Inactivation of integrase (IN) and reverse transcriptase (RT) can revoke the replication of HIV virions, and non-infectious HIV particles are desirable virus-like particle (VLP) vaccine candidates. Here, we produced inactive HIV-1 particles fit for vaccine and virological purposes by introducing a mutation into the pol sequence. Proviral DNA (pNL4-3) was cut at two points in the pol region using the BalI restriction enzyme and then religated. HEK 293T cells were transfected with the resultant plasmid (pmzNL4-3) to produce mutated virions. To confirm a production of VLPs and evaluate their biological activity the p24 load and syncytium formation (MT2 cells) were analyzed. The assay indicated that mzNL4-3 virions were assembled and contained functional envelope glycoproteins (ENV). In addition, mzNL4-3 virions were able to infect neither the MT2 nor HEK 293T cells. Furthermore, the immunogenicity of VLPs was investigated in a mouse model. According to the data on vaccinated mice, the titer of ENV-specific antibodies rose rapidly after a boosting injection. Moreover, lymphoid cells extracted from these mice proliferated after exposure to the antigen. The mzNL4-3 virus particles possessed immunogenic antigens of HIV and can effectively trigger humoral and CD4 immune responses. Non-infectious mzNL4-3 virions may also be used in biomedical experiments for improving the biological safety conditions. Moreover, the mzNL4-3 seems to be a promising candidate for further HIV-1 vaccine investigations.