In vitro and in vivo characterization of a recombinant rhesus cytomegalovirus containing a complete genome

Cytomegaloviruses (CMVs) are highly adapted to their host species resulting in strict species specificity. Hence, in vivo examination of all aspects of CMV biology employs animal models using host-specific CMVs. Infection of rhesus macaques (RM) with rhesus CMV (RhCMV) has been established as a representative model for infection of humans with HCMV due to the close evolutionary relationships of both host and virus. However, the only available RhCMV clone that permits genetic modifications is based on the 68–1 strain which has been passaged in fibroblasts for decades resulting in multiple genomic changes due to tissue culture adaptations. As a result, 68–1 displays reduced viremia in RhCMV-naïve animals and limited shedding compared to non-clonal, low passage isolates. To overcome this limitation, we used sequence information from primary RhCMV isolates to construct a full-length (FL) RhCMV by repairing all mutations affecting open reading frames (ORFs) in the 68–1 bacterial artificial chromosome (BAC). Inoculation of adult, immunocompetent, RhCMV-naïve RM with the reconstituted virus resulted in significant viremia in the blood similar to primary isolates of RhCMV and furthermore led to high viral genome copy numbers in many tissues at day 14 post infection. In contrast, viral dissemination was greatly reduced upon deletion of genes also lacking in 68–1. Transcriptome analysis of infected tissues further revealed that chemokine-like genes deleted in 68–1 are among the most highly expressed viral transcripts both in vitro and in vivo consistent with an important immunomodulatory function of the respective proteins. We conclude that FL-RhCMV displays in vitro and in vivo characteristics of a wildtype virus while being amenable to genetic modifications through BAC recombineering techniques.

(ORFs) for most species. Ribozyme profiling data suggests that the actual number of translated 91 viral mRNAs is likely significantly higher (5), however only a subset of these produce high levels 92 of protein during infection of fibroblasts (6, 7). Co-evolution of these viruses with their host 93 species over millions of years has led to a sequence relationship between CMV species that 94 generally mirrors that of their hosts while also resulting in strict species specificity (8,9). Hence, 95 HCMV does not replicate and is not pathogenic in immunocompetent animals, and animal models 96 of HCMV thus generally rely on studying infection of a given host with their respective animal 97 CMV. The most commonly used models are mice, rats, guinea pigs and rhesus macaques (RM). 98 The close evolutionary relationship of RM to humans (as compared to rodents) is mirrored in the 99 evolutionary relationship of the rhesus CMV (RhCMV) genome to HCMV as the overall genomic 100 organization is similar and most viral gene families are found in both CMV species (10). 101 Infection of RM with RhCMV has thus become a highly useful animal model for HCMV 102 including a model for congenital infection (11). In addition, RhCMV has been used extensively to a BAC-cloned RhCMV representative of primary isolates to enable studies that reflect circulating 133 RhCMV strains and recapitulate the pathogenesis of HCMV. In addition, such a tissue culture non- 134 adapted, but genetically modifiable RhCMV clone would also be a useful tool to model HCMV-135 based vaccine development for live-attenuated candidates derived from clinical isolates (34). 136 Here, we describe the construction of such a BAC-cloned RhCMV genome in which all 137 presumed mutations in 68-1 that result in altered ORFs were repaired thus closely reflecting a 138 clone of the original 68-1 isolate prior to tissue culture passage. We demonstrate that the resulting  154 Compared to circulating and low passage isolates, RhCMV strain 68-1 has acquired a large 155 inversion in the region homologous to one end of the HCMV "unique long" (UL) sequence of the  This is likely due to significant polymorphism across strains for these genes in RhCMV (39). To 173 create a BAC that most closely resembles a clone of the original 68-1 primary urine isolate we 174 therefore synthesized the entire gene region containing the inverted and missing genes in the UL 175 10 region in two overlapping fragments that were then inserted into RhCMV 68-1.2 by homologous 176 recombination (Fig. 1). Subsequently, we used en passant recombination to repair all point 177 mutation resulting in truncated ORFs as well as a nonsynonymous point mutation in Rh164 178 (UL141). Finally, we deleted a transposon from Rh167 (O14) that was inadvertently acquired 179 during the construction of the RhCMV 68-1.2 BAC. We confirmed the correct sequence of our 180 BAC by restriction digest and next generation sequencing (NGS) and termed the final construct 181 full length RhCMV (FL-RhCMV).

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To characterize the phylogenetic relationship of the 68-1 derived FL-RhCMV BAC clone 183 to related old world NHP CMV species, we cultured and sequenced new isolates from RhCMV, 184 cynomolgus CMV (CyCMV), Japanese macaque CMV (JaCMV) and baboon CMV (BaCMV) 185 from two different US primate centers. We also performed next generation sequencing on viral 186 DNA isolated from stocks of the extensively characterized RhCMV isolates UCD52 and UCD59 187 grown on epithelial cells and included these genome sequences into our analysis. For comparison, 188 we included all NHP CMV sequences of complete or mostly complete genomes deposited in 189 GenBank (Fig. 2). As expected, FL-RhCMV clustered with all other RhCMV isolates and was 190 more distantly related to the CMVs from other NHPs, with the evolutionary relationship of CMV 191 species tracing the evolutionary relationship between their corresponding host species.

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To ensure that FL-RhCMV contained the full ORFeome of all presently confirmed and 193 predicted viral genes of circulating RhCMV strains, we compared the full annotation of  RhCMV with that of other old world NHP CMVs (35, 40-42). All RhCMV genomes lack an 195 internal repeat sequence so that the genomic regions corresponding to the unique long (UL) or the 196 unique short (US) coding regions are fixed in a given orientation whereas HCMV and ChCMV 197 genomes can freely switch between four isomeric forms (Supplementary Fig. 1). Interestingly, indicating that the full genome content has been restored (Fig. 3). A closer examination of full 206 genome alignments of all known old world NHP CMV genome sequences additionally allowed us 207 to further refine our previously established annotations with changes largely comprising 208 reannotations of start codons and splice donor-and acceptor sites (Supplementary Table 1). 209 Comparing the viral ORFeomes across old world NHP CMV species revealed a very high degree 210 of conservation in the entire lineage of viruses so that the entire RhCMV annotation can almost 211 seamlessly be transferred to all related species. While our results are based on comparative 212 genomics and hence need to be confirmed experimentally by mass spectrometry or ribozyme 213 profiling, it is interesting to note that most ORFs that differ between NHP CMV species are due 214 to gene duplication events that occurred in six different loci across the genome (Supplementary 215 Fig. 2-7). Taken together we conclude that the FL-RhCMV clone we engineered is likely a 216 representative of the genomes contained in the original 68-1 isolate.

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In vitro characterization of FL-RhCMV 219 As we have reported earlier (10), one of the ORFs frequently mutated in passaged RhCMV and 220 other old world NHP CMV isolates is the RL11 family member Rh13.1 (Fig. 3) The cells were overlaid to prevent cell-free spread and upon recovery of virus we measured viral 232 plaques sizes after 18 days. As a control, we included a FL-RhCMV in which the Rh13.1 ORF had 233 been deleted (FL-RhCMVΔRh13.1). The development of plaques was severely impeded in TRFs 234 transfected with FL-RhCMV or FL-RhCMV/Rh13.1/tetO (Fig. 4A, B). In contrast, FL-235 RhCMVΔRh13.1 spread rapidly in TRF and expression of the tetR led to a partial rescue of plaque 236 formation by FL-RhCMV/Rh13.1/tetO (Fig. 4A, B). 237 As an alternative approach to conditionally express Rh13.1 we explored the use of 238 aptazyme riboswitches mediating the tetracycline dependent degradation of mRNAs in cis (45). 239 We inserted the Tc40 aptazyme sequence upstream and the Tc45 aptazyme sequence downstream 240 of the Rh13.1 coding region in FL-RhCMV and monitored the stability of Rh13.1 and the 241 surrounding genomic region by NGS upon recovery and propagation of virus in the presence or 242 absence of tetracycline. FL-RhCMV/Rh13.1/apt grown in the absence of tetracycline displayed 243 multiple mutations and deletions in this genomic region as early as passage 2 (Fig. 4C). In contrast, 244 13 by activating the aptazyme using tetracycline we were able to generate virus stocks that contained 245 an intact Rh13.1 sequence (Fig. 4C). These data are consistent with Rh13.1 being selected against 246 in FL-RhCMV similar to selection against RL13 in HCMV because these homologous proteins 247 impede spread in tissue culture. We further conclude that mutations in the Rh13.1 homologs found 248 in many old world NHP CMV genomes (Fig. 3) are due to rapid tissue culture adaptations whereas 249 the parental isolates likely contained an intact ORF. Thus, Rh13.1 and its homologs are preserved 250 in vivo, but are selected against in vitro.

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It was previously shown that repair of the PRC increased the ability of RhCMV 68-1.2 to 252 infect epithelial and endothelial cells without affecting growth characteristics in fibroblasts (25).

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Similarly, growth characteristics of FL-RhCMV/Rh13.1/apt were comparable to that of 68-1 and 254 PRC-repaired 68-1.2 in rhesus fibroblasts with respect to kinetics and peak titers in a multistep 255 growth curve (Fig. 5A). Since these comparable growth kinetics were observed in the absence of show any significant differences across the examined strains (Fig. 5C). We previously 281 demonstrated that Rh159 is an ER-resident glycoprotein that intracellularly retains NK cell 282 activating ligands, a function that is not shared with UL148 (51). However, these observations do 283 not rule out a role of Rh159 for PRC expression and cell tropism. While further work will be 284 required to establish this role, our results indicate that FL-RhCMV is remarkably similar to low  virus stock production (Fig. 6B). Since both experiments showed virtually the same development 300 and progression of plasma viremia after i.v. inoculation (Fig. 6C), we conclude that in vivo 301 replication of FL-RhCMV is comparable to that of low passage RhCMV isolates.   passaged strains appear capable of maintaining an intact RL13 ORF (57). RL13 seems to limit 430 viral spread, particularly in fibroblasts (57) but the exact mechanism of this inhibition is not clear.

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Rh13.1 belongs to the RL11 family of single transmembrane glycoproteins present in all old world 432 NHP CMVs, as well as great ape and HCMV, but not in CMVs of new world primates (Fig. 9). 433 The functional conservation of Rh13.1 and RL13 is surprising since the RL11 family is highly 434 diverse both within a given CMV species and especially when comparing family members between 435 great ape and old world monkey CMVs (10, 40, 58 However, we also observed that FL-RhCMV lacking Rh13.1 displayed substantial in vivo 445 spread that was significantly more pronounced than a mutant that lacked the UL128 and UL130-  (Fig. 9) primate CMVs (Supplementary Fig. 4) Since 68-1 lacks these highly expressed genes, they are not required for the establishment and

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The formation of plaques was then monitored by imaging for eGFP fluorescence at various 656 timepoints, using a Zeiss Axio Observer Z1.  Quantitative PCR (qPCR) analysis to assess mRNA expression levels.

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Primary rhesus fibroblasts were seeded in 6-well plates and infected either with FL-, 68-1, or 68-694 1.2 RhCMV at a MOI of 1. Total RNA was then isolated at 48 hours post infection (hpi).

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Uninfected rhesus fibroblasts were used as a negative control. After cDNA synthesis, the 696 quantitative PCR (q-PCR) assay was performed using primers and probes specific to each gene of 697 interest      Figure S8: Exploratory analysis to assess equivalency across RNA-seq data.