Identification of molecular determinants from Moloney leukemia virus 10 homolog (MOV10) protein for virion packaging and anti-HIV-1 activity

Abstract

Discovery of novel antiretroviral mechanism is essential for the design of innovative antiretroviral therapy. Recently, we and others reported that ectopic expression of Moloney leukemia virus 10 (MOV10) protein strongly inhibits retrovirus replication. MOV10, a putative RNA helicase, can be packaged into HIV-1 virions by binding to the nucleocapsid (NC) region of Gag and inhibit viral replication at a postentry step. Here, we report critical determinants for MOV10 virion packaging and antiviral activity. MOV10 has 1,003 amino acids and seven helicase motifs. We found that MOV10 packaging requires the NC basic linker, and Gag binds to the N-terminal amino acids 261-305 region of MOV10. Our predicted MOV10 three-dimensional structure model indicates that the Gag binding region is located in a structurally exposed domain, which spans amino acids 93-305 and is Cys-His-rich. Simultaneous mutation of residues Cys-188, Cys-195, His-199, His-201, and His-202 in this domain significantly compromised MOV10 anti-HIV-1 activity. Notably, although MOV10-Gag interaction is required, it is not sufficient for MOV10 packaging, which also requires its C-terminal all but one of seven helicase motifs. Moreover, we have mapped the minimal MOV10 antiviral region to amino acids 99-949, indicating that nearly all MOV10 residues are required for its antiviral activity. Mutations of residues Cys-947, Pro-948, and Phe-949 at the C terminus of this region completely disrupted MOV10 anti-HIV-1 activity. Taken together, we have identified two critical MOV10 packaging determinants and eight other critical residues for anti-HIV-1 activity. These results provide a molecular basis for further understanding the MOV10 antiretroviral mechanism.

FIGURE 1.

MOV10 is packaged into HIV-1 virions from human CD4⁺ T cells. 3 × 10⁵ H9, CEM-SS, or CEM-T4 cells were infected with NL HIV-1 equivalent to 20 ng of p24^Gag, respectively. Six days later, virions were purified from the culture supernatants by ultracentrifugation through a 20% sucrose cushion. Cells and virions were lysed, and proteins were analyzed by Western blotting. MOV10 and A3G were detected by anti-V5 antibodies, and Gag-CFP fusions were detected by anti-GFP antibodies.

FIGURE 2.

MOV10 packaging into HIV-1 requires the NC basic linker. A, schematic illustrates p55^Gag and NC for creating Gag-CFP fusion constructs. The number indicates the C-terminal AA residue expressed, with the first Gag residue considered 1. B, MOV10 or A3G was expressed with indicated Gag-CFP fusion proteins in 293T cells. Virions were purified through a 20% sucrose cushion via ultracentrifugation. Cellular and virion-associated proteins were determined by Western blotting with the indicated antibodies.

FIGURE 3.

HIV-1 Gag binds to the N-terminal AA 261–305 region of MOV10. A, schematic illustrates MOV10 N-terminal or C-terminal deletion mutations. Numbers indicate AA positions. The locations of the putative CH domain (see Fig. 6) and seven helicase motifs are indicated. The border of the putative CH domain, the first residue of the helicase motif I, and the last residue of the helicase motif VI are numbered. B, Gag binding domain of MOV10 was mapped by immunoprecipitation (IP). The indicated MOV10 deletion mutants were expressed with HA-tagged codon-optimized HIV-1 HXB2 Gag in 293T cells. Proteins were pulled down by anti-HA antibody and analyzed by Western blotting.

FIGURE 4.

Gag binding domain of MOV10 is not sufficient for virion packaging. The indicated MOV10 mutants were expressed with HIV-1 proviral construct in 293T cells. Virions were purified, and virion incorporation of these mutants was determined by Western blotting.

FIGURE 5.

All but one of seven helicase motifs are also required for MOV10 virion packaging. A, anti-HIV-1 activity and virion incorporation of MOV10 helicase mutants. The indicated MOV10 mutants were expressed with VSV-G pseudotyped pNL-Luc reporter viruses in 293T cells, and viral infectivity was analyzed in CEM-SS cells. Viral infectivity is presented as relative value, with the infectivity of viruses produced in the presence of a control vector as 100%. The S.E. were calculated from three independent experiments. In addition, virions were purified by ultracentrifugation, and cellular and virion proteins were analyzed by Western blotting. B, interaction of MOV10 helicase mutants with HIV-1 Gag, as determined by the same as in Fig. 3B.

FIGURE 6.

MOV10 three-dimensional structure model. A, ribbon structure model of human MOV10 protein from AA 122 to 968. It was generated from the x-ray structure of human UPF1 protein from AA 116 to 914 (Protein Data Bank code 2WJY). The C-terminal core structure of RNA helicase domain from residue 306 to 968 is shown in blue; the Gag binding domain from residue 261 to 305 is shown in light blue; and the N-terminal part of a potential CH-domain from residue 122 to 260 is shown in magenta. In this CH domain, histidines are shown in green, cysteines are shown in orange, and their position is indicated by the corresponding number. B, protein sequence of MOV10 from AA 93 to 305. Histidines are shown in green, cysteines are shown in orange, and the Gag binding sequence from AA 261 to 305 is underlined. Residues that were targeted for mutagenesis are also underlined. C, anti-HIV-1 activity of MOV10 CH domain mutants, as determined in Fig. 5A. Their cellular expression was determined by Western blotting.

FIGURE 7.

A and B, anti-HIV-1 activity of MOV10 deletion mutants, as determined in Fig. 5A. Protein expression in cells and virions in B was determined as in Fig. 4.