Introducing a Frameshift Mutation to the Pol Sequence of Hiv-1 Provirus and Evaluation of the Immunogenic Characteristics of the Mutated Virions (Rinnl4-3) Publisher



Zabihollahi R^{1, 2} ; Sadat SM¹ ; Vahabpour R¹ ; Salehi M³ ; Azadmanesh K³ ; Siadat SD¹ ; Saraji ARA^{1, 4} ; Pouriavali MH¹ ; Momen SB¹ ; Aghasadeghi MR¹ Show Affiliations
Source: Molecular Biology Published:2012

Abstract

Inactivation of the reverse transcriptase (RT) and integrase (IN) enzymes can abolish the replication of the human immunodeficiency virus (HIV) and, thus, its infectivity. Here, inactivated HIV particles convenient for designing virus-like particle (VLP) based vaccines have been produced. Inactivated HIV-provirus was created by introducing a frame shift mutation. HIV provirus DNA was cut in the pol region by Age I restriction enzyme, followed by filling of sticky ends using the Klenow fragment before ligation. The resulting plasmid was named as pRINNL4-3. HEK-293T cells were used as producer, after being transfected with the modified plasmid. Viral particle production and biological activity were assayed by virus capsid protein (p24) quantification and syncytium formation in MT2 cells, respectively. The immunogenicity of the RINNL4-3 virions was investigated in a mouse model. The mutation was expected to inactivate the virus RT and IN enzymes. The results showed that the VLPs were assembled, as measured by the p24 load of the culture supernatant, and contained functional envelope proteins (Env) as monitored by the syncytium formation. However, these VLPs had no ability to infect target MT2 cells, as well as their VSVG (vesicular stomatitis virus-glycoprotein) pseudotyped counterparts infected HEK-293T cells. A high level of antibody response was observed in immunized mice. Since RINNL4-3 virions are replication incompetent, they are convenient for production and use in biomedical studies. Also, RINNL4-3 is a candidate for a vaccine development due to it contains envelope and structural virus proteins which are crucial for triggering neutralizing antibodies and the cellular immune response. © 2012 Pleiades Publishing, Ltd.