Share this article on:

Defective HIV-1 provirus found in peripheral T lymphocytes and granulocytes in an AIDS patient imply viral infection of progenitor cells

Kaneda, Tsuguhiroa; Murakami, Takayaa; Hagiwara, Tomokoa; Hattori, Junkoa; Yamamoto, Kazukoa; Sato, Katsuhikob; Morishita, Takayukib; Utsumi, Makotoa


aDepartment of Clinical Research, Nagoya National Hospital (Tokai Area Central Hospital for AIDS Treatment and Research), Nagoya, 460-0001 Japan; and bDepartment of Microbiology, Aichi Prefectural Institute of Public Health, Nagoya, Japan.

Received: 21 December 2000;

revised: 1 February 2001; accepted: 6 February 2001.

The cells that simultaneously express both CD4 and chemokine receptor molecules, such as CD4 T lymphocytes, macrophages, dendritic, and microglial cells are the major target cells, and serve as reservoirs for HIV-1. For this reason, matured CD15 granulocytes or CD8 T lymphocytes in the peripheral blood should not be susceptible to HIV-1 because they do not express CD4 molecules on their cell surfaces. Nevertheless, in the present study, we found the HIV-1 provirus from CD8 T lymphocytes and CD15 granulocytes as well as from CD4 T lymphocytes by the polymerase chain reaction (PCR) method from one AIDS patient who manifested severe pancytopenia. The cellular DNA was prepared from CD8 and CD15 cells purified by an immuno-beads method. Subsequently, both reverse transcriptase (RT) and protease (P) genes were amplified by nested PCR, and then the PCR products were visualized by 1.2% agarose gel running. A contamination of CD4 cells into CD8 or CD15 cell preparations was suspected because the purification method used guarantees only a 95% purity. To clarify whether CD8 and CD15 cells possess HIV-1, we adopted two different methodologies; a DNA base sequence analysis of PCR products, and an HIV-1 detection by peptide nucleic acid-probed in-situ hybridization, which we established recently [1]. After DNA sequences of five cloned PCR products from each cell type were analysed, the amino acid sequences deduced from the DNA base sequence were compared. Some nonsense mutations were found in these genes. In the RT gene, nonsense mutations of W212# and W229# were found in four clones from CD4 cells. A K70# mutation was found in one clone from CD8 cells; however, no stop codons were found in any clone from CD15 cells. In the P gene, the same nonsense mutation of W42# was found in four clones from CD4 and CD15 cells. In contrast, no nonsense mutations were found in any clone from CD8 cells. These findings suggest that the majority of HIV-1 proviruses in CD4 and CD15 cells are not complete, at least in terms of possessing stop codons in RT or P genes. They also suggest that CD4 cells scarcely contaminate the CD8 cell or CD15 cell preparations because of the differences in the position of the codon where the nonsense mutation occurred. The peptide nucleic acid-probed in-situ hybridization method clearly detected the HIV-1 provirus in the nuclei of CD8 and CD15 cells. The percentages of the HIV-1 provirus-positive CD8 and CD15 cells were 7.0 and 6.0%, respectively. As the HIV-1 provirus-positive CD4 cells were 11.1%, slight contamination of CD4 cells to CD8 or CD15 cell preparations did not affect their positivities (see Table 1).

Several studies reported the existence of a defective HIV-1 provirus in periperal blood mononuclear cells [2–4]. Abnormalities such as nonsense mutation or deletion were frequently found, for example, in the Vif, Nef, and TaT genes. Therefore, the existence of the nonsense mutation also found in the RT or P genes in the present study is not considered to be rare.

Next, we would like to discuss the biological significance of the existence of a deficient HIV-1 provirus in CD8 or CD15 cells. Some studies have reported an HIV-1 infection of haematopoietic progenitor cells [5,6], especially CD34+CD4+ cells, and of the progenitor cells of colony-forming units–granulocyte/macrophage, which can differentiate to granulocytes or macrophages. In addition, the harbouring of HIV-1 in CD8 T lymphocytes was reported [7]. On the other hand, as for the fate of the HIV-1-infected cells, most of the cells attacked by HIV-1 may be destroyed [8,9]. However, some can still survive if HIV-1 obtains nonsense mutations for some reason in the genes that are essential for viral replication and survival. Such cells possessing a deficient HIV-1 provirus subsequently grow and differentiate to mature blood cells. The deficient HIV-1 provirus in matured CD8 or CD15 cells thus indicates a history of past infection by HIV-1. In other words, it serves as a marker for past HIV-1 infection. If this occurs, the population of stem cells or progenitor cells should be decreased compared with normal levels, and finally may become one of the serious causes of pancytopenia or haematopoiesis dysfunction. As the locations where nonsense mutations occurred were different between CD4, CD8, and CD15 cells, we speculate that, at least in this patient, HIV-1 infected progenitor cells of lymphocytes and granulocytes, rather than haematopoietic progenitor cells.

Tsuguhiro Kanedaa

Takaya Murakamia

Tomoko Hagiwaraa

Junko Hattoria

Kazuko Yamamotoa

Katsuhiko Satob

Takayuki Morishitab

Makoto Utsumia

Back to Top | Article Outline


1. Murakami T, Hagiwara T, Yamamoto K, et al. A novel method for detecting HIV-1 by non-radioactive in situ hybridization: application of peptide nucleic acid probe and catalyzed signal amplification. J Pathol 2001, in press.
2. Chang LJ, Zhang C. Infection and replication of Tat− human immunodeficiency viruses: genetic analyses of LTR and tat mutations in primary and long-term human lymphoid cells. Virology 1995, 211: 157 –169.
3. Tominaga K, Kato S, Negishi M, Takano T. A high frequency of defective vif genes in peripheral blood mononuclear cells from HIV type 1-infected individuals. AIDS Res Hum Retroviruses 1996, 12: 1543 –1549.
4. Salvi R, Garbuglia AR, Di Caro A, Pulciani S, Montella F, Benedetto A. Grossly defective nef gene sequences in a human immunodeficiency virus type 1-seropositive long-term nonprogressor. J Virol 1998, 72: 3646 –3657.
5. Zhao SF, Li W, Dornadula G. et al. Chemokine receptors and the molecular basis for human immunodeficiency virus type 1 entry into peripheral hematopoietic stem cells and their progeny. J Infect Dis 1998, 178: 1623 –1634.
6. Chelucci C, Hassan HJ, Locardi C. et al. In vitro human immunodeficiency virus-1 infection of purified hematopoietic progenitors in single-cell culture. Blood 1995, 85: 1181 –1187.
7. Semenzato G, Agostini C, Ometto L. et al. CD8+ T lymphocytes in the lung of acquired immunodeficiency syndrome patients harbor human immunodeficiency virus type 1. Blood 1995, 85: 2308 –2314.
8. Donahue RE, Johnson MM, Zon LI, Clark SC, Groopman JE. Suppression of in vitro haematopoiesis following human immunodeficiency virus infection. Nature 1987, 326: 200 –203.
9. Katsikis PD, Wunderlich ES, Smith CA, Herzenberg LA, Herzenberg LA. Fas antigen stimulation induces marked apoptosis of T lymphocytes in human immunodeficiency virus-infected individuals. J Exp Med 1995, 181: 2029 –2036.
© 2001 Lippincott Williams & Wilkins, Inc.