Research Article

Magnesium Functions as Superior Co-factor for Measuring Reverse Transcriptase Activity of HIV-1, HIV-2, and SIV

Salequl Islam
Jahangirnagar University, Bangladesh
* Corresponding author
Mohammad Ali Moni
School of Public Health and Community Medicine, UNSW Sydney, Australia
Atsushi Tanaka
Gunma University Graduate School of Medicine, Japan
Hiroo Hoshino
Gunma University Graduate School of Medicine, Japan
DOI icon 10.24018/ejmed.2020.2.4.433

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Submitted 2020-08-09
Published 2020-08-23

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Article Main Content

This study compared different detection methods of human/simian immunodeficiency virus (HIV/SIV) infections in the cell line systems; notably, i) Indirect immunofluorescence assay (IFA), ii) integrated proviral DNA detection, iii) detection of syncytia, iv) measurement of reverse transcriptase (RT) activity. RTs of various retroviruses require cations, including Mg2+, Mn2+, Ni2+, and Cu2+, for their enzyme-activities. The study further compared the roles of Mg2+ and Mn2+ as cofactors for RT activities of freshly harvested HIV-1, HIV-2, and SIV. The NP-2/CD4/coreceptor cells were seeded for overnight and infected with viral inoculums at a multiplicity of infection (MOI) 1.0. The cells were passaged regularly in a 2-3 days interval and maintained up to 2 weeks.  Infected cells were detected by indirect immunofluorescence assay (IFA). Multinucleated giant cells (MGC) in syncytia were quantified by Giemsa-staining. Proviral DNA was detected by PCR, and reverse transcriptase (RT) activity was measured.  Two different cations, Mg2+ and Mn2+ were used as cofactors for RT assay. We found all the strains of HIV-1, HV-2 and SIV to infection in the cell line conveniently. IFA had identified all the viral infections in the infected cells. Proviral DNA detection, syncytia formation was observed in the infected cells. We found a better performance of Mg2+ as cofactor over Mn2+ in RT assay for HIV-1, HIV-2, SIV. Different four detection techniques of HIV/SIV infections show high level of agreement in the NP-2-based cell line system. Mg2+ remains a better cofactor for RT.

Keywords: HIV/SIV, RT assay, IFA, Proviral DNA, Syncytia

References

  1. H. zur Hausen, Viruses in human cancers. Eur J Cancer, 1999. 35(14): p. 1878-85.
    DOI  |   Google Scholar
  2. P.A. Lazo, Structure, DNaseI hypersensitivity and expression of integrated papilloma virus in the genome of HeLa cells. Eur J Biochem, 1987. 165(2): p. 393-401.
    DOI  |   Google Scholar
  3. Y. Soda, et al., Establishment of a new system for determination of coreceptor usages of HIV based on the human glioma NP-2 cell line. Biochem Biophys Res Commun, 1999. 258(2): p. 313-21.
    DOI  |   Google Scholar
  4. A.G. Dalgleish, et al., The CD4 (T4) antigen is an essential component of the receptor for the AIDS retrovirus. Nature, 1984. 312(5996): p. 763-7.
    DOI  |   Google Scholar
  5. J.S. Allan, J. Strauss, and D.W. Buck, Enhancement of SIV infection with soluble receptor molecules. Science, 1990. 247(4946): p. 1084-8.
    DOI  |   Google Scholar
  6. H. Choe, et al., The beta-chemokine receptors CCR3 and CCR5 facilitate infection by primary HIV-1 isolates. Cell, 1996. 85(7): p. 1135-48.
    DOI  |   Google Scholar
  7. T. Dragic, et al., HIV-1 entry into CD4+ cells is mediated by the chemokine receptor CC-CKR-5. Nature, 1996. 381(6584): p. 667-73.
    DOI  |   Google Scholar
  8. S. Islam, et al., CKR-L3, a deletion version CCR6-isoform shows coreceptor-activity for limited human and simian immunodeficiency viruses. BMC Infect Dis, 2014. 14: p. 354.
    DOI  |   Google Scholar
  9. B. Forghani, J.W. Hurst, and C.S. Chan, Advantages of a human immunodeficiency virus type 1 (HIV-1) persistently infected HeLa T4+ cell line for HIV-1 indirect immunofluorescence serology. J Clin Microbiol, 1991. 29(10): p. 2266-72.
    DOI  |   Google Scholar
  10. R.S. Blumberg, et al., Detection of human T-cell lymphotropic virus type III-related antigens and anti-human T-cell lymphotropic virus type III antibodies by anticomplementary immunofluorescence. J Clin Microbiol, 1986. 23(6): p. 1072-7.
    DOI  |   Google Scholar
  11. S. Islam, et al., X4-tropic human immunodeficiency virus IIIB utilizes CXCR4 as coreceptor, as distinct from R5X4-tropic viruses. Microbiol Immunol, 2013. 57(6): p. 437-44.
    DOI  |   Google Scholar
  12. J.D. Lifson, et al., AIDS retrovirus induced cytopathology: giant cell formation and involvement of CD4 antigen. Science, 1986. 232(4754): p. 1123-7.
    DOI  |   Google Scholar
  13. A. Sylwester, et al., HIV-induced T cell syncytia are self-perpetuating and the primary cause of T cell death in culture. J Immunol, 1997. 158(8): p. 3996-4007.
     Google Scholar
  14. A.B. van 't Wout, H. Schuitemaker, and N.A. Kootstra, Isolation and propagation of HIV-1 on peripheral blood mononuclear cells. Nat Protoc, 2008. 3(3): p. 363-70.
    DOI  |   Google Scholar
  15. H. Kuno, et al., A simple and rapid reverse transcriptase assay for the detection of retroviruses in cell cultures. Cytotechnology, 1999. 29(3): p. 221-7.
    DOI  |   Google Scholar
  16. S. Gupta, et al., Can HIV reverse transcriptase activity assay be a low-cost alternative for viral load monitoring in resource-limited settings? BMJ Open, 2016. 6(1): p. e008795.
    DOI  |   Google Scholar
  17. A. Herschhorn and A. Hizi, Retroviral reverse transcriptases. Cell Mol Life Sci, 2010. 67(16): p. 2717-47.
    DOI  |   Google Scholar
  18. S.P. Goff. Retroviral reverse transcriptase: synthesis, structure, and function. J Acquir Immune Defic Syndr (1988), 1990. 3(8): p. 817-31.
     Google Scholar
  19. A.G. Filler and A.M. Lever, Effects of cation substitutions on reverse transcriptase and on human immunodeficiency virus production. AIDS Res Hum Retroviruses, 1997. 13(4): p. 291-9.
    DOI  |   Google Scholar
  20. K.J. Fenstermacher and J.J. DeStefano, Mechanism of HIV reverse transcriptase inhibition by zinc: formation of a highly stable enzyme-(primer-template) complex with profoundly diminished catalytic activity. J Biol Chem, 2011. 286(47): p. 40433-42.
    DOI  |   Google Scholar
  21. J.F. Sears, R. Repaske, and A.S. Khan, Improved Mg2+-based reverse transcriptase assay for detection of primate retroviruses. J Clin Microbiol, 1999. 37(6): p. 1704-8.
    DOI  |   Google Scholar
  22. Y. Kawakami, et al., Bruton's tyrosine kinase regulates apoptosis and JNK/SAPK kinase activity. Proc Natl Acad Sci U S A, 1997. 94(8): p. 3938-42.
    DOI  |   Google Scholar
  23. C. Nishiyama, et al., Overproduction of PU.1 in mast cell progenitors: its effect on monocyte- and mast cell-specific gene expression. Biochem Biophys Res Commun, 2004. 313(3): p. 516-21.
    DOI  |   Google Scholar
  24. K. Yamaguchi and R.A. Byrn, Clinical isolates of HIV-1 contain few pre-existing proteinase inhibitor resistance-conferring mutations. Biochim Biophys Acta, 1995. 1253(2): p. 136-40.
    DOI  |   Google Scholar
  25. L. Ratner et al., Complete nucleotide sequences of functional clones of the AIDS virus. AIDS Res Hum Retroviruses, 1987. 3(1): p. 57-69.
    DOI  |   Google Scholar
  26. N. Shimizu et al., A formylpeptide receptor, FPRL1, acts as an efficient coreceptor for primary isolates of human immunodeficiency virus. Retrovirology, 2008. 5: p. 52.
    DOI  |   Google Scholar
  27. M. Guyader, et al., Genome organization and transactivation of the human immunodeficiency virus type 2. Nature, 1987. 326(6114): p. 662-9.
    DOI  |   Google Scholar
  28. Y.M. Naidu et al., Characterization of infectious molecular clones of simian immunodeficiency virus (SIVmac) and human immunodeficiency virus type 2: persistent infection of rhesus monkeys with molecularly cloned SIVmac. J Virol, 1988. 62(12): p. 4691-6.
    DOI  |   Google Scholar
  29. A. Jinno, et al., Identification of the chemokine receptor TER1/CCR8 expressed in brain-derived cells and T cells as a new coreceptor for HIV-1 infection. Biochem Biophys Res Commun, 1998. 243(2): p. 497-502.
    DOI  |   Google Scholar
  30. H. Hoshino, et al., Establishment and characterization of 10 cell lines derived from patients with adult T-cell leukemia. Proc Natl Acad Sci U S A, 1983. 80(19): p. 6061-5.
    DOI  |   Google Scholar
  31. H.S. Iqbal, et al., Use of an HIV-1 reverse-transcriptase enzyme-activity assay to measure HIV-1 viral load as a potential alternative to nucleic acid-based assay for monitoring antiretroviral therapy in resource-limited settings. J Med Microbiol, 2007. 56(Pt 12): p. 1611-1614.
    DOI  |   Google Scholar
  32. Poiesz, B.J., et al., Detection and isolation of type C retrovirus particles from fresh and cultured lymphocytes of a patient with cutaneous T-cell lymphoma. Proc Natl Acad Sci U S A, 1980. 77(12): p. 7415-9.
    DOI  |   Google Scholar
  33. M.K. Kiptoo, S.S. Mpoke, and Z.W. Ng'ang'a, New indirect immunofluorescence assay as a confirmatory test for human immunodeficiency virus type 1. East Afr Med J, 2004. 81(5): p. 222-5.
    DOI  |   Google Scholar
  34. W. Heneine, et al., Detection of reverse transcriptase by a highly sensitive assay in sera from persons infected with human immunodeficiency virus type 1. J Infect Dis, 1995. 171(5): p. 1210-6.
    DOI  |   Google Scholar
  35. H. Pyra, J. Boni, and J. Schupbach, Ultrasensitive retrovirus detection by a reverse transcriptase assay based on product enhancement. Proc Natl Acad Sci U S A, 1994. 91(4): p. 1544-8.
    DOI  |   Google Scholar
  36. J. Silver, et al., An RT-PCR assay for the enzyme activity of reverse transcriptase capable of detecting single virions. Nucleic Acids Res, 1993. 21(15): p. 3593-4.
    DOI  |   Google Scholar
  37. E.C. Bolton, A.S. Mildvan, and J.D. Boeke, Inhibition of reverse transcription in vivo by elevated manganese ion concentration. Mol Cell, 2002. 9(4): p. 879-89.
    DOI  |   Google Scholar
  38. H. Huang, et al., Structure of a covalently trapped catalytic complex of HIV-1 reverse transcriptase: implications for drug resistance. Science, 1998. 282(5394): p. 1669-75.
    DOI  |   Google Scholar