A single amino acid substitution of the human immunodeficiency virus type 1 capsid protein affects viral sensitivity to TRIM5α
© Kuroishi et al; licensee BioMed Central Ltd. 2010
Received: 8 February 2010
Accepted: 7 July 2010
Published: 7 July 2010
Human immunodeficiency virus type 1 (HIV-1) productively infects only humans and chimpanzees but not Old World monkeys, such as rhesus and cynomolgus (CM) monkeys. To establish a monkey model of HIV-1/AIDS, several HIV-1 derivatives have been constructed. We previously reported that efficient replication of HIV-1 in CM cells was achieved after we replaced the loop between α-helices 6 and 7 (L6/7) of the capsid protein (CA) with that of SIVmac239 in addition to the loop between α-helices 4 and 5 (L4/5) and vif. This virus (NL-4/5S6/7SvifS) was supposed to escape from host restriction factors cyclophilin A, CM TRIM5α, and APOBEC3G. However, the replicative capability of NL-4/5S6/7SvifS in human cells was severely impaired.
By long-term cultivation of human CEMss cells infected with NL-4/5S6/7SvifS, we succeeded in rescuing the impaired replicative capability of the virus in human cells. Sequence analysis of the CA region of the adapted virus revealed a G-to-E substitution at the 116th position of the CA (G116E). Introduction of this substitution into the molecular DNA clone of NL-4/5S6/7SvifS indeed improved the virus' replicative capability in human cells. Although the G116E substitution occurred during long-term cultivation of human cells infected with NL-4/5S6/7SvifS, the viruses with G116E unexpectedly became resistant to CM, but not human TRIM5α-mediated restriction. The 3-D model showed that position 116 is located in the 6th helix near L4/5 and L6/7 and is apparently exposed to the protein surface. The amino acid substitution at the 116th position caused a change in the structure of the protein surface because of the replacement of G (which has no side chain) with E (which has a long negatively charged side chain).
We succeeded in rescuing the impaired replicative capability of NL-4/5S6/7SvifS and report a mutation that improved the replicative capability of the virus. Unexpectedly, HIV-1 with this mutation became resistant to CM TRIM5α-mediated restriction.
Human immunodeficiency virus type 1 (HIV-1) productively infects only humans and chimpanzees, but not Old World monkeys (OWM) such as cynomolgus (CM) and rhesus (Rh) monkeys . Unlike the replication of simian immunodeficiency virus isolated from macaques (SIVmac), HIV-1 replication is blocked early after viral entry, before the establishment of a provirus in OWM cells [1–3]. To establish a monkey model of HIV-1/AIDS, several viruses that are chimeras of HIV-1 and SIVmac (SHIV) have been constructed and tested for replicative capability in simian cells [4, 5]. The host range of HIV-1 was limited because of some intrinsic restriction factors in simian cells, such as ApoB mRNA editing catalytic subunit (APOBEC) 3G , cyclophilin A (CypA) [7–9], BST-2 (CD317; tetherin) [10, 11] and TRIM5α, a member of the tripartite motif (TRIM) family proteins . Rh and CM TRIM5α restrict HIV-1, but not SIVmac [13, 14]. A lack of functional TRIM5α expression in pig-tailed monkey enabled Hatziioannou et al. to construct a SHIV strain that differs from HIV-1 only in the vif gene and can efficiently replicate in pig-tailed monkeys . Although this virus was designed to escape from monkey APOBEC3G mediated restriction, this virus failed to grow in Rh and CM cells. Kamada et al. attempted to evade the restrictions mediated by CypA in OWM cells by replacing the loop between α-helices 4 and 5 (L4/5) of the HIV-1 capsid (CA) with that of SIVmac in addition to vif because CypA fails to bind to the L4/5 of SIVmac. However, this was not enough to escape from TRIM5α-mediated restriction .
TRIM5α consists of RING, B-box 2, coiled-coil, and SPRY (B30.2) domains . TRIM5α recognizes the multimerized CA of an incoming virus by its α-isoform specific SPRY domain [18–20]. Studies on chimeric TRIM5αs have shown that the determinant of the species-specific restriction against viral infection resides in the variable regions of the SPRY domain [21, 22]. On the other hand, we previously identified a single amino acid of the surface-exposed loop between α-helices 6 and 7 (L6/7) of the HIV-2 CA as a determinant of the susceptibility of HIV-2 to CM TRIM5α. On the basis of this finding, we have succeeded in improving simian-tropic HIV-1, which was generated by Kamada et al. , by replacing L6/7 of CA with those of SIVmac239 in addition to L4/5 and vif ; the new resultant virus has more efficient replication in CM cells. The resultant virus, NL-ScaVR6/7S, showed efficient replicative capability in CM cells; however, the replicative capability of this virus in human cells was severely impaired.
In the present report, we describe our efforts to rescue the impaired replicative capability of NL-ScaVR6/7S after long-term cultivation in human CEMss cells, and we report on the amino acid mutation that improved the replicative capability of this virus.
Materials and methods
For viral adaptation in human cells, 100 ng of p24 of NL-ScaVR6/7S , renamed in this report as NL-4/5S6/7SvifS, was inoculated into 1 × 106 of human T cell line CEMss cells. The infected culture was gradually expanded to keep the cell concentration at 1 × 106/mL. The culture supernatants were collected periodically, and p24 levels were measured with an ELISA kit (ZeptoMetrix, Buffalo, NY). Virus in the culture supernatant at day 42 after infection was designated NL-4/5S6/7SvifSd42, and inoculated into fresh CEMss cells. Six days after re-infection, the matrix (MA)-CA region of the integrated provirus was amplified by PCR from the genomic DNA of infected cells and cloned into pCR 2.1-TOPO vector (Invitrogen, Carlsbad, CA) to generate pTopo-MA-CAadp42. Nucleotide sequences of 6 independent clones were determined by ABI Prism 3100 Genetic Analyzer (Applied Biosystems, USA).
Cells and virus propagation
The human kidney adherent 293T cells were cultured in Dulbecco's modified Eagle medium supplemented with 10% heat-inactivated fetal bovine serum (FBS). The human T cell lines CEMss and MT4 were maintained in RPMI 1640 medium supplemented with 10% FBS. Virus stocks were prepared by transfection of 293T cells with HIV-1 NL4-3 and its derivatives using the calcium phosphate co-precipitation method. Viral titers were measured with an ELISA kit.
A cell line stably expressing CM or humanTRIM5α was established as described previously . Briefly, a pCEP4 plasmid (Invitrogen) encoding CM or human TRIM5α fused with HA tag in its C-terminus was transfected into TK-ts13 hamster cells. Transfected cells were then cultured in the presence of 0.3 mg/ml of hygromycin B (Gibco) for 14 days to remove untransfected cells. The expression of TRIM5α was confirmed by Western blot analysis of cell lysate with anti-HA antibody (HA High Affinity, Roch).
CEMss or MT4 cells (1 × 105) were infected with 20 ng of p24 of NL-4/5SvifS, NL-4/5S6/7SvifS, or NL-4/5SG116E6/7SvifS. The culture supernatants were collected periodically, and p24 levels were measured with an ELISA kit. To analyze the viral sensitivity to TRIM5α, 1 × 105 CEMss cells were first infected with SeV expressing each of the TRIM5αs at a multiplicity of infection of 10 plaque-forming units per cell and incubated at 37°C for 9 hours. Cells were then superinfected with 20 ng of p24 of HIV-1 NL4-3 or its derivatives. The culture supernatants were collected periodically, and the levels of p24 were measured with an ELISA kit.
For the single-round infection assay, CEMss or canine Cf2Th cells were infected with SeV expressing TRIM5α as described above, and super-infected with vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped HIV-1 clones expressing GFP. In case of TK-ts13 hamster cells stably expressing CM, human or CM-SPRY(-) TRIM5α, cells were infected with VSV-G pseudotyped lentivector expressing GFP under the control of cytomegalovirus promoter. Two days after infection, the cells were fixed by formaldehyde, and GFP expressing cells were counted with a flow-cytometer. The percentage of the GFP-positive cells in the presence of TRIM5α was divided by the percentage of GFP-positive cells in the presence of CM-SPRY (-) to define the percent of infection. The differences in percent infection between WT-GFP and G116E-GFP, or 4/5S6/7S-GFP and 4/5SG116E6/7S-GFP were statistically evaluated by using the unpaired t test.
Particle purification and Western blotting
The culture supernatants of 293T cells transfected with plasmids encoding HIV-1 NL4-3 derivatives were clarified by low-speed centrifugation. Nine milliliters of the resultant supernatants were layered onto a 2 mL cushion of 20% sucrose in phosphate buffered saline (PBS) and centrifuged at 35,000 rpm for 2 hours in a Beckman SW41 rotor. After centrifugation, the virion pellets were resuspended in PBS, and p24 antigen concentrations were measured by ELISA. Fifty nanograms of p24 of HIV-1 derivatives were applied to SDS-polyacrylamide gel electrophoresis, and the virion-associated proteins were transferred to a PVDF membrane. CA and CypA proteins were visualized with the anti-p24 antibody (Abcam) and anti-CypA antibody (Affinity BioReagents, Golden, CO), respectively.
The structure of the N-terminal domain of the HIV-1 CA protein (PDB number 1GWP)  was used as a template for building the domain model with the G116E substitution. The model was built using Modeller 9v4  and visualized with PyMOL v1.0r2 (The PyMOL Molecular Graphics System, http://pymol.sourceforge.net/).
A virus with SIVmac CA L4/5, L6/7, and vif gained efficient replicative capability after adaptation in human T cell line
Analysis of 95 HIV-1 strains in the Los Alamos HIV sequence databases http://www.hiv.lanl.gov/, including subtypes A to K of group M, revealed that there was no HIV-1 strain carrying glutamic acid at the 116th position of the CA, although this position was occupied with variable amino acid residues (35 strains carried glycine; 36, alanine; 9, threonine; 7, arginine; 6, glutamine; 1 each, isoleucine or aspartic acid).
A single amino acid substitution in CA rescued impaired replicative capability in human cells
The amino acid residue at the 116th position of the CA affects viral growth in the presence of TRIM5α
With respect to viral sensitivity to human TRIM5α, the growth of both NL-G116EvifS and NL-4/5SG116E6/7SvifS was slightly impaired compared with that of NL-vifS in CEMss cells over-expressing human TRIM5α. The growth of the NL4-3 virus was not affected by human TRIM5α, while that of NL-G116E was slightly suppressed by human TRIM5α. These results suggest that the viruses with G116E substitution were more sensitive to human TRIM5α although the G116E substitution occurred during long-term cultivation of human cells infected with NL-4/5S6/7SvifS. This excludes a possibility that the improved replicative capability of human cell-adapted virus is the result of escape from human TRIM5α-mediated restriction.
A G116E substitution affects viral sensitivity to CM TRIM5α-mediated restriction in a single-round infection assay
On the contrary, the GFP-expressing virus with G116E was more sensitive to humanTRIM5α expressed from the SeV in CEMss cells than the wild type virus (Figure 5B, right). These results again confirmed the results in the live virus replication experiments shown in Figure 4. In the case of TK-ts13, cells stably expressing human TRIM5α in which TRIM5α expression is in more physiological levels; however, the difference in sensitivity to human TRIM5α between the wild type and G116E lentivector was not observed (Figure 5C and 5D). Furthermore, when we used TRIM5α knockout Jurkat cells, we also failed to detect the difference in sensitivity to human TRIM5α between the wild type and G116E virus (data not shown). These results indicated that the effect of G116E substitution is virtually negligible at physiological levels of endogenous human TRIM5α, although this substitution increases the susceptibility of HIV-1 to human TRIM5α.
A G-to-E substitution at the 116th position did not affect the association between CA and CypA or Gag processing
Structural model of the capsid protein
By long-term cultivation of human CEMss cells infected with NL-ScaVR6/7S (NL-4/5S6/7SvifS), a simian tropic HIV-1 that could grow efficiently in CM cells but inefficiently in human cells, we succeeded in rescuing the impaired replicative capability of the virus in human cells. Sequence analysis of the MA-CA region of the adapted virus revealed that the there was a G-to-E single amino acid substitution at the 116th position of the CA. Introduction of this substitution into the molecular DNA clone of NL-4/5S6/7SvifS indeed improved the virus' replicative capability in human cells. We thus concluded that the recovered replicative capability in human cells was mainly the result of acquisition of the single amino acid substitution at the 116th position of the CA, although small effects of mutations in regions other than the MA-CA cannot be fully excluded at present.
Although the 116th position of the CA is highly variable among natural HIV-1 strains from subtypes A to K, no virus with E at the 116th position was found in the Los Alamos HIV sequence database 2009 http://www.hiv.lanl.gov/. On the other hand, most HIV-2 and SIVmac strains have glutamine, which has a long side chain similar to E, at this position, and some strains have E. It is possible that the combination of the amino acid residue at the 116th position and L6/7 is important for viral growth. Consistent with this hypothesis, NL-4/5SG116EvifS, a virus with an HIV-1 derived L6/7 and the G116E substitution, showed impaired growth in MT4 cells (data not shown).
The precise reasons for the impaired replicative capability of NL-4/5S6/7SvifS and effect of G116E in human cells remain to be elucidated. Analysis of a series of CA mutants shown in Figures 4 and 5 clearly excluded the possibility that the impaired replicative capability of NL-4/5S6/7SvifS in human cells resulted from an increased sensitivity to human TRIM5α because a virus with the SIVmac L4/5 and L6/7 (4/5S6/7S) showed similar infectivity to the wild-type virus in the presence of human TRIM5α, and a virus with the SIVmac L4/5, L6/7, and G116E substitution (4/5SG116E6/7S) became more sensitive to human TRIM5α (Figure 5B). On the other hand, the virus with the SIVmac L4/5 and L6/7 showed slightly impaired cleavage of p55 Gag precursors, although p24 mature CA proteins were clearly detected (Figure 6). However, the addition of G116E substitution did not facilitate the cleavage of Gag, and a small defect in Gag processing could only partially explain the attenuated growth of NL-4/5S6/7SvifS. Another possibility is that NL-4/5S6/7SvifS was restricted by a certain intrinsic restriction factor that was previously suggested to be present in human cells [13, 14], and that the adapted virus could escape from this restriction by G116E substitution, since the G116E was acquired through the adaptation in human cells. It is thus necessary to conduct further analysis to substantiate this unidentified restriction factor.
Although the G116E substitution occurred during long-term cultivation of human cells infected with NL-4/5S6/7SvifS, the viruses with G116E unexpectedly became resistant to CM TRIM5α-mediated restriction (Figures 4 and 5). Replacing the HIV-1 L6/7 (HNPPIP) of the CA with that of SIVmac239 (RQQNPIP) resulted in elongation of the loop by one amino acid, and it is reasonable to assume that the G116E substitution occurred to compensate the structural warp caused by the extended L6/7. This compensatory substitution occurred at the central position of the surface composed of L4/5 and L6/7, a structure considered to be important for TRIM5α binding . The amino acid substitution of G with E at the 116th position caused an important change in the structure of the surface composed of L4/5 and L6/7 because G, which has no side chain, was replaced by E, which has a long, negatively charged side chain as shown in Figure 7. This change in the conformational structure of L4/5 and L6/7 might affect the interaction between the CA and TRIM5α. Alternatively, this single amino acid substitution might influence the configuration of surrounding loops by the changes in the side chain and polarity without directly involving the binding site of TRIM5α.
We succeeded in rescuing the impaired replicative capability of simian tropic HIV-1 NL-4/5S6/7SvifS and unexpectedly identified a single amino acid substitution in the CA that affects viral sensitivity to CM TRIM5α-mediated restriction. This finding will increase our understanding of the detailed molecular interactions between the CA and TRIM5α.
human immunodeficiency virus type 1
simian immunodeficiency virus isolated form macaque
HIV-1/SIV chimeric virus
green fluorescence protein
vesicular stomatitis virus glycoprotein
a loop between α-helices 4 and 5
a loop between α-helices 6 and 7.
The TRIM5α-KD Jurkat and Luci-siRNA Jurkat cells were kindly provided by Dr. Jeremy Luban. The authors wish to thank Ms. Setsuko Bandou and Ms. Noriko Teramoto for their helpful assistance. This work was supported by grants from the Health Science Foundation, the Ministry of Education, Culture, Sports, Science, and Technology, and the Ministry of Health, Labour and Welfare, Japan.
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