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Functional analysis of human cytomegalovirus UL/b' region using SCID-hu mouse model
Kalpana Dulal1, Tong Cheng2, Lianwei Yang2
1Department of Microbiology and Molecular Genetics, Rutgers-New Jersey Medical School, Newark, New Jersey.
Abstract:
Human cytomegalovirus (HCMV) attenuated strains, Towne, and AD169, differ from prototypic pathogenic strains, such as Toledo, in that they are missing a ∼15-kb segment in the UL/b' region. In contrast to the attenuated strains, Toledo can replicate in human tissue implants in SCID (SCID-hu) mice. Thus, this model provides a unique in vivo system to study the mechanism of viral pathogenesis. Twenty-two ORFs have been annotated in the UL/b' region, including tissue-tropic genes encoded in a pentameric gH/gl complex. To differentiate the role of the pentameric gH/gl complex versus the functions of other ORFs in the 15-kb region in supporting viral growth in vivo, a series of recombinant viral strains were constructed and their ability to replicate in SCID-hu mice was tested. The mutations in the Towne and AD169 strains were repaired to restore their pentameric gH/gl complex and it was found that these changes did not rescue their inability to replicate in the SCID-hu mice. Subsequently four deletion viruses (D1, D2, D3, and D4) in the 15-kb region from the Toledo strain were created. It was demonstrated that D2 and D3 were able to grow in SCID-hu mice, while D1 and D4 were not viable. Interestingly, co-infection of the implant with the D1 and D4 viruses could compensate their respective growth defect in vivo. The results demonstrated that rescuing viral epithelial tropism is not sufficient to revert the attenuation phenotype of AD169 or Towne, and pathogenic genes are located in the segments missing in D1 and D4 viruses. J. Med. Virol. 88:1417-1426, 2016. © 2016 Wiley Periodicals, Inc.
Insights
Human cytomegalovirus (HCMV) pathogenesis was studied in SCID-hu mice. Key pathogenic genes missing in attenuated strains were identified within the UL/b' region, crucial for in vivo viral replication.
Area of Science:
- Virology
- Immunology
- Genetics
Background:
- Attenuated human cytomegalovirus (HCMV) strains (Towne, AD169) lack a 15-kb UL/b' region segment present in pathogenic strains like Toledo.
- The SCID-hu mouse model allows in vivo study of HCMV pathogenesis, differentiating viral replication capabilities.
Purpose of the Study:
- To investigate the role of the UL/b' region, specifically the pentameric gH/gL complex and other ORFs, in HCMV's in vivo replication and pathogenesis.
- To identify specific genes within the 15-kb UL/b' deletion region responsible for pathogenic viral growth.
Main Methods:
- Construction of recombinant HCMV strains with repaired pentameric gH/gL complexes in attenuated strains.
- Generation of deletion mutants (D1-D4) within the 15-kb UL/b' region of the pathogenic Toledo strain.
- Testing the replication ability of engineered viral strains in human tissue implants within SCID-hu mice.
Main Results:
- Repairing the pentameric gH/gL complex in Towne and AD169 did not restore their SCID-hu mouse replication.
- HCMV deletion mutants D2 and D3 replicated in SCID-hu mice, while D1 and D4 were non-viable.
- Co-infection with D1 and D4 viruses rescued their individual growth defects in vivo, indicating functional complementation.
Conclusions:
- Restoring epithelial tropism alone is insufficient to reverse the attenuation of AD169 or Towne HCMV strains.
- Essential pathogenic genes for in vivo replication are located within the deleted UL/b' segments of the D1 and D4 mutants.
