Background:  GB virus C (GBV-C) infection is associated with prolonged survival among HIV-infected individuals, and GBV-C inhibits HIV replication in vitro by inducing chemokines. We previously demonstrated that some, though not all, GBV-C NS5A proteins inhibit PKR-mediated eIF-2a phosphorylation, and this may help the virus avoid clearance by cellular antiviral response mechanisms. Overexpression of GBV-C NS5A in Jurkat cells led to increased levels of SDF-1 release and subsequent inhibition of X4-tropic HIV replication. To characterize this observation further, we expressed a series of NS5A deletion mutants in Jurkat cells and measured HIV replication in these cell lines.

Methods:  Stable Jurkat cell lines expressing PKR-inhibiting and non-inhibiting NS5A proteins were generated, as were a series of C-terminal deletion mutants. These cell lines expressed NS5A proteins of 123, 161, 181, 250, 314, and 363 amino acids. All constructs had stop codons after NS5A, followed by the EMC IRES and GFP. A control cell line expressing only GFP served as a negative control, and NS5A and GFP expression were regulated by tetracycline. HIV replication was measured by p24 antigen release into culture supernatant or by measuring infectivity.

Results:  Jurkat cell lines demonstrated regulated expression of NS5A and deletion mutants as shown by Western blot and GPF expression. The NS5A and GFP genes were shown to remain linked by real-time polymerase chain reaction (RT-PCR) of cellular DNA from recombinant cell lines. Expression of either PKR-inhibiting or non-inhibiting NS5A proteins resulted in HIV inhibition (> 95% reduction in p24 antigen), thus the HIV- and PKR-inhibiting functions are independent. All deletion mutants of 250 amino acids or larger retained HIV-inhibiting effects, whereas those with 181 amino acids or smaller did not.

Conclusions:  Expression of GBV-C NS5A protein inhibits HIV replication independently of its effect on PKR-mediated eIF-2a phosphorylation. A region between amino acids 181 and 250 is required for HIV inhibition. N-terminal deletion mutants have been generated, and cell lines are being selected to further determine the protein requirements for this effect, as are cell lines expressing the NS5A proteins from related viruses (HCV, GBV-B, BVDV, and dengue). Studies on the effects of NS5A mutant proteins on gene expression, chemokine, and chemokine receptor expression are underway. The GBV-C NS5A protein may provide novel, cellular-based approaches to inhibiting HIV replication.