Here we describe a high-resolution functional profile of the MHV-68 RTA locus in the context of the viral genome. Using a high-throughput mutational analysis system, potential RTA promoter cis-elements and RTA protein subdomains critical for virus replication were identified. The high-resolution profiling analysis of herpesvirus, however, has been challenging due to the large viral genome size (140 to 240 kb). Advances in recombination technology, capillary electrophoresis, optical detection, and bioinformatics made this large-scale mutational analysis study possible. We have identified a total of 1,229 independent insertions in the RTA locus, of which 393, 282, and 554 insertions were critically impaired, attenuated, and tolerated, respectively, for viral replication. The RTA locus profiling was conducted by selecting the mutant library for several rounds of replication in cell culture. During the selection, the insertion mutants with low replication fitness were out-competed by viruses with normal levels of replication fitness. This resulted in critically impaired or attenuated phenotypes.
Transcomplementation among mutants during population selection is an issue that can potentially affect the outcome of the functional profiling. During the reconstitution step, the possibility of transcomplementation was high, as the mutant RTA locus fragments and RTA locus null MHV-68 genomes were transfected into the cells. Thus, most of the mutant viruses can be generated. To avoid transcomplementation during subsequent selection steps, however, we used a low MOI of 0.05 to infect fibroblast cells. Furthermore, we subjected the mutant viral library to an additional five rounds of selection after reconstitution. During each round of selection, the chance of two transcomplementing mutants in a population of over 1,000 different mutant viruses coinfecting the same cell is very remote. Thus, these selection conditions greatly minimized the effect of transcomplementation in the final observed mutant phenotypes. Moreover, we were able to reproduce the functional profiling phenotypes by using individual mutants.
We have introduced 15-bp random insertions in the RTA locus using a mini-Mu transposon by in vitro mutagenesis. We have used the optimum concentrations of transposon donor DNA and RTA locus target plasmids to have a single insertion per RTA locus plasmid. Sequencing and restriction digestion analysis of randomly picked RTA locus plasmids revealed only a single transposon insertion. Since the insertions are random, the frequency of the mutant plasmids with two insertions in identical sites is extremely low. If both or one of the insertions is lethal, that mutant will be negatively selected. If both of the insertions are tolerated, that mutant will be subjected to neutral selection. The frequencies of double insertions that can significantly change the phenotypes are negligible. Therefore, mutants with more than one insertion have a low possibility of influencing the phenotypes observed in the functional profiling.
Recently, we reported a functional profiling study of the hepatitis C virus genome (9.6 kb) (1
). The large genome size of herpesviruses (ranging from 120 to 240 kb) poses unique challenges for a genome-scale functional profiling study. To introduce a 15-bp insertion, the drug-resistant gene of the transposon that is inserted into the viral genome has to be removed by restriction digestion and religation. This step is technically less difficult for viruses with small genomes. We have observed that after digesting the 140- to 150-kb MHV-68 BAC plasmid with the restriction enzyme, it was very difficult to religate the BAC plasmid ends together. The intramolecular ligation was extremely inefficient. We have tested several approaches and selected a piecemeal approach in which the herpesvirus genome can be profiled as segments. Each segment can be individually subjected to transposon insertion, restriction digestion, and religation to introduce the 15-bp insertions. Subsequently, the mutated segment can be recombined into a virus lacking that particular segment using Flp recombination. We constructed MHV-68 BAC clones with a FRT site replacing each segment of the genome for the herpesviral genome-scale functional profiling. We observed that the Flp recombination step was very efficient in BHK-21 cells for MHV-68 viral reconstitution. We utilized the Flp recombination strategy for RTA
locus functional profiling. Under cell culture selection conditions, about 45% of 15-bp insertions in the herpesviral RTA
locus did not affect the virus replication, whereas only about 16% of insertions did not affect replication of hepatitis C virus (1
). These results suggest that larger-genome-containing organisms can accommodate the genetic insertions without deleterious effects on survival fitness.
The MHV-68 RTA
locus contains ORF48, ORF49, and ORF50. Due to the compact nature of the viral genome, the promoter and coding regions of genes have a great deal of overlap. Thus, we are aware that some of the insertions may disrupt the function of protein domains and/or the cis
-elements that control the expression of neighboring genes. The promoter cis
-elements located in protein coding regions will need to be precisely identified by comparing the profiles of the mutant library that has been selected in a parental cell line and a cell line expressing the disrupted protein. Nevertheless, because ORF48 is not essential for viral replication in vitro, the critical regions identified in the ORF48 coding region most likely contain cis
-elements that regulate RTA transcription. The transcription initiation sites of RTA were mapped between nt 66469 and 66503 using an RNase protection assay (25
). We observed that most of the insertions upstream of the RTA transcription start site (between nt 66255 and 66439) exhibited the critically impaired phenotypes (see Fig. S1 in the supplemental material). These insertions can possibly interrupt the cis
-element sequences essential for the binding of transcription factors involved in RTA mRNA synthesis, thus impairing the viral replication. Hence, in a separate study, we are exploring the mechanism of activation of these RTA promoter cis
-elements by identifying and characterizing the trans
-factors and the upstream signaling pathways involved.
RTA, as the master regulator for viral lytic replication, executes its function by interacting with various cellular and viral proteins. The MHV-68 RTA has 43% amino acid similarity with KSHV RTA (10
). The N terminal of RTA is conserved between these two viruses with limited amino acid conservation in the C-terminal TA domain. We have identified many deleterious insertions in the regions corresponding to the RTA N-terminal DBD and C-terminal TA domain. These insertions may result in RTA mutant proteins that fail to bind DNA or interact with other critical proteins. The functional profiling phenotypes of the RTA protein were verified by examining several individual mutants having amino acid substitutions and insertions. Substituting RTA N-terminal DBD residues KD (Fig. ) and C-terminal TA domain residue SLYD resulted in a reduction in transactivating function; however, the KD mutant was incompetent in transcomplementing RTA-null virus. This interesting observation could be a result of the failure of the KD mutant to bind to the RTA target promoter elements, thus rendering it defective in transactivating the lytic genes and completing lytic viral replication, whereas the SLYD mutant could bind to the RTA target DNA elements and could partially bind to the transactivating factors, resulting in initiation of the viral lytic gene expression cascade despite a lower level compared to that of wild-type virus. These results suggest that the DNA binding domain is absolutely critical for RTA function. The different results could also be due to the different ratios of RTA protein to the viral promoter DNA in the reporter assay and in the transcomplementation assay. Insertions in RTA amino acid residue 37 resulted in lethal and attenuated phenotypes, and insertions at residue 38 resulted in tolerated phenotypes. Analysis of the mutant RTA with alanine substitutions of residues 37 and 38 (RTA-QQ) exhibited a lethal phenotype. This result suggests a critical role for RTA residue 37 during viral replication.
The present study is the first comprehensive high-resolution mutational analysis of a 3.8-kb viral genome locus in the context of the viral genome. We have identified many of the RTA subdomains that were nonessential for virus replication in cell culture; however, these subdomains might play critical roles during in vivo lytic and latent infections. Thus, profiling the mutant library in wild-type and knockout mice, as well as in mice with various genetic backgrounds, would provide greater insight into the role of RTA in virus-host interactions. We have obtained a functional profile of the RTA locus during infection in BALB/c mice (unpublished data). We have shown that the mutant viral library can be recovered from the lung tissues to generate a profile, which sets the stage for our future in vivo studies. The viral promoter and protein are both positively and negatively regulated by viral and cellular factors. Selecting the mutant viral library in the presence or absence of these regulatory factors in cell culture would enable the identification of the viral subdomains that interact with these factors.
In the future, this approach can be expanded to genome-scale profiling. To demonstrate the feasibility, we have successfully reconstituted up to 21-kb viral fragments into the viral genome and efficiently recovered infectious viruses. Thus, the whole herpesvirus genome can be profiled by mutating and reconstituting overlapping viral fragments to cover the entire length of the genome. Functional domain mapping can complement structural biology studies of viral proteins. The whole genome library can be used to elucidate the function of viral subdomains involved in tissue tropism, immune regulation, autophagy, apoptosis, cell survival, signal transduction, and other cellular processes. This approach will greatly expedite the functional genomics studies of herpesviruses.