Characterization of the Near Full-Length Genome of a Novel HIV-1 CRF01_AE/CRF07_BC Recombinant in an Injection Drug User from Southern Taiwan.

HIV-1 CRF07_BC became prevalent in Taiwan after the epidemic among injection drug users (IDUs). We describe a unique recombinant form (URF) consisting of CRF01_AE and CRF07_BC (named URF_0107-H8) genes detected from an IDU. The 8.8 kb near full-length genome of URF_0107-H8 had a CRF01_AE backbone with two CRF07_BC fragments in the reverse transcriptase and integrase region [RT-Int; HXB2 nucleotide (nt) positions 2942-4709] and within the envelop (nt 8467-8722) gene. Phylogenetic analyses revealed that its 1.8 kb RT-Int sequence clustered with those of CRF07_BC strains from Taiwan, while sequences of CRF01_AE portions were more similar to those of Central African origin than contemporaneous CRF01_AE isolates in Taiwan or prevalent in East or Southeast Asia. Recombination breakpoints and phylogenetic relationships of URF_0107-H8 were different from those of CRF01_AE/CRF07_BC URFs previously reported from China. This highlighted the importance of continual monitoring of genetic evolution of HIV strains and the emergence of new recombinants.

Autographa californica multiple nucleopolyhedrovirus LEF-2 is a capsid protein required for amplification but not initiation of viral DNA replication.

The late expression factor 2 gene (lef-2) of baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) has been identified as one of the factors essential for origin-dependent DNA replication in transient expression assays and has been shown to be involved in late/very late gene expression. To study the function of lef-2 in the life cycle of AcMNPV, lef-2 knockout and repair bacmids were generated by homologous recombination in Escherichia coli. Growth curve analysis showed that lef-2 was essential for virus production. Interestingly, a DNA replication assay indicated that lef-2 is not required for the initiation of viral DNA replication and that, rather, it is required for the amplification of DNA replication. lef-2 is also required for the expression of late and very late genes, as the expression of these genes was abolished by lef-2 deletion. Temporal and spatial distributions of LEF-2 protein in infected cells were also analyzed, and the data showed that LEF-2 protein was localized to the virogenic stroma in the nuclei of the infected cells. Analysis of purified virus particles revealed that LEF-2 is a viral protein component of both budded and occlusion-derived virions, predominantly in the nucleocapsids of the virus particles. This observation suggests that LEF-2 may be required immediately after virus entry into host cells for efficient viral DNA replication.

Stimulation of baculovirus transcriptome expression in mammalian cells by baculoviral transcriptional activators.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the type species of the family Baculoviridae, is an insect-specific virus that can enter a variety of mammalian cells. The potential of this versatile virus for protein expression or gene therapy in mammalian cells has become the focus of many studies. In most mammalian cells, transduced AcMNPV genes are either not expressed or expressed at an extremely low level. Here, we studied the effects of the two major AcMNPV trans-activators, IE1 and IE2, on the activation of AcMNPV genome in Vero E6 cells. Microarray analysis showed that when IE1 was overexpressed, it significantly activated genes gp64 and pe38, and upregulated ie2, he65, pcna, orf16, orf17 and orf25. Although, there were only two genes, pe38 and orf17, that were activated by IE2, we discovered interestingly that the combination of IE1 and IE2 factors had a synergistic effect on activation of the AcMNPV genome in mammalian cells, and activated around 38 %, or 59 out of the 155 genes placed on the microarray. This is the first detailed study of baculoviral transcription regulation in mammalian cells, and it shows that the baculoviral genome can be activated in a mammalian system, and also that the two major trans-activators, IE1 and IE2, play a central role in this activation.