Defining stable reference genes in HIV latency reversal experiments

Cristina Ceriani; Gabrielle S Streeter; Kena J Lemu; Katherine S James; Simon Ghofrani; Brigitte Allard; Bonnie E Shook-Sa; David M Margolis; Nancie M Archin

doi:10.1128/JVI.02305-20

Defining stable reference genes in HIV latency reversal experiments

J Virol. 2021 May 10;95(11):e02305-20. doi: 10.1128/JVI.02305-20. Epub 2021 Mar 24.

Authors

Cristina Ceriani^{1

2}, Gabrielle S Streeter³, Kena J Lemu¹, Katherine S James^{1

2}, Simon Ghofrani^{1

2}, Brigitte Allard^{1

2}, Bonnie E Shook-Sa³, David M Margolis^{1

2

4}, Nancie M Archin^{5

2}

Affiliations

¹ UNC HIV Cure Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27514, United States.
² Department of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27154, United States.
³ Biostatistics Core, Center for AIDS Research, University of North Carolina, Chapel Hill, NC 27514, USA.
⁴ Department of Microbiology and Immunology, The University of North Carolina at Chapel Hill, Chapel Hill, NC, United States.
⁵ UNC HIV Cure Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27514, United States nancie_archin@med.unc.edu.

Abstract

Quantification of cell associated HIV RNA (ca-RNA) is one of the most important and commonly used methods to evaluate the performance of latency-reversing agents (LRAs). Copies of HIV RNA measured by qPCR, are often normalized to the input RNA or cell number. However, these could be affected by biological variability and/or technical errors, which can be avoided by using an internal reference gene. To obtain reliable data, it is essential to select stable reference genes (RGs) of which the expression is not influenced by biological variability, the type of cells, or the LRAs used. However, to date, no study has carefully evaluated RG stability following LRA exposure. We analyzed the stability of six widely used RGs (GAPDH, TBP, YWHAZ, UBE2D2, HPRT1 and RPL27A) in human PBMC and CD4+ T cells. LRA exposure significantly influenced the stability of these RGs. Overall, TBP, UBE2D2, and RPL27A were the most stable RGs in all tested conditions. TBP was generally the most stable RG whereas GAPDH varied the most. Finally, we evaluated the impact of applying different RG normalizers to host genes and HIV ca-RNA data. Altered results were observed both in host and HIV gene expression when unstable RGs were used. Our data underline the importance of testing the stability of RGs utilized to evaluate LRA-induced HIV ca-RNA expression. To our knowledge, this is the first careful evaluation of the stability of RGs after LRA exposure and will significantly contribute to the quality of data analysis in regard to gene expression.IMPORTANCELatency-reversing agents (LRAs) are ubiquitously used in the "shock-and-kill" HIV cure strategy and their performance is often evaluated by ex-vivo quantification of cell associated HIV RNA. HIV RNA, measured by qPCR, is often normalized to internal reference genes, but the expression of these genes should not be influenced by the experimental settings. We found that treatment of human PBMC and CD4+ T cells with LRAs significantly altered the expression of several commonly used reference genes, such as GAPDH. Finally, we evaluate the impact of different reference genes on normalization of host genes and HIV cell associated RNA expression and demonstrated that using unstable reference genes dramatically altered experimental outcome. Our data highlight the importance of using reference genes that are unaffected by LRAs under study to correctly evaluate host gene and cell associated HIV RNA expression induced by latency-reversing agents.

Abstract

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