Dataset Information

A Homolog Pentameric Complex Dictates Viral Epithelial Tropism, Pathogenicity and Congenital Infection Rate in Guinea Pig Cytomegalovirus.

ABSTRACT: In human cytomegalovirus (HCMV), tropism to epithelial and endothelial cells is dependent upon a pentameric complex (PC). Given the structure of the placenta, the PC is potentially an important neutralizing antibody target antigen against congenital infection. The guinea pig is the only small animal model for congenital CMV. Guinea pig cytomegalovirus (GPCMV) potentially encodes a UL128-131 HCMV PC homolog locus (GP128-GP133). In transient expression studies, GPCMV gH and gL glycoproteins interacted with UL128, UL130 and UL131 homolog proteins (designated GP129 and GP131 and GP133 respectively) to form PC or subcomplexes which were determined by immunoprecipitation reactions directed to gH or gL. A natural GP129 C-terminal deletion mutant (aa 107-179) and a chimeric HCMV UL128 C-terminal domain swap GP129 mutant failed to form PC with other components. GPCMV infection of a newly established guinea pig epithelial cell line required a complete PC and a GP129 mutant virus lacked epithelial tropism and was attenuated in the guinea pig for pathogenicity and had a low congenital transmission rate. Individual knockout of GP131 or 133 genes resulted in loss of viral epithelial tropism. A GP128 mutant virus retained epithelial tropism and GP128 was determined not to be a PC component. A series of GPCMV mutants demonstrated that gO was not strictly essential for epithelial infection whereas gB and the PC were essential. Ectopic expression of a GP129 cDNA in a GP129 mutant virus restored epithelial tropism, pathogenicity and congenital infection. Overall, GPCMV forms a PC similar to HCMV which enables evaluation of PC based vaccine strategies in the guinea pig model.

SUBMITTER: Coleman S

PROVIDER: S-EPMC4936736 | biostudies-literature | 2016 Jul

REPOSITORIES: biostudies-literature

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A Homolog Pentameric Complex Dictates Viral Epithelial Tropism, Pathogenicity and Congenital Infection Rate in Guinea Pig Cytomegalovirus.

Coleman Stewart S Choi K Yeon KY Root Matthew M McGregor Alistair A

PLoS pathogens 20160707 7

In human cytomegalovirus (HCMV), tropism to epithelial and endothelial cells is dependent upon a pentameric complex (PC). Given the structure of the placenta, the PC is potentially an important neutralizing antibody target antigen against congenital infection. The guinea pig is the only small animal model for congenital CMV. Guinea pig cytomegalovirus (GPCMV) potentially encodes a UL128-131 HCMV PC homolog locus (GP128-GP133). In transient expression studies, GPCMV gH and gL glycoproteins intera ...[more]

PMID: 27387220

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Project description:The guinea pig is the only small animal model for congenital CMV but requires species-specific guinea pig cytomegalovirus (GPCMV). Tegument protein GP83 is the presumed homolog of HCMV pp65 but gene duplication in the UL82-UL84 homolog locus in various animal CMV made it unclear if GP83 was a functional homolog. A GP83 null deletion mutant GPCMV (GP83dPC+) generated in the backdrop of glycoprotein pentamer complex (PC) positive virus, required for non-fibroblast infection, had normal growth kinetics on fibroblasts but was highly impaired on epithelial and trophoblast cells. GP83dPC+ virus was highly sensitive to IFN-I suggesting GP83 had an innate immune evasion function. GP83 interacted with cellular DNA sensors guinea pig IFI16 and cGAS indicating a role in the cGAS/STING pathway. Ectopically expressed GP83 in trophoblast cells restored GP83dPC+ virus growth. Additionally, mutant virus growth was restored in epithelial cells by expression of bovine viral diarrhea virus (BVDV) NPRO protein targeting IRF3 as part of the cGAS/STING pathway or alternatively by expression of fibroblast cell receptor PDGFRA. HCMV pp65 is a T cell target antigen and a recombinant adenovirus encoding GP83 was evaluated as a vaccine. In GPCMV challenge studies, vaccinated animals had varying levels of protection against wild type virus with a protective response against 22122 prototype strain but little protection against a novel clinical strain of GPCMV (TAMYC), despite 100% identity in GP83 protein sequences. Overall, GP83 is a functional pp65 homolog with novel importance for epithelial cell infection but a GP83 T cell response provides limited vaccine efficacy.ImportanceCongenital CMV (cCMV) is a leading cause of cognitive impairment and deafness in newborns and a vaccine is a high priority. The guinea pig is the only small animal model for cCMV but requires guinea pig cytomegalovirus (GPCMV). The translational impact of GPCMV research is potentially reduced if the virus does not encode functional HCMV homolog proteins. This study demonstrates that tegument protein GP83 (pp65 homolog) is involved in innate immune evasion and highly important for infection of non-fibroblast cells via the viral glycoprotein pentamer complex (PC)-dependent endocytic entry pathway. The PC pathway is highly significant for virus dissemination and disease in the host, including cCMV. A GP83 candidate Ad-vaccine strategy in animals induced a cell-mediated response but failed to provide cross strain protection against a novel clinical strain of GPCMV. Results suggest that the pp65 antigen provides very limited efficacy as a stand-alone vaccine, especially in cross strain protection.

Project description:UnlabelledGuinea 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.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.

Project description:The development of a vaccine against congenital human cytomegalovirus (HCMV) infection is a major priority. The pentameric complex (PC) of virion envelope proteins gH, gL, UL128, UL130, and UL131A is a key vaccine target. To determine the importance of immunity to the homologous PC encoded by guinea pig cytomegalovirus (GPCMV) in preventing congenital CMV, PC-intact and PC-deficient live-attenuated vaccines were generated and directly compared for immunogenicity and efficacy against vertical transmission in a vertical transmission model. A virulent PC-intact GPCMV (PC/intact) was modified by galK mutagenesis either to abrogate PC expression (PC/null; containing a frame-shift mutation in GP129, homolog of UL128) or to delete genes encoding three MHC Class I homologs and a protein kinase R (PKR) evasin while retaining the PC (3DX/Δ145). Attenuated vaccines were compared to sham immunization in a two-dose preconception subcutaneous inoculation regimen in GPCMV seronegative Hartley guinea pigs. Vaccines induced transient, low-grade viremia in 5/12 PC/intact-, 2/12 PC/null-, and 1/11 3DX/Δ145-vaccinated animals. Upon completion of the two-dose vaccine series, ELISA titers for the PC/intact group (geometic mean titer (GMT) 13,669) were not significantly different from PC/null (GMT 8127) but were significantly higher than for the 3DX/Δ145 group (GMT 6185; p < 0.01). Dams were challenged with salivary gland-adapted GPCMV in the second trimester. All vaccines conferred protection against maternal viremia. Newborn weights were significantly lower in sham-immunized controls (84.5 ± 2.4 g) compared to PC/intact (96 ± 2.3 g), PC/null (97.6 ± 1.9 g), or 3DX/Δ145 (93 ± 1.7) pups (p < 0.01). Pup mortality in sham-immunized controls was 29/40 (73%) and decreased to 1/44 (2.3%), 2/46 (4.3%), or 4/40 (10%) in PC/intact, PC/null, or 3DX/Δ145 groups, respectively (all p < 0.001 compared to control). Congenital GPCMV transmission occurred in 5/44 (11%), 16/46 (35%), or 29/38 (76%) of pups in PC/intact, PC/null, or 3DX/Δ145 groups, versus 36/40 (90%) in controls. For infected pups, viral loads were lower in pups born to vaccinated dams compared to controls. Sequence analysis demonstrated that infected pups in the vaccine groups had salivary gland-adapted GPCMV and not vaccine strain-specific sequences, indicating that congenital transmission was due to the challenge virus and not vaccine virus. We conclude that inclusion of the PC in a live, attenuated preconception vaccine improves immunogenicity and reduces vertical transmission, but PC-null vaccines are equal to PC-intact vaccines in reducing maternal viremia and protecting against GPCMV-related pup mortality.

Dataset Information

A Homolog Pentameric Complex Dictates Viral Epithelial Tropism, Pathogenicity and Congenital Infection Rate in Guinea Pig Cytomegalovirus.

Publications

A Homolog Pentameric Complex Dictates Viral Epithelial Tropism, Pathogenicity and Congenital Infection Rate in Guinea Pig Cytomegalovirus.

Similar Datasets

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