18 research outputs found
Proteolytic processing of the nonstructural proteins of dengue 2 virus
The genomes of many positive stranded RNA viruses and of all
retroviruses are translated as large polyproteins which are proteolytically
processed by cellular and viral proteases. Viral proteases are structurally
related to two families of cellular proteases, the pepsin-like and trypsin-like
proteases. This thesis describes the proteolytic processing of several
nonstructural proteins of dengue 2 virus, a representative member of the
Flaviviridae, and describes methods for transcribing full-length genomic
RNA of dengue 2 virus. Chapter 1 describes the in vitro processing of the
nonstructural proteins NS2A, NS2B and NS3. Chapter 2 describes a system
that allows identification of residues within the protease that are directly or
indirectly involved with substrate recognition. Chapter 3 describes
methods to produce genome length dengue 2 RNA from cDNA templates.
The nonstructural protein NS3 is structurally related to viral trypsinlike
proteases from the alpha-, picorna-, poty-, and pestiviruses. The
hypothesis that the flavivirus nonstructural protein NS3 is a viral
proteinase that generates the termini of several nonstructural proteins was
tested using an efficient in vitro expression system and antisera specific for
the nonstructural proteins NS2B and NS3. A series of cDNA constructs
was transcribed using T7 RNA polymerase and the RNA translated in
reticulocyte lysates. Proteolytic processing occurred in vitro to generate
NS2B and NS3. The amino termini of NS2B and NS3 produced in vitro
were found to be the same as the termini of NS2B and NS3 isolated from
infected cells. Deletion analysis of cDNA constructs localized the protease
domain necessary and sufficient for correct cleavage to the first 184 amino
acids of NS3. Kinetic analysis of processing events in vitro and experiments
to examine the sensitivity of processing to dilution suggested that an
intramolecular cleavage between NS2A and NS2B preceded an
intramolecular cleavage between NS2B and NS3. The data from these
expression experiments confirm that NS3 is the viral proteinase
responsible for cleavage events generating the amino termini of NS2B and
NS3 and presumably for cleavages generating the termini of NS4A and NS5
as well.
Biochemical and genetic experiments using viral proteinases have
defined the sequence requirements for cleavage site recognition, but have
not identified residues within proteinases that interact with substrates. A
biochemical assay was developed that could identify residues which were
important for substrate recognition. Chimeric proteases between yellow
fever and dengue 2 were constructed that allowed mapping of regions
involved in substrate recognition, and site directed mutagenesis was used
to modulate processing efficiency.
Expression in vitro revealed that the dengue protease domain
efficiently processes the yellow fever polyprotein between NS2A and NS2B
and between NS2B and NS3, but that the reciprocal construct is inactive.
The dengue protease processes yellow fever cleavage sites more efficiently
than dengue cleavage sites, suggesting that suboptimal cleavage efficiency
may be used to increase levels of processing intermediates in vivo. By
mutagenizing the putative substrate binding pocket it was possible to
change the substrate specificity of the yellow fever protease; changing a
minimum of three amino acids in the yellow fever protease enabled it to
recognize dengue cleavage sites. This system allows identification of
residues which are directly or indirectly involved with enzyme-substrate
interaction, does not require a crystal structure, and can define the
substrate preferences of individual members of a viral proteinase family.
Full-length cDNA clones, from which infectious RNA can be
transcribed, have been developed for a number of positive strand RNA
viruses, including the flavivirus type virus, yellow fever. The technology
necessary to transcribe genomic RNA of dengue 2 virus was developed in
order to better understand the molecular biology of the dengue subgroup. A
5' structural region clone was engineered to transcribe authentic dengue
RNA that contains an additional 1 or 2 residues at the 5' end. A 3'
nonstructural region clone was engineered to allow production of run off
transcripts, and to allow directional ligation with the 5' structural region
clone. In vitro ligation and transcription produces full-length genomic
RNA which is noninfectious when transfected into mammalian tissue
culture cells. Alternative methods for constructing cDNA clones and
recovering live dengue virus are discussed.</p
Processing of nonstructural proteins NS4A and NS4B of dengue 2 virus in vitro and in vivo
The production, from polyprotein precursors, of two hydrophobic nonstructural proteins of dengue 2 (DEN2) virus, NS4A and NS4B, was analyzed both in cell-free systems and in infected cells. In DEN2-infected cells, NS4B is first produced as a peptide of apparent size 30 kDa; NS4B is then post-translationally modified, in an unknown way, to produce a polypeptide of apparent size 28 kDa. The rate and extent of NS4B modification was found to be cell-dependent; in BHK cells the half-time for the conversion of the 30-kDa form to the 28-kDa form was 90 min. N-terminal sequence analysis of NS4B suggests that the N-terminus is produced by an enzyme with a specificity similar to that of signalase. Low levels of a putative polyprotein, NS4AB, were also found in mammalian cells, but not mosquito cells, infected with DEN2, suggesting that a small proportion of DEN2 4A/4B cleavage can occur post-translationally or that some nonstructural polyproteins escape normal processing. Cleavage of the 4A/4B bond in infected cells required expression of DEN2 sequences in addition to those in NS4A and NS4B, as NS4AB produced in cells by a vaccinia expression system was not cleaved. NS4AB produced in cells by a vaccinia expression system was modified post-translationally, presumably in the same way as NS4B. We show that upon translation of DEN2 polyproteins in a cell-free system, the N-terminus of NS4A is generated by cleavage by the viral nonstructural proteinase NS3 and that processing of DEN2 polyproteins occurs with a preferred, but nonobligatory order
Dengue 2 Virus NS2B and NS3 Form a Stable Complex That Can Cleave NS3 within the Helicase Domain
Flavivirus genomic RNA is translated into a large polyprotein that is proceSsed into structural and nonstructural proteins. The N-termini of several nonstructural proteins are produced by cleavage at dibasic sites by a two-component viral proteinase consisting of NS2B and NS3. NS3 contains a trypsin-like serine proteinase domain at its N-terminus, whereas the function of NS2B in proteolysis is yet to be determined. We have used an NS3-specific antiserum, under nondenaturing conditions, to demonstrate that NS2B and NS3 form a complex both in vitro and in vivo. The N-terminal 184 residues of NS3 are sufficient to form the complex with NS28. The complex forms efficiently when the NS2B and NS3 are translated from two different 1nRNAs as well as when NS28 and NS3 are translated as a polyprotein from the same mRNA. A chimeric complex can be formed between yellow fever NS2B and a chimeric yellow fever-dengue 2 NS3. Using anti-NS3 antisera, we also found that a 50-kDa fragment of NS3, consisting of the N-terminal approximately 460 residues, is produced in infected mammalian cells. This fragment is not produced in infected mosquito cells, but will form in Triton X-100 lysates of mosquito cells. The cleavage of NS3 to form this fragment is catalyzed by the NS3 proteinase itself and proteolysis requires NS28. Examination of the amino acid sequence of NS3 reveals a potential conserved cleavage site that resembles other sites cleaved by the NS3/NS2B proteinase; this site occurs within a conserved RNA helicase sequence motif. The importance of this alternatively processed form of NS3 and its role in the replication cycle of dengue virus remain to be determined
A pre-steady state and steady state kinetic analysis of the N-ribosyl hydrolase activity of hCD157
Genetic Ablation of CD38 Protects against Western Diet-Induced Exercise Intolerance and Metabolic Inflexibility.
Nicotinamide adenine dinucleotide (NAD+) is a key cofactor required for essential metabolic oxidation-reduction reactions. It also regulates various cellular activities, including gene expression, signaling, DNA repair and calcium homeostasis. Intracellular NAD+ levels are tightly regulated and often respond rapidly to nutritional and environmental changes. Numerous studies indicate that elevating NAD+ may be therapeutically beneficial in the context of numerous diseases. However, the role of NAD+ on skeletal muscle exercise performance is poorly understood. CD38, a multi-functional membrane receptor and enzyme, consumes NAD+ to generate products such as cyclic-ADP-ribose. CD38 knockout mice show elevated tissue and blood NAD+ level. Chronic feeding of high-fat, high-sucrose diet to wild type mice leads to exercise intolerance and reduced metabolic flexibility. Loss of CD38 by genetic mutation protects mice from diet-induced metabolic deficit. These animal model results suggest that elevation of tissue NAD+ through genetic ablation of CD38 can profoundly alter energy homeostasis in animals that are maintained on a calorically-excessive Western diet
CD38 KO mice retain sensitivity to beta-adrenergic signaling.
<p>(A) Lysates from WAT of C57Bl6 mice fed with either ND or HFHSD were immunoblotted with indicated antibodies. (B) Lysates from WAT of WT or CD38 KO mice on HFHSD were immunoblotted with indicated antibodies.</p
CD38 KO mice are protected from HFHSD- induced obesity.
<p>(A) Body weight was measured for WT (grey) and CD38 KO (black) during the 4 months of ND (dashed lines) or HFHSD (solid lines) treatment from age of 2months old. n = 13–15. **, p value<0.01 (WT vs KO). (B) Fat mass was measured by qNMR for WT (grey) and CD38 KO (black) during the 4 months of ND (dashed lines) or HFHSD (solid lines) treatment from age of 2months old. n = 13–15. **, p value<0.01 (WT vs KO). (C) Tissue weights were measured for WT (grey) and CD38 KO (black) after animals were dissected after 4 months of HFHSD. n = 8 **, p value<0.01 (WT vs KO).</p
Parameters of body weight, body composition and serum profile for mice on normal diet or high-fat high-sucrose diet for 4 months
<p>N = 10–12 per group.</p><p>†, p value<0.05 (ND vs HFHS)</p><p>††, p value<0.01 (ND vs HFHSD)</p><p>Parameters of body weight, body composition and serum profile for mice on normal diet or high-fat high-sucrose diet for 4 months</p
Comparison of exercise capacity with different diets
<p>††, p value<0.01 (ND vs diet).</p><p>¶, p value <0.05 (HFD vs HFHSD)</p><p>Comparison of exercise capacity with different diets</p