29 research outputs found
Nuclear Retention of Multiply Spliced HIV-1 RNA in Resting CD4(+) T Cells
HIV-1 latency in resting CD4(+) T cells represents a major barrier to virus eradication in patients on highly active antiretroviral therapy (HAART). We describe here a novel post-transcriptional block in HIV-1 gene expression in resting CD4(+) T cells from patients on HAART. This block involves the aberrant localization of multiply spliced (MS) HIV-1 RNAs encoding the critical positive regulators Tat and Rev. Although these RNAs had no previously described export defect, we show that they exhibit strict nuclear localization in resting CD4(+) T cells from patients on HAART. Overexpression of the transcriptional activator Tat from non-HIV vectors allowed virus production in these cells. Thus, the nuclear retention of MS HIV-1 RNA interrupts a positive feedback loop and contributes to the non-productive nature of infection of resting CD4(+) T cells. To define the mechanism of nuclear retention, proteomic analysis was used to identify proteins that bind MS HIV-1 RNA. Polypyrimidine tract binding protein (PTB) was identified as an HIV-1 RNA-binding protein differentially expressed in resting and activated CD4(+) T cells. Overexpression of PTB in resting CD4(+) T cells from patients on HAART allowed cytoplasmic accumulation of HIV-1 RNAs. PTB overexpression also induced virus production by resting CD4(+) T cells. Virus culture experiments showed that overexpression of PTB in resting CD4(+) T cells from patients on HAART allowed release of replication-competent virus, while preserving a resting cellular phenotype. Whether through effects on RNA export or another mechanism, the ability of PTB to reverse latency without inducing cellular activation is a result with therapeutic implications
NlpC/P60 domain-containing proteins of Mycobacterium avium subspecies paratuberculosis that differentially bind and hydrolyze peptidoglycan
A subset of proteins containing NlpC/P60 domains are bacterial peptidoglycan hydrolases that cleave noncanonical peptide linkages and contribute to cell wall remodeling as well as cell separation during late stages of division. Some of these proteins have been shown to cleave peptidoglycan in Mycobacterium tuberculosis and play a role in Mycobacterium marinum virulence of zebra fish; however, there are still significant knowledge gaps concerning the molecular function of these proteins in Mycobacterium avium subspecies paratuberculosis (MAP). The MAP genome sequence encodes five NlpC/P60 domain-containing proteins. We describe atomic resolution crystal structures of two such MAP proteins, MAP_1272c and MAP_1204. These crystal structures, combined with functional assays to measure peptidoglycan cleavage activity, led to the observation that MAP_1272c does not have a functional catalytic core for peptidoglycan hydrolysis. Furthermore, the structure and sequence of MAP_1272c demonstrate that the catalytic residues normally required for hydrolysis are absent, and the protein does not bind peptidoglycan as efficiently as MAP_1204. While the NlpC/P60 catalytic triad is present in MAP_1204, changing the catalytic cysteine-155 residue to a serine significantly diminished catalytic activity, but did not affect binding to peptidoglycan. Collectively, these findings suggest a broader functional repertoire for NlpC/P60 domain-containing proteins than simply hydrolases
MAP1272c Encodes an NlpC/P60 Protein, an Antigen Detected in Cattle with Johne’s Disease
The protein encoded by MAP1272c has been shown to be an antigen of Mycobacterium avium subsp. paratuberculosis that con- tains an NlpC/P60 superfamily domain found in lipoproteins or integral membrane proteins. Proteins containing this domain have diverse enzymatic functions that include peptidases, amidases, and acetyltransferases. The NlpC protein was examined in comparison to over 100 recombinant proteins and showed the strongest antigenicity when analyzed with sera from cattle with Johne’s disease. To further localize the immunogenicity of NlpC, recombinant proteins representing defined regions were ex- pressed and evaluated with sera from cattle with Johne’s disease. The region from amino acids 74 to 279 was shown to be the most immunogenic. This fragment was also evaluated against a commercially available enzyme-linked immunosorbent assay (ELISA). Two monoclonal antibodies were produced in mice immunized with the full-length protein, and each recognized a dis- tinct epitope. These antibodies cross-reacted with proteins from other mycobacterial species and demonstrated variable sizes of the proteins expressed from these subspecies. Both antibodies were further analyzed, and their interaction with MAP1272c and MAP1204 was characterized by a solution-based, luminescent binding assay. These tools provide additional means to study a strong antigen of M. avium subsp. paratuberculosis
MAP1272c Encodes an NlpC/P60 Protein, an Antigen Detected in Cattle with Johne’s Disease
The protein encoded by MAP1272c has been shown to be an antigen of Mycobacterium avium subsp. paratuberculosis that con- tains an NlpC/P60 superfamily domain found in lipoproteins or integral membrane proteins. Proteins containing this domain have diverse enzymatic functions that include peptidases, amidases, and acetyltransferases. The NlpC protein was examined in comparison to over 100 recombinant proteins and showed the strongest antigenicity when analyzed with sera from cattle with Johne’s disease. To further localize the immunogenicity of NlpC, recombinant proteins representing defined regions were ex- pressed and evaluated with sera from cattle with Johne’s disease. The region from amino acids 74 to 279 was shown to be the most immunogenic. This fragment was also evaluated against a commercially available enzyme-linked immunosorbent assay (ELISA). Two monoclonal antibodies were produced in mice immunized with the full-length protein, and each recognized a dis- tinct epitope. These antibodies cross-reacted with proteins from other mycobacterial species and demonstrated variable sizes of the proteins expressed from these subspecies. Both antibodies were further analyzed, and their interaction with MAP1272c and MAP1204 was characterized by a solution-based, luminescent binding assay. These tools provide additional means to study a strong antigen of M. avium subsp. paratuberculosis
NlpC/P60 domain-containing proteins of Mycobacterium avium subspecies paratuberculosis that differentially bind and hydrolyze peptidoglycan
A subset of proteins containing NlpC/P60 domains are bacterial peptidoglycan hydrolases that cleave noncanonical peptide linkages and contribute to cell wall remodeling as well as cell separation during late stages of division. Some of these proteins have been shown to cleave peptidoglycan in Mycobacterium tuberculosis and play a role in Mycobacterium marinum virulence of zebra fish; however, there are still significant knowledge gaps concerning the molecular function of these proteins in Mycobacterium avium subspecies paratuberculosis (MAP). The MAP genome sequence encodes five NlpC/P60 domain-containing proteins. We describe atomic resolution crystal structures of two such MAP proteins, MAP_1272c and MAP_1204. These crystal structures, combined with functional assays to measure peptidoglycan cleavage activity, led to the observation that MAP_1272c does not have a functional catalytic core for peptidoglycan hydrolysis. Furthermore, the structure and sequence of MAP_1272c demonstrate that the catalytic residues normally required for hydrolysis are absent, and the protein does not bind peptidoglycan as efficiently as MAP_1204. While the NlpC/P60 catalytic triad is present in MAP_1204, changing the catalytic cysteine-155 residue to a serine significantly diminished catalytic activity, but did not affect binding to peptidoglycan. Collectively, these findings suggest a broader functional repertoire for NlpC/P60 domain-containing proteins than simply hydrolases
MAP1272c Encodes an NlpC/P60 Protein, an Antigen Detected in Cattle with Johne’s Disease
The protein encoded by MAP1272c has been shown to be an antigen of Mycobacterium avium subsp. paratuberculosis that con- tains an NlpC/P60 superfamily domain found in lipoproteins or integral membrane proteins. Proteins containing this domain have diverse enzymatic functions that include peptidases, amidases, and acetyltransferases. The NlpC protein was examined in comparison to over 100 recombinant proteins and showed the strongest antigenicity when analyzed with sera from cattle with Johne’s disease. To further localize the immunogenicity of NlpC, recombinant proteins representing defined regions were ex- pressed and evaluated with sera from cattle with Johne’s disease. The region from amino acids 74 to 279 was shown to be the most immunogenic. This fragment was also evaluated against a commercially available enzyme-linked immunosorbent assay (ELISA). Two monoclonal antibodies were produced in mice immunized with the full-length protein, and each recognized a dis- tinct epitope. These antibodies cross-reacted with proteins from other mycobacterial species and demonstrated variable sizes of the proteins expressed from these subspecies. Both antibodies were further analyzed, and their interaction with MAP1272c and MAP1204 was characterized by a solution-based, luminescent binding assay. These tools provide additional means to study a strong antigen of M. avium subsp. paratuberculosis
Induction of Virus Production by PTB or Tat
<p>High-level expression of PTB or Tat in resting CD4<sup>+</sup> T cells from patients on HAART induces virus production that is comparable to that seen following PHA stimulation. Resting CD4<sup>+</sup> T cells purified from patients on HAART were transfected as described above with expression vectors for PTB or Tat, or as a positive control, were stimulated with PHA. For all transfections, supernatant HIV-1 levels were measured at 72 h post-stimulation. For PHA-stimulated cells, supernatant levels of HIV-1 RNA were measured every day for a 6-d period. Levels shown reflect peak virus release over the 6-d period.</p
PTB Binds Specifically to MS HIV-1 RNA and Is Upregulated upon Cellular Stimulation
<div><p>(A) PTB binds specifically to MS HIV-1 RNA. Recombinant PTB was pre-incubated with increasing molar equivalents (0X, 1X, 2X, 5X) of non-biotinylated MS HIV-1 RNA (relative to biotinylated MS HIV-1 RNA) or a 5-fold mass excess of non-specific (NS) competitor, total yeast RNA; and then incubated with biotinylated, in vitro-transcribed MS HIV-1 RNA bound to streptavidin-conjugated magnetic particles. PTB protein eluted by RNase digestion was analyzed by Western blot.</p><p>(B) PTB expression increases in response to T-cell stimulation. Expression of PTB was measured by Western blotting in resting CD4<sup>+</sup> T cells cultured for 72 h without activating stimuli (−) or with (+) anti-CD3/anti-CD28. Vinculin served as a loading control.</p></div