Background:  We characterized a novel anti-HIV gene identified by differential Affymetrix microarray screening of an HIV permissive and an HIV non-permissive CD4 T-cell clone.

Methods:  The ability of G9 to suppress HIV production was assessed in 293T producer cells. Plasmid DNA encoding HA-tagged G9 or control empty vector was transfected into 293T cells along with HIV proviral DNA (NL4-3/YU2). The titre (infectious particles / mL) and infectivity (infectious particles / ng p24) of the resultant virus stock was assessed on indicator lines (CD4⁺CCR5⁺Ghost and CD4⁺CXCR4⁺CEM.G37) that expressed GFP upon productive HIV infection. The ability of G9 to inhibit early steps of infection was assessed by engineering expression of G9 (by retroviral transduction) linked to a marker gene (red fluorescent protein RFP) in CD4⁺CXCR4⁺CCR5⁺G9^low Jurkat T-cells. RFP⁺G9^high or RFP⁺G9^low empty vector Jurkat cells were challenged with replication-competent wild type or VSVg-pseudotyped HIV and HIV RU5 DNA accumulation measured by quantitative polymerase chain reaction within 12 hours of infection.

Results:  p24 concentration of 293-T virus stock produced in the presence of G9 was as much as 15-fold lower than that produced in the presence of empty vector control; the titre of the resultant virus was also lower (as much as 9-fold inhibition noted in a target-dependent manner). However, G9 only moderately reduced progeny virion infectivity (maximum 2-fold inhibition). Western blot assays of purified virus prepared in the presence of HA-tagged G9 probed with anti-HA antibody revealed no detectable incorporation of G9 into progeny virions. Lysates taken from producer cells and probed with an anti-Gag antibody by Western blot showed a reduction in total Gag production. In addition, G9 blocked incoming virus infection. HIV RU5 DNA in G9⁺ Jurkat cells was as much as 19-fold lower than empty vector control Jurkat cells 4 hours post-infection with wild type HIV. This inhibition was reduced to <2-fold when the same cells were challenged with VSVg/HIV and not associated with down-regulation of HIV receptor/co-receptors.

Conclusions:  G9 blocks 2 stages of the HIV lifecycle:  it inhibits HIV production prior to release of virus particles, and it inhibits an entry-dependent step at or prior to reverse transcription. G9 is a small molecular weight, secreted protein; some members of the extended G9 family of proteins have known innate immune activity. Further studies will reveal the mechanisms of the G9 anti-HIV effect and its immunotherapeutic potential.