Infection by Diverse HIV-1 Subtypes Leads to Different Elevations in HERV-K Transcriptional Levels in Human T Cell Lines

Abstract

Human endogenous retroviruses (HERVs) make up ~8% of the human genome, and for millions of years, they have been subject to strict biological regulation. Many HERVs do not participate in normal physiological activities in the body. However, in some pathological conditions, they can be abnormally activated. For example, HIV infection can cause abnormal activation of HERVs, and under different infection conditions, HERV expression may be different. We observed significant differences in HERV-K transcription levels among HIV-1 subtype-infected individuals. The transcriptional levels in the HERV-K gag region were significantly increased in HIV-1 B subtype-infected patients, while the transcriptional levels in the HERV-K pol region were significantly increased in CRF01_AE and CRF07_BC subtype-infected patients. In vitro, the transcriptional levels of HEVR-K were increased 5-fold and 15-fold in MT2 cells transfected with two different HIV-1 strains (B and CRF01_AE, respectively). However, there was no significant difference in transcriptional levels among regions of HERV-K. When MT2 cells were infected with different subtypes of HIV-1 Tat proteins (B, CRF01_AE), which is constructed by lentiviruses, and the transcription levels of HERV-K were increased 4-fold and 2-fold, respectively. Thus, different subtypes of HIV-1 have different effects on HERV-K transcription levels, which may be caused by many factors, not only Tat protein.

Keywords: CRF01_AE; HERV-K (HML-2); HIV-1; RNAscope; Tat.

Figure 1

(A) Blue represents HIV-1B subtype-infected persons, red represents HIV-1 CRF01_AE subtype-infected persons, purple represents HIV-1 CRF07_BC-infected persons, yellow represents other CRFs and URFS-infected persons, and green represents the healthy population control group. The Y-axis is ΔCT = CT_HERV−K−CT_β−actin. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001. (B) Red represents transcriptional levels in the HERV-K gag region, blue represents transcriptional levels in the HERV-K pol region, and green represents transcriptional levels in the HERV-K env region. The Y-axis is ΔCT = CT_HERV−K−CT_β−actin. The X-axis represents log¹⁰ of the viral load in each infected person. Connecting curves are indicated.

Figure 2

(A) Red represents MT2 cells, blue represents TZM-bl cells, black represents H9 cells, and green represents 293T cells. The X-axis is the time of infection. The Y-axis represents log¹⁰ of the HIV-1 pol load, and the units are indicated in the title of the Y-axis. Connecting curves are indicated. (B) Red represents MT2 cells, blue represents TZM-bl cells, black represents H9 cells, and green represents 293T cells. The X-axis is the time of infection. The Y-axis shows the fold increase in the transcription level in the HERV-K env region calculated by the 2^−ΔΔCT method. Standard errors for triplicate experiments are indicated. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001. All comparisons were relative to uninfected control cells.

Figure 3

(A) DAPI was used to stain and label the nuclei, green fluorescent proteins (GFP) with a special probe were used to stain and label HEVR-K env region mRNA, and red fluorescent proteins (RFPs) with a special probe were used to stain and label HIV-1 gag-pol region mRNA. The positive and negative controls used standard control probes provided in the kit (ACD). (B) MT2 cells in both the IIIB group and GX002 group exhibited notable cytopathic effects (CPEs) after 48 h of infection. The microscopic view of uninfected MT2 cells is shown.

Figure 4

(A) Red represents the GX002 group, blue represents the IIB group, green represents the NL4-3 group. The X-axis is the time of infection. The Y-axis represents log¹⁰ of the HIV-1 pol load, and the units are indicated in the title of the Y-axis. Standard errors for triplicate experiments are indicated. (B) Red represents the GX002 group, blue represents the IIB group, green represents the NL4-3 group. The X-axis is the time of infection. The Y-axis represents the value of p24 antigen, and the units are indicated in the title of the Y-axis. Standard errors for triplicate experiments are indicated. (C) The bars represent the groups indicated in the figure. The X-axis is the time of infection. The Y-axis shows the fold increase in transcription level in the HERV-K gag, pol, env region calculated by the 2^−ΔΔCT method. Standard errors for triplicate experiments are indicated. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001. All comparisons were relative to uninfected control cells.

Figure 5

(A) pNL4-3 and pGX002 in the DOX(+) groups expressed large amounts of Tat protein; MT2 cells in the uninfected group and the blank plasmid-transfected group DOX(+) did not express Tat protein; all MT2 cells in the DOX(-) groups did not express Tat protein. (B) Red represents the pGX002 group, blue represents the pNL4-3 group. The Y-axis represents log¹⁰ of the Tat gene load, and the units are indicated in the title of the Y-axis. Standard errors for triplicate experiments are indicated. (C) Blue represents HERV-K gag, red represents HERV-K pol, green represents HERV-K env. The X-axis is Tat subtype groups. The Y-axis shows the fold increase in transcription level in the HERV-K gag, pol, env region calculated by the 2^−ΔΔCT method. Standard errors for triplicate experiments are indicated. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.