Construction and characterization of chimeric BHIV (BIV/HIV-1) viruses carrying the bovine immunodeficiency virus gag gene

Abstract

Aim: To explore the possibility of the replacement of the gag gene between human immunodeficiency virus and bovine immunodeficiency virus, to achieve chimeric virions, and thereby gain a new kind of AIDS vaccine based on BHIV chimeric viruses.

Methods: A series of chimeric BHIV proviral DNAs differing in the replacement regions in gag gene were constructed, and then were transfected into 293T cells. The expression of chimeric viral genes was detected at the RNA and protein level. The supernatant of 293T cell was ultra centrifuged to detect the probable chimeric virion. Once the chimeric virion was detected, its biological activities were also assayed by infecting HIV-sensitive MT4 cells.

Results: Four chimeric BHIV proviral DNAs were constructed. Genes in chimeric viruses expressed correctly in transfected 293T cells. All four constructs assembled chimeric virions with different degrees of efficiency. These virions had complete structures common to retroviruses and packaged genomic RNAs, but the cleavages of the precursor Gag proteins were abnormal to some extent. Three of these virions tested could attach and enter into MT4 cells, and one of them could complete the course of reverse transcription. Yet none of them could replicate in MT4 cells.

Conclusion: The replacement of partial gag gene of HIV with BIV gag gene is feasible. Genes in chimeric BHIVs are accurately expressed, and virions are assembled. These chimeric BHIVs (proviral DNA together with virus particles) have the potential to become a new kind of HIV/AIDS vaccine.

Figure 1

The genomic structure of four chimeric BHIV DNAs. Yellow: CMV promoter; white: HIV-1 gene; blue: BIV gene.

Figure 2

RT-PCR results in 293T cells. A: HIV-1 tat: 1.DL2000, 2. Negative control 3. pCG1, 4. pcG2, 5. pcG3, 6. pcG5; B: BIV gag: 1. Negative control, 2. pCG1, 3. pCG2, 4. pCG3, 5. pCG5.

Figure 3

Western blot of 293T cells transfected by chimeric proviral DNAs. Western blot with rabbit anti-BIV CA serum. Lane 1: Marker; lane 2: 293T cell protein as negative control; lane 3: pCG1; lane 4: pCG2; lane 5: pCG3; lane 6: pCG5.

Figure 4

Chimeric virus particles observed by EM.

Figure 5

Western blot of chimeric virions assembled from 293T cells after transfection. First antibody was anti-BIV CA. Lane 1: Marker; lane 2: 293T cell protein as negative control; lane 3: pCG1; lane 4: pCG2; lane 5: pCG3; lane 6: pCG5.

Figure 6

Relative reverse transcriptase activities of chimeric virions in 1 mL supernatant of 293T cells (mean±SD, n = 4). All samples are positive to negative control (^bP<0.01 vs negative control). The RT activity of pCG1 virion was lower than the other three chimeric virions and HIV positive control (^dP<0.01 vs pCG1).

Figure 7

HIV-1 env gene RT-PCR results of chimeric virions. 1. pCG1; 2. pCG2; 3. pCG3, 4. pCG5; 5. DL2000 marker; 6-9: Sample of pCG1, pCG2, pCG3, pCG5 RNAs without adding reverse transcriptase as negative control.

Figure 8

Western blot of chimeric virions infecting MT4 cells. First antibody was anti-BIV CA. Lane 1: pCG5 virion as positive control; lane 2: MT4 cell protein as negative control; lane 3: pCG2; lane 4: pCG3; lane 5: pCG5.

Figure 9

HIV-1 env gene PCR results of MT4 genomic DNA after infecting chimeric virions. 1. pCG2; 2. pCG3; 3. pCG5; 4. DL2000 marker; 5-7: Culture medium of pCG2, pCG3, pCG5 infecting MT4 (in last 8 h) as negative control.

Figure 10

Sketch map of deduced sequential cleavage of chimeric Gag precursor by HIV-1 protease (p1 between NC and p6 is not concerned); numbers show the sequence of the cleavage. Blue: from BIV; white: from HIV.