Improving Tuberculosis (TB) and Human Immunodeficiency Virus (HIV) Treatment Monitoring in South Africa: Evaluation of an Advanced TB/HIV Course for Healthcare Workers

Abstract

The Autographa californica multiple nucleocapsid nucleopolyhedrovirus (AcMNPV) alkaline nuclease (AN) associates with the baculovirus single-stranded DNA binding protein LEF-3 and possesses both a 5؅33؅ exonuclease and an endonuclease activity. These activities are thought to be involved in DNA recombination and replication. To investigate the role of AN in AcMNPV replication, the ␭ Red system was used to replace the an open reading frame with a chloramphenicol acetyltransferase gene (cat) and a bacmid containing the AcMNPV genome in Escherichia coli. The AcMNPV an knockout bacmid (vAcAN-KO/GUS) was unable to propagate in Sf9 cells, although an an-rescued bacmid (vAcAN-KO/GUS-Res) propagated normally. In addi-tion, the mutant did not appear to produce budded virions. These data indicated that an is an essential baculovirus gene. Slot blot and DpnI assays of DNA replication in Sf9 cells transfected with vAcAN-KO/GUS, vAcAN-KO/GUS-Res, and a wild-type bacmid showed that the vAcAN-KO/GUS bacmid was able to replicate to levels similar to those seen with the vAcAN-KO/GUS-Res and wild-type bacmids at early stages posttrans-fection. However, at later time points DNA did not accumulate to the levels seen with the repaired or wild-type bacmids. Northern analysis of Sf9 cells transfected with bacmid vAcAN-KO/GUS showed that transcription of late and very late genes was lower at later times posttransfection relative to the results seen with wild-type and vAcAN-KO/GUS-Res bacmids. These data suggest that the an gene might be involved in the maturation of viral DNA or packaging of the DNA into virions. Members of the Baculoviridae family have double-stranded circular DNA genomes of 100 to 180 kb, depending on the strain of virus (6). The Autographa californica multiple nucle-opolyhedrovirus (AcMNPV) is the type NPV species and is widely used for the generation of recombinant viruses for ex-pression of foreign genes. Production of such expression vec-tors is based on efficient homologous recombination of a gene of interest into the virus genome (21). Although homologous recombination of the AcMNPV genome has been widely em-ployed, its mechanism is unknown and the viral gene products involved in this process have not been identified. In previous reports, it was shown that alkaline nuclease (AN) encoded by AcMNPV (open reading frame [ORF] 133 [ORF133]) associ-ates with the baculovirus single-stranded DNA (ssDNA)-bind-ing (SSB) protein, LEF-3, and that this complex possesses a 5Ј-to-3Ј nuclease activity (11, 18). Homologs of baculovirus ANs are widely distributed in eu-bacteria and archaea and are also found in eukaryotes (1, 2). These enzymes participate in the repair of double-strand breaks and in homologous recombination and therefore play a vital role in the maintenance of genome integrity. The best-studied recombination system is from bacteriophage ␭ and is called the Red (for " recombination defective ") system (for reviews, see references 9, 25, and 29). It includes ␭ exonuclease (Red␣), which degrades double-stranded DNA (dsDNA) from the 5Ј ends, producing 3Ј overhangs which serve as intermedi-ates in recombination (13). The ␭ exonuclease forms a toroid-shaped trimer in solution with a channel in the center that can accommodate dsDNA at one end but only ssDNA at the other (8). During recombination, ␭ exonuclease interacts with a SSB protein (Red␤), which promotes renaturation of complemen-tary strands, thereby mediating strand annealing and strand exchange reactions (3, 12). In studies of herpes simplex virus type 1 (HSV-1) when the gene encoding AN was deleted, viral DNA synthesis and late viral protein expression appeared to be normal, but the number of infectious progeny virus was re-duced (31). In this report, we describe the deletion of the AcMNPV an gene by use of the ␭ Red homologous recombination system (4). After the gene deletion was performed, we then investi-gated DNA replication and gene expression by this construct. The an knockout bacmid produced no measurable levels of infectious virus progeny after transfection into Spodoptera fru-giperda 9 (Sf9) cells. However, up to 48 h posttransfection (p.t.), the DNA replication rate of the an knockout bacmid was similar to that of the wild-type bacmid or a repaired bacmid. In addition, Northern blot analysis indicated that expression lev-els of early, late, and very late genes of the wild-type, an knockout, and rescued bacmids were also similar until 48 h p.t. These data suggest that an is essential for virus progeny pro-duction but is not essential for DNA replication and transcrip-tion. With our previous biochemical studies, these data strongly suggested that an might be involved in maturation of replicated DNA or packaging of viral genome into virions.