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CG dinucleotide codon removal improves expression of HIV-1 reporter viruses in humanized mice

Benitez Moreno, Mariana A (2021) CG dinucleotide codon removal improves expression of HIV-1 reporter viruses in humanized mice. Master's Thesis, University of Pittsburgh. (Unpublished)

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Abstract

Two replication-competent CCR5-tropic HIV-1 reporter constructs were developed that encode either nanoluciferase (nLuc) or a near-infrared fluorescent protein (iRFP) for in vivo imaging of HIV-1 infection in humanized mice. However, the original versions of these reporters had reduced expression after 5 weeks post-infection. We hypothesized that CG dinucleotides present in the reporter genes could lead to reduced expression. Thus, we eliminated all CG dinucleotides with synonymous mutations in the reporter genes. Our objectives were to measure the intensities of HIV-1 p24 and reporter expression in the spleens of humanized mice that were infected with the original or codon-optimized (CO) reporter viruses. We hypothesized that CO reporter viruses would have improved reporter expression and would correlate better with HIV-1 p24 staining. Spleens were collected from humanized mice that had been infected for 7-15 weeks with one of the original reporter viruses or one of the CO reporter viruses. Thin sections of spleen were stained with antibodies against HIV-1 p24 and nLuc. Immunofluorescence of p24, nLuc, and iRFP was measured by confocal microscopy and analyzed for fluorescence intensity and co-localization. The intensity of p24 expression was similar in the spleens of mice infected with original or the CO HIV-1 reporter viruses. This was consistent with plasma viremia levels remaining high for animals infected with either virus. In contrast, the intensity of reporter expression was greater in the spleens of humanized mice infected with CO HIV-1 reporter viruses compared to mice infected with original HIV-1 reporter viruses. The difference was greater for iRFP virus, which originally had 138 CG dinucleotides in the reporter gene, than for nLuc virus, which had 38 CG dinucleotides in the reporter gene. These results correlated with whole animal reporter imaging. These findings suggest that CG dinucleotide codon removal improves the expression of the reporter genes in HIV-infected spleen cells in humanized mice. The fluorescence imaging analysis successfully quantified the HIV-1 p24 and reporter gene expression in HIV-1 infected mice spleens that can be applicable in other studies. Future in vivo imaging studies will be performed using CO reporter viruses.


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Details

Item Type: University of Pittsburgh ETD
Status: Unpublished
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Benitez Moreno, Mariana Amab612@pitt.edumab612
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Thesis AdvisorAmbrose, Zandreazaa4@pitt.eduzaa4
Committee MemberMartinson, Jeremyjmartins@pitt.edujmartins
Committee MemberMailliard, Robbiermb19@pitt.edurmb19
Date: 11 May 2021
Date Type: Publication
Defense Date: 16 April 2021
Approval Date: 11 May 2021
Submission Date: 29 April 2021
Access Restriction: No restriction; Release the ETD for access worldwide immediately.
Number of Pages: 36
Institution: University of Pittsburgh
Schools and Programs: Graduate School of Public Health > Infectious Diseases and Microbiology
Degree: MPH - Master of Public Health
Thesis Type: Master's Thesis
Refereed: Yes
Uncontrolled Keywords: cg dinucleotide, HIV-1, humanized mice, reporter virus,
Date Deposited: 11 May 2021 20:16
Last Modified: 14 May 2021 20:12
URI: http://d-scholarship.pitt.edu/id/eprint/41062

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  • CG dinucleotide codon removal improves expression of HIV-1 reporter viruses in humanized mice. (deposited 11 May 2021 20:16) [Currently Displayed]

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