**1. Introduction**

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. Because HCMV proliferates in human cells specifically, 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]. However, the murine cytomegalovirus (MCMV) provides an excellent animal model for studying the biology of cytomegaloviruses (CMV) through its specific infection of mice. An MCMV genome of 230 kb is predicted to encode more than 170 open reading frames (ORFs), 78 of which have an extensive homology to those of HCMV [1–3]. Moreover, the pathogenesis of MCMV infection in mice is very similar to that of HCMV infection in humans in several aspects such as active infections, establishment of latency, and reactivation after latent infections [1,3–5]. A complete understanding of the biology of MCMV and the function of its genes may provide insights into the pathogenesis of HCMV.

As a structural protein, the HCMV UL80 assembly protein is presumed to function in packaging DNA. A protease is encoded by the N-terminal region of UL80 that cleaves the assembly protein precursor at a site near the C terminus. The MCMV homolog (M80) of the assembly protein of HCMV (UL80) conserves the domain structure and cleavage sites present in HCMV UL80. MCMV is different in that the conserved CD1 and CD2 domains are separated by 100 amino acids, whereas in all other sequenced herpes viruses the two domains are separated by 80 to 84 residues [2]. The MCMV homolog (M80.5) of HCMV, UL80.5 ORF, is referred to code for protease. The ORFs of MCMV M80 and M80.5 are required for the assembly of proteins and proteases (capsid synthesis) and further virion production. The transcriptions of adjoining MCMV M80 and M80.5 ORFs have different start sites; however, they have the same stop site. Therefore, the overlapping region of MCMV M80 and M80.5 (M80/80.5) is an appropriate target site for virus inhibition.

Transcription activator-like effectors (TALEs) are proteins secreted by the plant pathogenic bacteria *Xanthomonas* via a type III secretion system where they infect various plant species [6]. These proteins can bind promoter sequences in the host plant and activate the expression of host genes that aid bacterial infections. TALEs are important virulence factors that act as transcriptional activators in the plant cell nucleus, where they directly bind to DNA via a central domain of tandem repeats [6]. Each TALE contains a central repetitive region consisting of varying numbers of repeat units (about 17.5 repeats) of 34 amino acids [6,7]. The DNA-binding domain contains a highly conserved 34-amino acid sequence, with the exception of the 12th and 13th amino acids [6]. Only the 12th and 13th amino acids in TALEs are changeable and variable; the other amino acids are constant and stable. These two locations' RVDs (Repeat Variable Di-residues) are highly variable and show a strong correlation with a specific nucleotide recognition by different frequencies, for example NI recognizes A (55%), NG recognizes T (50%), NN recognizes G (7%) and HD recognizes C (69%) [6]. DNA transcribes RNA according to the complementary base pairing rule (A = U, G ≡ C) and RNA translates protein according to the standard genetic code (RNA codon table). After the breaking of the code of the DNA-binding specificity of TALEs [6,7], we know that two amino acids can also recognize one nucleotide. The restriction endonuclease *Fok* I, naturally found in the bacterium *Flavobacterium okeanokoites*, consists of an N-terminal specific DNA-binding domain and a C-terminal nonspecific DNA cleavage domain. The DNA-binding domain recognizes the non-palindromic sequence 5- -GGATG-3 when the catalytic domain cleaves double-stranded DNA nonspecifically at a fixed distance of 9 and 13 nucleotides downstream of the recognition site. *Fok* I exists as an inactive monomer and becomes an active dimer upon binding to its target DNA and in the presence of specific divalent metals [7]. The DNA cleavage domain of *Fok* I functions as a homodimer, requiring two constructs with unique DNA-binding domains for sites in the target genome with proper orientation and spacing. Transcription activator-like effector nucleases (TALENs) are artificial restriction enzymes generated by fusing the specific TALE DNA-binding domain to a nonspecific *Fok* I DNA cleavage domain [8,9] (Figure 1).

TALENs were shown to be a valuable tool for precise genome engineering with low toxicity [10]. Additionally, they have been successfully applied in the genetic engineering of human pluripotent cells [11,12], and the generation of knockout animals, such as nematodes (*Caenorhabditis elegans*) [8], rats [13], and zebrafish [14,15]. TALENs can be used to edit genomes by making double-stranded breaks (DSBs), which cells respond to with two repair mechanisms: non-homologous end joining (NHEJ) or homologous recombination (HR). DNA can be introduced into a genome through NHEJ in the presence of exogenous double-stranded DNA fragments. HR can also introduce foreign DNA at the DSB as the transfected double-stranded sequences are used as templates for the repair enzymes. However, TALENs have some potential problems. If TALENs do not specifically target a unique site within the genome of interest, off-target cleavage may occur [11]. Such off-target cleavage may lead to the production of enough DSB to overcome the repair machinery and consequently result in chromosomal rearrangements and/or cell death [8].

**Figure 1.** Transcription activator-like effector nuclease (TALEN) targeting and cleavage on the target site. The specific repeat variable di-residue (RVD) used to recognize each base is indicated by shading, as defined in the key (NI: A, NG: T, NN: G, HD: C). The cleavage site is in the adjoining region between the left and right target site. The left transcription activator-like effector (TALE) and the right TALE of TALEN plasmids recognized their target sequence and allowed their associated *Fok* I endonucleases to work as homodimers to cleave the sense strand 9 bp and antisense strand 13 bp downstream of the binding site. The binding of the TALEs to the target sites allows *Fok* I to dimerize and create a double-stranded break (DSB) with sticky ends within the spacer.

Previous reports have suggested that TALENs can be engineered to adapt for an antiviral strategy. For example, TALENs were known to be effective in the inactivation of Hepatitis B virus (HBV) replication in cultured cells and in vivo [16], and in the targeting of the HBV genome [17]. Also, Epstein–Barr virus (EBV)-encoded nuclear antigen-1 (EBNA1) plays a crucial role in EBV episome replication and persistence. TALEN-mediated targeted disruption of EBNA1 was shown to inhibit the growth of EBV-infected cells, hinting at a possible therapeutic application for EBV-associated disorders [18]. TALENs may also provide a new strategy for the treatment of CMV infections.

Latency is a specific phase in viral life cycles, in which viral particles stop producing after infection, but the viral genome has not been completely removed. Proviral latency and episomal latency are two known viral latency models. CMV belongs to the episomal latency model which is essentially quiescent in myeloid progenitor cells, and is reactivated by differentiation, inflammation, immunosuppression or critical diseases [19]. CMV latency has been defined as the absence of infectious viruses, despite the presence of viral DNA. Although the molecular mechanisms by which latency is established and maintained have not been clear, transcriptional control of viral gene expression is very important in controlling viral latency and reactivation. Viral replication is initiated by the expression of *ie* (immediately early) genes. Studies with CMV have suggested that latency is established through the repression of *ie-1* gene expression. *Ie-1* proteins, the first proteins expressed by the virus during productive infection, are transcriptionally regulatory proteins that are required for the induction of early and late gene expression, viral DNA synthesis, and virion production [20–22]. Since MCMV *ie-1* proteins are required for viruses to start replication from latency, the *ie-1* gene is one of the key targets for latency in animal studies. In this study, our goal is to develop an effective strategy to inhibit the growth of MCMV in cell culture and animals specifically, particularly for the removal of latent viruses.
