Repair of a Mutation Disrupting the Guinea Pig Cytomegalovirus Pentameric Complex Acquired during Fibroblast Passage Restores Pathogenesis in Immune-Suppressed Guinea Pigs and in the Context of Congenital Infection

Abstract

Guinea pig cytomegalovirus (GPCMV) provides a valuable model for congenital cytomegalovirus transmission. Salivary gland (SG)-passaged stocks of GPCMV are pathogenic, while tissue culture (TC) passage in fibroblasts results in attenuation. Nonpathogenic TC-derived virus N13R10 (cloned as a bacterial artificial chromosome [BAC]) has a 4-bp deletion that disruptsGP129, which encodes a subunit of the GPCMV pentameric complex (PC) believed to govern viral entry into select cell types, and GP130, an overlapping open reading frame (ORF) of unknown function. To determine if this deletion contributes to attenuation of N13R10, markerless gene transfer in Escherichia coli was used to construct virus r129, a variant of N13R10 in which the 4-bp deletion is repaired. Virions from r129 were found to contain GP129 as well as two other PC subunit proteins, GP131 and GP133, whereas these three PC subunits were absent from N13R10 virions. Replication of r129 in fibroblasts appeared unaltered compared to that of N13R10. However, following experimental challenge of immunocompromised guinea pigs, r129 induced significant weight loss, longer duration of viremia, and dramatically higher (up to 1.5 ؋ 10 6 -fold) viral loads in blood and end organs compared to N13R10. In pregnant guinea pigs, challenge with doses of r129 virus of >5 ؋ 10 6 PFU resulted in levels of maternal viremia, congenital transmission, pup viral loads, intrauterine growth restriction, and pup mortality comparable to that induced by pathogenic SG virus, although higher doses of r129 were required. These results suggest that the GP129-GP130 mutation is a significant contributor to attenuation of N13R10, likely by abrogating expression of a functional PC. IMPORTANCETissue culture adaptation of cytomegaloviruses rapidly selects for mutations, deletions, and rearrangements in the genome, particularly for viruses passaged in fibroblast cells. Some of these mutations are focused in the region of the genome encoding components of the pentameric complex (PC), in particular homologs of human cytomegalovirus (HCMV) proteins UL128, UL130, and UL131A. These mutations can attenuate the course of infection when the virus is reintroduced into animals for vaccine and pathogenesis studies. This study demonstrates that a deletion that arose during the process of tissue culture passage can be repaired, with subsequent restoration of pathogenicity, using BAC-based mutagenesis. Restoration of pathogenicity by repair of a frameshift mutation in GPCMV gene GP129 using this approach provides a valuable genetic platform for future studies using the guinea pig model of congenital CMV infection. Infection with human cytomegalovirus (HCMV) is a leading cause of disability in newborns, and development of an effective vaccine is a major public health priority (1, 2). Preclinical modeling of vaccines against congenital infection must rely on the study of species-specific CMVs, since HCMV will not infect nonhuman cells (3, 4). The study of guinea pig cytomegalovirus (GPCMV) is particularl...

The pentameric complex of human Cytomegalovirus: cell tropism, virus dissemination, immune response and vaccine development

“…Yamada et al [117] first described a region of the GPCMV genome which appeared to contain homologues of HCMV PC and, more recently, Auerbach et al [118] concluded that the guinea pig genes GP129, GP131 and GP 133 are the homologues of the HCMV genes UL128, UL130 and UL131, and that the relevant recombinant proteins are immunoreactive with convalescent sera from infected animals [119]. Thus, the protein products of GPCMV GP129, 131 and 133 locus were referred to as the GP PC, and the mutations acquired during passaging in fibroblasts attenuated virus pathogenicity [120], whereas repair resulted in congenital transmission, intrauterine growth restriction and elevated pup mortality. Thus, it was concluded that a vaccine aimed at preventing congenital infection should contain PC.…”

MVA-Vectored Pentameric Complex (PC) and gB Vaccines Improve Pregnancy Outcome after Guinea Pig CMV Challenge, but Only gB Vaccine Reduces Vertical Transmission

“…The GPCMV PC, in particular the GP129, also plays a role in macrophage-mediated dissemination of virus in vivo [59], possibly mediated through a putative CC chemokine function [54]. The GPCMV PC is also required for pathogenesis in experimentally challenged, non-pregnant animals [60]. Future work will be required to more fully characterize whether vaccination against the GPCMV PC exerts a beneficial effect by eliciting virus-neutralizing activity, or perhaps instead by inducing antibody responses that target an immunomodulatory or chemokine-like activity, conferred by the GP129 protein.…”

A Guinea pig cytomegalovirus resistant to the DNA maturation inhibitor BDCRB

“…Two-step galactokinase (galK)-mediated recombineering in E. coli (Warming et al, 2005) was used to modify BAC clone N13R10r129, which contains the GPCMV strain 22122 genome (McVoy et al, 2016), to include an expression cassette for NanoLuc ® luciferase. Sequences for all oligonucleotides used are given in Table 1.…”