Abstract
Cytomegalovirus (CMV) infections are still a global health problem, because the latent viruses persist in humans and cause recurring diseases. Currently, there are no therapies for CMV latent infections and the therapies for active infections are limited by side effects and other problems. It is impossible to eradicate latent viruses in animals. HCMV (human CMV) is specific to human diseases; however, it is difficult to study HCMV due to its host specificity and long life cycle. Fortunately, MCMV (murine CMV) provides an excellent animal model. Here, three specific pairs of transcription activator-like effector nuclease (TALEN) plasmids (MCMV1–2, 3–4, and 5–6) were constructed to target the MCMV M80/80.5 sequence in order to test their efficacy in blocking MCMV lytic replication in NIH3T3 cell culture. The preliminary data showed that TALEN plasmids demonstrate specific targeting and cleavage in the MCMV M80/80.5 sequence and effectively inhibit MCMV growth in cell culture when the plasmid transfection is prior to the viral infection. The most specific pairs of TALEN plasmids (MCMV3–4) were further used to confirm the negative regulation of latent MCMV replication and gene expression in Balb/c mice. The injection of specific TALEN plasmids caused significant inhibition in the copy number level of immediately early gene (ie-1) DNA in five organs of mice, when compared with the controls. The result demonstrated that TALENs potentially provide an effective strategy to remove latent MCMV in animals.
Highlights
Studies on the functions of viral genes in human cytomegalovirus (HCMV) replication in vivo and understanding of viral pathogenesis are essential for developing novel drugs and strategies to treat the viral infections, but there are no suitable animal models for HCMV infection at present
The Transcription activator-like effectors (TALEs) DNA sequences of two nonspecific pairs of transcription activator-like effector nuclease (TALEN) plasmids W1FS-W7R1 and KSHV1-2 were both proved to be nonhomologous to murine cytomegalovirus (MCMV)
When the specific TALEN plasmid (MCMV1-2, 3-4 and 5-6) transfection was prior to the viral infection, we found that the viral titers decreased by about 65%, 75%, and 25%, 1, 3 and 5 days post infection, respectively, compared with the controls, using lipofectamine as a transfection reagent (Figure 4A–C)
Summary
Studies on the functions of viral genes in human cytomegalovirus (HCMV) replication in vivo and understanding of viral pathogenesis are essential for developing novel drugs and strategies to treat the viral infections, but there are no suitable animal models for HCMV infection at present. HCMV proliferates in human cells grows slowly and has a very long lytic replication cycle in humans, it is quite difficult to study HCMV (genome size 235 kb) gene function and pathogenesis [1]. The murine cytomegalovirus (MCMV) provides an excellent animal model for studying the biology of cytomegaloviruses (CMV) through its specific infection of mice. A complete understanding of the biology of MCMV and the function of its genes may provide insights into the pathogenesis of HCMV
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