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. 2010:4:225-32.
doi: 10.2174/1874431101004010225. Epub 2010 Dec 3.

In Vivo validation of a bioinformatics based tool to identify reduced replication capacity in HIV-1

Christina M R Kitchen  1 , Paul Krogstad, Scott G Kitchen
Affiliations

In Vivo validation of a bioinformatics based tool to identify reduced replication capacity in HIV-1

Christina M R Kitchen et al. Open Med Inform J. 2010.

Abstract

Although antiretroviral drug resistance is common in treated HIV infected individuals, it is not a consistent indicator of HIV morbidity and mortality. To the contrary, HIV resistance-associated mutations may lead to changes in viral fitness that are beneficial to infected individuals. Using a bioinformatics-based model to assess the effects of numerous drug resistance mutations, we determined that the D30N mutation in HIV-1 protease had the largest decrease in replication capacity among known protease resistance mutations. To test this in silico result in an in vivo environment, we constructed several drug-resistant mutant HIV-1 strains and compared their relative fitness utilizing the SCID-hu mouse model. We found HIV-1 containing the D30N mutation had a significant defect in vivo, showing impaired replication kinetics and a decreased ability to deplete CD4+ thymocytes, compared to the wild-type or virus without the D30N mutation. In comparison, virus containing the M184V mutation in reverse transcriptase, which shows decreased replication capacity in vitro, did not have an effect on viral fitness in vivo. Thus, in this study we have verified an in silico bioinformatics result with a biological assessment to identify a unique mutation in HIV-1 that has a significant fitness defect in vivo.

Keywords: Bayesian; HIV-1; bioinformatics; exchangeable on subsets; in vivo validation.; prior model selection; replication capacity; variable selection.

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Figures

Fig. (1)
Fig. (1)
Prior illustration for the models. For each codon position j, the column shows δj, the prior 0/1 indicator if codon j is important to the fitness phenotype. The curve shows the prior distribution on the regression coefficient βj. (A) Model 1, the uninformative prior. All codon positions have the same prior. (B) The Exchangeable on Subsets Prior (ESP). In the figure, 4 subsets are shown. Each codon within a subset has the same prior δj and prior distribution on βj. Different subsets have different values for the priors on the δj’s and βj’s. The four subsets are shown in different colors.
Fig. (2)
Fig. (2)
Replication kinetics of wild-type, M184V-containing, D30N-containing, and M184V and D30N-containing viruses in PHA-stimulated PBMC. Viral p24 antigen production was assessed at days 5 and 8 post infection. The results shown are representative of 3 independent experiments with PBMC derived from different donors.
Fig. (3)
Fig. (3)
Viral replication of wild-type, M184V-containing, D30N-containing, and M184V and D30N-containing viruses in vivo. Thy/liv implants were infected, separately (n=5 mice per group), with identical amounts of infectious units of each virus. Implants were analyzed following biopsy of infected tissue by quantitative PCR for HIV proviral DNA at 3, 5, and 7 weeks following infection. The amounts of proviral DNA are provided as copies of full length proviral DNA per 100,000 cells, as determined by the use of HIV and human β-globin specific primers and quantitative comparison to known controls. Mock infected controls were negative for HIV proviral DNA (not shown).
Fig. (4)
Fig. (4)
Depletion of CD4-bearing cells following infection with wild-type, M184V-containing, D30N-containing, and M184V and D30N-containing viruses in vivo. (A) CD4 versus CD8 flow cytometry staining profiles were determined on biopsied thy/liv tissue at the indicated times following infection with one of the four indicated viruses. Cells were stained with monoclonal antibodies (and their corresponding fluorochromes) to CD45 (FITC), CD8 (ECD), and CD4 (APC). CD4 and CD8 profiles of this population are given by gating on the human CD45+ population of cells. The percentages of cells in each quadrant are indicated. The data is representative of one mouse per group (total of 5 mice per group) of a total of three experiments. (B) Graphic representation of the percentage of total CD4+ cells in the respective thy/liv implants over time.
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