Recombinant HIV-1 Vif protein (HXB2)

Acquired immunodeficiency syndrome (AIDS) is an infectious disease caused by the human immunodeficiency virus (HIV). Since the first case was reported in 1981, AIDS has spread rapidly throughout the world. HIV-1 is an RNA virus belonging to the subgenus primate immunodeficiency virus of the family Retroviridae and the genus lentivirus. The HIV-1 virus is approximately 120 nm in diameter and approximately spherical in shape. The outer layer of the virus is an envelope in which the viral envelope proteins gp120 and gp41 are embedded. Between them, gp41 is a transmembrane protein, while gp120 is on the surface, and the two are bound by non-covalent interactions. Inside is a spherical matrix formed by the p17 protein and a conical capsid formed by the p24 protein. The capsid contains the viral RNA genome, enzymes (reverse transcriptase, integrase, protease) and other host cell components. The genetic material of the HIV-1 virus is RNA, and two single-stranded positive-stranded RNAs are joined by hydrogen bonds at the 5' end to form a dimer. The HIV-1 genome is approximately 9.7Kb in size and consists of structural genes, regulatory genes, helper genes and long terminal repeats (LTR) at both ends. The structural genes include gag, pol and env, which mainly encode viral core proteins, polymerases and envelope proteins; the regulatory genes include tat and rev, whose main function is to promote viral gene expression; the helper genes include nef, vpr, vif and vpu, all of which play an important role in HIV-1 replication.

Figure 1. Schematic representation of the mechanisms of action of the HIV-1 Vif protein and APOBEC3. (Sources: Jónsson SR, et al. 2013)

HIV-1 Vif is a basic protein encoded by the conservative vif gene. It plays a major role in the production of infectious viral particles, increasing the infectivity of the virus by 10 to 1000 times. If the Vif protein mutates or its function is restricted, the production of HIV-1 viral particles will be significantly reduced. Therefore, the research on Vif protein has attracted more and more attention. HIV-1 Vif protein contains 192 amino acid residues and its molecular weight is 23kDa. It contains several important functional domains: N-terminal tryptophan-rich extension region, conserved zinc-binding hydrophobic HCCH region, downstream SOCS-box region and polymerization region. In HIV-1 Vif, the N-terminal tryptophan-rich extension region (residues 1 to 21) is highly conserved. The amino acids at positions 63 to 70 and 86 to 89 are conserved and play a decisive role in maintaining normal expression levels of Vif. In HIV-1, the conserved glutamic acid at position 88 and the conserved tryptophan at position 89 are located in a charged hydrophilic region 89EWRKKR93, which is thought to enhance the steady-state expression of Vif in host cells. The conserved HCCH region (residues 108-139) includes two conserved His/Cys pairs, which are predicted to be flanked by an a-helix containing a cluster of conserved hydrophobic residues. This motif coordinates a zinc ion through conserved residues H108, C114, C113, and H139 and directly binds to Cullin5. The highly conserved 144SLQYLA149 motif, the BC-box region, is essential for the inactivation of the APOBEC3 protein. This BC-box motif is primarily responsible for binding to ElonginC, which further targets the APOBEC3 antiviral factor to the proteasome. There is a high degree of similarity between the conserved SLQ(Y/F) LAFFFF motif of Vif (F represents a hydrophobic residue) and the SOCS-box of SOCS proteins (suppressor of cytokine signaling). In HIV-1 Vif, the conserved 161PPLP164 region plays a key role in multimerization, also known as the multimerization region. Vif releases Pr55Gag at the C-terminus through multimerization of the 161PPLP164 motif. In HIV-1, multimerization of Vif is essential for viral infectivity and for preventing APOBEC3G from being incorporated into viral particles. However, this multimerization site is not conserved in HIV-2 and SIV. Post-translationally phosphorylated viral proteins can regulate the infectivity of HIV-1. In the life cycle of the virus, many viral proteins are regulated by phosphorylation at different stages. HIV-1 Vif can be phosphorylated by cellular kinases in vivo and in vitro, and phosphorylation of Vif is crucial for the replication of HIV-1. Four major phosphorylation sites have been identified: T96, S144, T155 and T188, the latter three of which are located at the C-terminus of Vif. Among them, T96 and S144 are highly conserved in all lentiviruses. Mutation of T96 can cause a significant loss of Vif activity and inhibit HIV-1 replication. Mutation of S144 to alanine can cause loss of Vif activity, indicating that phosphorylation at this site plays an important regulatory role in HIV-1 replication and infectivity. The corresponding phosphorylation sites of the synthetic Vif polypeptide cannot be phosphorylated by MAPK, suggesting that MAPK recognition of these sites may require phosphorylation sites other than structural factors.

Studies have shown that the main function of HIV-1 Vif protein is to regulate the processes of virus invasion, assembly, budding, and maturation. Vif protein can also specifically interact with the antiviral factor APOBEC3G in the body to enhance the infectivity of the virus. Its specific functions are as follows: 1. HIV-1 Vif protein binds to the RNA of the virus and is packaged into the virus particle. Studies have found that HIV-1 Vif protein can bind to the RNA of the virus both in vitro and in vivo, and this binding is regulated by the N-terminal region and core region of Vif protein. The Vif protein packaged into the virus core can enhance the binding of matrix protein and reverse transcriptase to the virus core. Mutations in the zinc finger structure of the nucleocapsid protein in the core region of the virus can completely prevent Vif protein from being packaged into the virus particle. Further studies have shown that Vif protein is packaged into the virus particle by binding to the RNA of the virus through amino acid residues at positions 75 to 114, while mutations in amino acid residues at positions 75 to 114 prevent Vif protein from binding to viral RNA. 2. HIV-1 Vif protein can cause G2 phase cell arrest. The vif gene of HIV-1 enhances the infectivity of viral particles by inactivating antiviral factors in cells, supporting high-yield viral replication in primary human CD4+T cells and non-permissive T cell lines. Vif protein can also promote G2 phase arrest of HIV-1 infected cells. Viruses lacking Vif or Vpr have a much lower ability to induce cell cycle arrest than wild-type viruses, while viruses lacking both Vif and Vpr have the same cell cycle as uninfected cells. In addition, the expression of Vif alone induces G2 phase cell arrest with a cumulative effect. Cell apoptosis and G2 phase cell arrest are essential for the onset of AIDS. 3. HIV-1 Vif protein participates in and promotes viral reverse transcription. Cells can be divided into "permissive cells" and "non-permissive cells" based on whether HIV-1 lacking Vif can replicate in them. In the study of the reverse transcription of HIV-1 virus lacking Vif protein in non-permissive cells, it was found that Vif protein specifically prevented the production of negative and positive strands of viral DNA and inhibited the synthesis of full-length viral DNA. Vif protein binds to viral RNA and forms a polymer through its own C-terminus. Vif protein is activated by phosphorylation of cellular kinases and is essential for promoting viral infection. 4. HIV-1 Vif protein regulates viral assembly and budding. The infectivity of viruses lacking Vif protein is significantly weaker than that of wild HIV-1 virus. In these viruses lacking Vif protein, the amount of envelope protein packaged into the viral core is also significantly reduced, and the amount of core Gag and Pol is also reduced. Although in non-permissive cells, the amount of viral RNA contained in viruses lacking Vif protein is basically the same as that of wild HIV-1 virus, the former cannot synthesize viral DNA after infecting target cells.

Vif
HIV-1 Vif
Viral infectivity factor
SOR protein