Latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus recruits uracil DNA glycosylase 2 at the terminal repeats and is important for latent persistence of the virus.

Latency-associated nuclear antigen (LANA) of KSHV is expressed in all forms of Kaposi's sarcoma-associated herpesvirus (KSHV)-mediated tumors and is important for TR-mediated replication and persistence of the virus. LANA does not exhibit any enzymatic activity by itself but is critical for replication and maintenance of the viral genome. To identify LANA binding proteins, we used a LANA binding sequence 1 DNA affinity column and determined the identities of a number of proteins associated with LANA. One of the identified proteins was uracil DNA glycosylase 2 (UNG2). UNG2 is important for removing uracil residues yielded after either misincorporation of dUTP during replication or deamination of cytosine. The specificity of the 'LANA-UNG2 interaction was confirmed by using a scrambled DNA sequence affinity column. Interaction of LANA and UNG2 was further confirmed by in vitro binding and coimmunoprecipitation assays. Colocalization of these proteins was also detected in primary effusion lymphoma (PEL) cells, as well as in a cotransfected KSHV-negative cell line. UNG2 binds to the carboxyl terminus of LANA and retains its enzymatic activity in the complex. However, no major effect on TR-mediated DNA replication was observed when a UNG2-deficient (UNG(-/-)) cell line was used. Infection of UNG(-/-) and wild-type mouse embryonic fibroblasts with KSHV did not reveal any difference; however, UNG(-/-) cells produced a significantly reduced number of virion particles after induction. Interestingly, depletion of UNG2 in PEL cells with short hairpin RNA reduced the number of viral genome copies and produced infection-deficient virus.

Sol-gel modified poly(dimethylsiloxane) microfluidic devices with high electroosmotic mobilities and hydrophilic channel wall characteristics.

Using a sol-gel method, we have fabricated poly(dimethylsiloxane) (PDMS) microchips with SiO2 particles homogeneously distributed within the PDMS polymer matrix. These particles are approximately 10 nm in diameter. To fabricate such devices, PDMS (Sylgard 184) was cast against SU-8 molds. After curing, the chips were carefully removed from the mold and sealed against flat, cured pieces of PDMS to form enclosed channel manifolds. These chips were then solvated in tetraethyl orthosilicate (TEOS), causing them to expand. Subsequently, the chips were placed in an aqueous solution containing 2.8% ethylamine and heated to form nanometer-sized SiO2 particles within the cross-linked PDMS polymer. The water contact angle for the PDMS-SiO2 chips was approximately 90.2 degrees compared to a water contact angle for Sylgard 184 of approximately 108.5 degrees . More importantly, the SiO2 modified PDMS chips showed no rhodamine B absorption after 4 h, indicating a substantially more hydrophilic and nonabsorptive surface than native PDMS. Initial electroosmotic mobilities (EOM) of (8.3+/-0.2)x10(-4) cm2/(V.s) (RSD=2.6% (RSD is relative standard deviation); n=10) were measured. This value was approximately twice that of native Sylgard 184 PDMS chips (4.21+/-0.09)x10(-4) cm2/(V.s) (RSD=2.2%; n=10) and 55% greater than glass chips (5.3+/-0.4)x10(-4) cm2/(V.s) (RSD=7.7%; n=5). After 60 days of dry storage, the EOM was (7.6+/-0.3)x10(-4) cm2/(V.s) (RSD=3.9%; n=3), a decrease of only 8% below that of the initially measured value. Separations performed on these devices generated 80,000-100,000 theoretical plates in 6-14 s for both tetramethylrhodamine succidimidyl ester and fluorescein-5-isothiocyanate derivatized amino acids. The separation distance was 3.5 cm. Plots of peak variance vs analyte migration times gave diffusion coefficients which indicate that the separation efficiencies are within 15% of the diffusion limit.