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Cytomegalovirus UL128 homolog mutants that form a pentameric complex produce virus with impaired epithelial and trophoblast cell tropism and altered pathogenicity in the guinea pig.

ABSTRACT: Guinea pig cytomegalovirus (GPCMV) encodes a homolog pentameric complex (PC) for specific cell tropism and congenital infection. In human cytomegalovirus, the PC is an important antibody neutralizing target and GPCMV studies will aid in the development of intervention strategies. Deletion mutants of the C-terminal domains of unique PC proteins (UL128, UL130 and UL131 homologs) were unable to form a PC in separate transient expression assays. Minor modifications to the UL128 homolog (GP129) C-terminal domain enabled PC formation but viruses encoding these mutants had altered tropism to renal and placental trophoblast cells. Mutation of the presumptive CC chemokine motif encoded by GP129 was investigated by alanine substitution of the CC motif (codons 26-27) and cysteines (codons 47 and 62). GP129 chemokine mutants formed PC but GP129 chemokine mutant viruses had reduced epitropism. A GP129 chemokine mutant virus pathogenicity study demonstrated reduced viral load to target organs but highly extended viremia.

SUBMITTER: Coleman S

PROVIDER: S-EPMC5739588 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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Cytomegalovirus UL128 homolog mutants that form a pentameric complex produce virus with impaired epithelial and trophoblast cell tropism and altered pathogenicity in the guinea pig.

Coleman Stewart S Choi K Yeon KY McGregor Alistair A

Virology 20170623

Guinea pig cytomegalovirus (GPCMV) encodes a homolog pentameric complex (PC) for specific cell tropism and congenital infection. In human cytomegalovirus, the PC is an important antibody neutralizing target and GPCMV studies will aid in the development of intervention strategies. Deletion mutants of the C-terminal domains of unique PC proteins (UL128, UL130 and UL131 homologs) were unable to form a PC in separate transient expression assays. Minor modifications to the UL128 homolog (GP129) C-ter ...[more]

PMID: 28651121

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Project description:UNLABELLED: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 disrupts GP129, 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. IMPORTANCE:Tissue 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.

Project description:BackgroundCytomegalovirus (CMV) is a leading infectious cause of neurologic deficits, both in the settings of congenital and perinatal infection, but few animal models exist to study neurodevelopmental outcomes. This study examined the impact of neonatal guinea pig CMV (GPCMV) infection on spatial learning and memory in a Morris water maze (MWM) model.MethodsNewborn pups were challenged intraperitoneally (i.p.) with a pathogenic red fluorescent protein-tagged GPCMV, or sham inoculated. On days 15-19 post infection (p.i.), pups were tested in the MWM. Viral loads were measured in blood and tissue by quantitative PCR (qPCR), and brain samples collected at necropsy were examined by histology and immunohistochemistry.ResultsViremia (DNAemia) was detected at day 3 p.i. in 7/8 challenged animals. End-organ dissemination was observed, by qPCR, in the lung, liver, and spleen. CD4-positive (CD4+) and CD8-positive (CD8+) T cell infiltrates were present in brains of challenged animals, particularly in periventricular and hippocampal regions. Reactive gliosis and microglial nodules were observed. Statistically significant spatial learning and memory deficits were observed by MWM, particularly for total maze distance traveled (p < 0.0001).ConclusionNeonatal GPCMV infection in guinea pigs results in cognitive defects demonstrable by the MWM. This neonatal guinea pig challenge model can be exploited for studying antiviral interventions.ImpactCMV impairs neonatal neurocognition and memory in the setting of postnatal infection. The MWM can be used to examine memory and learning in a guinea pig model of neonatal CMV infection. CD4+ and CD8+ T cells infiltrate the brain following neonatal CMV challenge. This article demonstrates that the MWM can be used to evaluate memory and learning after neonatal GPCMV challenge. The guinea pig can be used to examine central nervous system pathology caused by neonatal CMV infection and this attribute may facilitate the study of vaccines and antivirals.

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Cytomegalovirus UL128 homolog mutants that form a pentameric complex produce virus with impaired epithelial and trophoblast cell tropism and altered pathogenicity in the guinea pig.

Publications

Cytomegalovirus UL128 homolog mutants that form a pentameric complex produce virus with impaired epithelial and trophoblast cell tropism and altered pathogenicity in the guinea pig.

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