphate, regenerating GTP, or to the 5'-diphosphate end of poly(A), forming a cap structure G(5')pppA(pA)n. The GMP-polypeptide has a molecular weight of 65,000 and is stable to heating in the presence of sodium dodecyl sulfate. On the basis of the alkali-stable and acid-labile nature of the bond and its susceptibility to nucleophilic attack by hydroxylamine at low pH, the GMP-polypeptide linkage appears to be a phosphoamine bond. After digestion with trypsin, a single GMP-peptide was resolved by two dimensional electrophoresis and chromatography.

Research paper thumbnail of Resistance to human respiratory syncytial virus (RSV) infection induced by immunization of cotton rats with a recombinant vaccinia virus expressing the RSV G glycoprotein

Proceedings of the National Academy of Sciences, 1986

A cDNA copy of the G glycoprotein gene of human respiratory syncytial virus (RSV) was placed unde... more A cDNA copy of the G glycoprotein gene of human respiratory syncytial virus (RSV) was placed under control of a vaccinia virus promoter and inserted into the thymidine kinase locus of the vaccinia virus genome. The recombinant vaccinia virus retained infectivity and expressed a 93-kDa protein that migrated with the authentic RSV G glycoprotein upon polyacrylamide gel electrophoresis. Glycosylation of the expressed protein and transport to the cell surface were demonstrated in the absence of other RSV proteins. Cotton rats that were inoculated intradermally with the infectious recombinant virus produced serum antibody to the G glycoprotein that neutralized RSV in vitro. Furthermore, the vaccinated animals were resistant to lower respiratory tract infection upon intranasal inoculation with RSV and had reduced titers of RSV in the nose.

Research paper thumbnail of Preparation and properties of a poly A resin and its use in the isolation of naturally occurring poly U sequences

Nucleic Acids Research, 1976

A method for the covalent attachment of poly A, as well as other nucleic acids and nucleosides, t... more A method for the covalent attachment of poly A, as well as other nucleic acids and nucleosides, to a methylene dianiline derivative of starch is described. The properties of this poly A resin and its use for the recovery of poly U sequences from both nuclear and cytoplasmic extracts of HeLa cells is described.

Research paper thumbnail of Amino acid sequence of human respiratory syncytial virus nucleocapsid protein

Nucleic Acids Research, 1983

Amino add sequence of the human respiratory syncytial (RS) virus nudeocapsid (NC) protein, deduce... more Amino add sequence of the human respiratory syncytial (RS) virus nudeocapsid (NC) protein, deduced from the DNA sequence of a recombinant plasmid, is presented. The cDNA plasmid (pRSBll) has 1412 bp of RS viral NC sequence and lacks six nucleotides of the 5'end of mRNA. There is a single long open reading frame encoding 467 amino acids. This 51540 dal protein is rich in basic amino acids and has no homologies with other known viral capsid proteins.

Research paper thumbnail of Respiratory syncytial virus envelope glycoprotein (G) has a novel structure

Nucleic Acids Research, 1985

ASTno acid sequence of human respiratory syncytiai virus envelope glycoprotein (G) was deduced fr... more ASTno acid sequence of human respiratory syncytiai virus envelope glycoprotein (G) was deduced from the DNA sequence of a recombinant plasmid and confirmed by limited amino add microsequendng of purified 90K G protein. The calculated molecular mass of the protein encoded by the only long open reading frame of 298 amino acids was 32,588 daltons and was somewhat smaller than the 36K polypeptide translated J[n vitro from mRNA selected by this plasmid. Inspection of the sequence revealed a single hydrophobic domain of 23 amino acids capable of membrane Insertion at 41 residues from the N-term1nus. There was no N-terminal signal sequence and the hydrophHic N-term1nal 20 residues probably represent the cytoplasmic tall of the protein. The N-terminally oriented membrane Insertion was somewhat analogous to paramyxovims hemagglutinin-neuraminidase (HN) and Influenza neuraminidase (NA). The protein was moderately hydrophilic and rich 1n hydroxy-amino adds. It was both N-and O-glycosylated with the latter contributing significantly to the net molecular mass 90K.

Research paper thumbnail of Cholesterol is obligatory for polarization and chemotaxis but not for endocytosis and associated signaling from chemoattractant receptors in human neutrophils

Journal of Biomedical Science, 2008

Plasma membrane cholesterol is critical for neutrophil chemotaxis, although how cholesterol affec... more Plasma membrane cholesterol is critical for neutrophil chemotaxis, although how cholesterol affects chemotactic signaling pathway has not been clearly delineated. Here we demonstrate that cholesterol was absolutely required for polarized redistribution of key chemotactic mediators in human neutrophils in response to all chemoattractants tested (fMet-Leu-Phe, and the chemokines CXCL1, CXCL8 and CXCL12). In particular, PI3K and phosphatidylinositol-3,4,5 triphosphate (PIP 3) failed to accumulate at the front and phosphatase and tensin homolog (PTEN) at the back of chemoattractant-stimulated neutrophils after cholesterol depletion. Cholesterol depletion did not affect early chemoattractant signaling events such as G-protein activation, intracellular calcium flux or G-protein-independent endocytosis-linked signaling, including the activation of mitogen-activated protein kinase (MAPK), Hck and Fgr transduced by b-arrestin. During cell polarization, F-actin assemblies redistributed the cholesterol-rich microdomains and cytoskeletonanchored proteins, including CD16 and CD44 from the leading edge. These data suggest that spatial polarization of chemotactic mediators is orchestrated by protein:protein interactions that organize cholesterol-rich domains of the plasma membrane. Keywords Neutrophils Á Chemotaxis Á Cholesterol Á Lipid rafts Á Chemoattractants Á G-proteins Á Endocytosis Á Arrestin Á Degranulation Á Protein kinases Á F-actin Abbreviations APC Allophycocyanin DRMs Detergent-resistant membranes 123

Research paper thumbnail of Real-time Kinetics of HIV-1 Rev-Rev Response Element Interactions

Journal of Biological Chemistry, 1999

The kinetics of interaction between the human immunodeficiency virus-1 Rev protein and its RNA ta... more The kinetics of interaction between the human immunodeficiency virus-1 Rev protein and its RNA target, Rev response element (RRE) RNA was determined in vitro using a biosensor technique. Our results showed that the primary Rev binding site is a core stem-loop RNA molecule of 30 nucleotides that bound Rev at a 1:1 ratio, whereas the 244-nucleotide full-length RRE bound four Rev monomers. At high Rev concentrations, additional binding of Rev to RRE was observed with ratios of more than 10:1. Because RRE mutants that lacked the core binding site and were inactive in vivo bound Rev nonspecifically at these concentrations, the real stoichiometric ratio of Rev-RRE is probably closer to 4:1. Binding affinity of Rev for RRE was approximately 10 ؊10 M, whereas the affinity for the core RNA was about 10 ؊11 M, the difference being due to the contribution of low affinity binding sites on the RRE. Mathematical analysis suggested cooperativity of Rev binding, probably mediated by the Rev oligomerization domains. C-terminal deletions of Rev had no effect on RRE binding, but truncation of the N terminus by as few as 11 residues significantly reduced binding specificity. This method was also useful to rapidly evaluate the potential of aminoglycoside antibiotics, to inhibit the Rev-RRE interaction.

Research paper thumbnail of Reduced Cell Surface Expression of CCR5 in CCR5Δ32 Heterozygotes Is Mediated by Gene Dosage, Rather Than by Receptor Sequestration

Journal of Biological Chemistry, 2001

Research paper thumbnail of Structure and Replication of Vaccinia Virus Telomeres

Cold Spring Harbor Symposia on Quantitative Biology, 1983

Page 1. Structure and Replication of Vaccinia Virus Telomeres BM BAROUDY, S. VENKATESAN, AND B. M... more Page 1. Structure and Replication of Vaccinia Virus Telomeres BM BAROUDY, S. VENKATESAN, AND B. MOSS Laboratory of Biology of Viruses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20205 ...

Research paper thumbnail of Incompletely base-paired flip-flop terminal loops link the two DNA strands of the vaccinia virus genome into one uninterrupted polynucleotide chain

Cell, 1982

The nature of the ends of the vaccinia virus genome was determined by nucleotide sequencing. Our ... more The nature of the ends of the vaccinia virus genome was determined by nucleotide sequencing. Our finding of terminal hairpins indicated that the linear double-stranded DNA molecule consists of a single continuous polynucleotide chain. The 104 nucleo tide apex of the hairpin contains predominantly A and T residues and is incompletely base-paired. These loops exist in two forms, which when inverted with respect to each other are complementary in sequence. Both forms of the 104 nucleotide loop are present in nearly equimolar amounts at each end of the genome. A set of 13 tandem 70 bp repeats begins 87 bp from the proximal segment of the terminal loop, followed by a unique sequence of 325 bp, and then by a second set of 18 tandem 70 bp repeats. The sequence of the 70 bp repeats reveals a 13 bp internal redundancy. Self-priming and de novo start replication models, which involve a site-specific nick in one DNA strand proximal to the 104 nucleotlde loop, account for the observed sequence inversions and incomplete base-pairing. Similar mechanisms may be involved in replication of the ends of the eucaryotic chromosome.