HRP2 determines the efficiency and specificity of HIV-1 integration in LEDGF/p75 knockout cells but does not contribute to the antiviral activity of a potent LEDGF/p75-binding site integrase inhibitor

Abstract

The binding of integrase (IN) to lens epithelium-derived growth factor (LEDGF)/p75 in large part determines the efficiency and specificity of HIV-1 integration. However, a significant residual preference for integration into active genes persists in Psip1 (the gene that encodes for LEDGF/p75) knockout (KO) cells. One other cellular protein, HRP2, harbors both the PWWP and IN-binding domains that are important for LEDGF/p75 co-factor function. To assess the role of HRP2 in HIV-1 integration, cells generated from Hdgfrp2 (the gene that encodes for HRP2) and Psip1/Hdgfrp2 KO mice were infected alongside matched control cells. HRP2 depleted cells supported normal infection, while disruption of Hdgfrp2 in Psip1 KO cells yielded additional defects in the efficiency and specificity of integration. These deficits were largely restored by ectopic expression of either LEDGF/p75 or HRP2. The double-KO cells nevertheless supported residual integration into genes, indicating that IN and/or other host factors contribute to integration specificity in the absence of LEDGF/p75 and HRP2. Psip1 KO significantly increased the potency of an allosteric inhibitor that binds the LEDGF/p75 binding site on IN, a result that was not significantly altered by Hdgfrp2 disruption. These findings help to rule out the host factor-IN interactions as the primary antiviral targets of LEDGF/p75-binding site IN inhibitors.

Allosteric integrase inhibitor potency is determined through the inhibition of HIV-1 particle maturation

“…S5). Consistent with results using mouse knockout cells (17), LEDGF/p75 knockdown yielded a significant 29-fold increase in BI-D potency during the acute phase of HIV-1 infection, whereas RAL potency was unaffected (Table 2). By contrast, LEDGF/p75 depletion did not significantly alter BI-D potency during HIV-1 production.…”

“…S3B), combined with the apparent inability of LEDGF/p75 to engage HIV-1 during virus egress, determines ALLINI potency (EC 50 and slope parameter). Because LEDGF/p75 depletion from target cells sharply increases BI-D EC 50 values, the host factor competes with the drug for binding to the CCD dimer interface during the acute phase of infection (17). The slope of the dose-response curve remains close to 1 under this condition (Table 2) consider that inhibition of multiple viral replication steps (core formation, reverse transcription, and integration) could also contribute to the multimode mechanism of ALLINI action.…”

“…Small molecules that compete for LEDGF/p75 binding enhance IN multimerization in the absence of viral DNA and accordingly allosterically inhibit IN activity (14)(15)(16). Although various terms, including LEDGINs for LEDGF/p75-IN inhibitors, have been coined for these investigational compounds, we prefer "allosteric IN inhibitor" or ALLINI to highlight the mechanistic basis of compound action (14,17).…”

A Critical Role of the C-terminal Segment for Allosteric Inhibitor-induced Aberrant Multimerization of HIV-1 Integrase

“…When added to target cells, ALLINIs block 3Ј-processing as well as reduce LEDGF/p75-mediated integration into active transcription units (39,46). Knockdown or knock-out of endogenous LEDGF/p75 significantly enhanced the potencies of these inhibitors in target cells, suggesting that there is competition between the cellular protein and ALLINIs for binding to HIV-1 IN during early steps of viral replication (12,47,48). However, the ALLINI antiviral potency is determined primarily through inhibiting the late stage of HIV-1 replication (12,41,42,44,45,48).…”

“…In producer cells, ALLINIs potently promote aberrant, higher-order multimerization of IN during virus maturation resulting in eccentric, non-infectious cores reminiscent to the phenotype seen with some class II IN mutants (8 -12, 44, 45). In contrast with target cells, the absence of LEDGF/p75 had no effect on ALLINI potencies in the virus producer cells (12,44,47,48). The recent discovery of pyridine-based multimerization selective IN inhibitors, which potently promoted aberrant IN multimerization in vitro and in infected cells but were not effective inhibitors of IN-LEDGF/p75 interactions, have further highlighted IN multimerization as a plausible antiviral target (46).…”