Abstract
BackgroundThe identification and characterization of several interferon (IFN)-induced cellular HIV-1 restriction factors, defined as host cellular proteins or factors that restrict or inhibit the HIV-1 life cycle, have provided insight into the IFN response towards HIV-1 infection and identified new therapeutic targets for HIV-1 infection. To further characterize the mechanism underlying restriction of the late stages of HIV-1 replication, we assessed the ability of IFNbeta-induced genes to restrict HIV-1 Gag particle production and have identified a potentially novel host factor called HECT domain and RCC1-like domain-containing protein 5 (HERC5) that blocks a unique late stage of the HIV-1 life cycle.ResultsHERC5 inhibited the replication of HIV-1 over multiple rounds of infection and was found to target a late stage of HIV-1 particle production. The E3 ligase activity of HERC5 was required for blocking HIV-1 Gag particle production and correlated with the post-translational modification of Gag with ISG15. HERC5 interacted with HIV-1 Gag and did not alter trafficking of HIV-1 Gag to the plasma membrane. Electron microscopy revealed that the assembly of HIV-1 Gag particles was arrested at the plasma membrane, at an early stage of assembly. The mechanism of HERC5-induced restriction of HIV-1 particle production is distinct from the mechanism underlying HIV-1 restriction by the expression of ISG15 alone, which acts at a later step in particle release. Moreover, HERC5 restricted murine leukemia virus (MLV) Gag particle production, showing that HERC5 is effective in restricting Gag particle production of an evolutionarily divergent retrovirus.ConclusionsHERC5 represents a potential new host factor that blocks an early stage of retroviral Gag particle assembly. With no apparent HIV-1 protein that directly counteracts it, HERC5 may represent a new candidate for HIV/AIDS therapy.
Highlights
The identification and characterization of several interferon (IFN)-induced cellular HIV-1 restriction factors, defined as host cellular proteins or factors that restrict or inhibit the HIV-1 life cycle, have provided insight into the IFN response towards HIV-1 infection and identified new therapeutic targets for HIV-1 infection
There was no significant difference in the viability of cells expressing HECT domain and Regulator of Chromosome Condensation 1 (RCC1)-like domain-containing protein 5 (HERC5) compared to the control cells (90.2% ± 4.4% SD versus 92.0% ± 4.3% SD respectively; P > 0.05, paired t test)
To determine if the inhibition of Gag particle production by the CS alone resembled the inhibition of Gag particle production induced by CS + HERC5, we examined transmission electron microscopy (TEM) micrographs of cells expressing replicationcompetent HIV-1 (R9) + CS only
Summary
HERC5 inhibits HIV-1 particle production HERC5 is the main cellular E3 ligase that conjugates ISG15 to proteins and can target newly synthesized proteins, including foreign proteins [6,13,21]. HOS-CD4/CXCR4 cells expressing HERC5 released 10.8-fold less infectious HIV1 particles into the supernatant after multiple rounds of replication (P = 0.001, paired t test) (Figure 1B). Similar levels of inhibition of HIV-1 particle release after single and multiple rounds of replication were observed by Western blot analysis (Figure 1C). Western blot analysis revealed that cells expressing HERC5 + CS released substantially less Gag-only particles into the supernatant than the control cells. Western blot analysis of the purified proteins using anti-p24CA revealed the presence of several species of Gag ranging from ~55 kDa to ~200 kDa in size (Figure 2B), including the ~85 kDa and ~110 kDa bands detected in the whole cell lysate (Figure 2A). Quantitative Western blot analysis of Gag particles released into the supernatant showed that Ubp abolished HERC5-induced restriction, despite the presence of similar levels of intracellular Gag (Figure 2C). As normally seen phenotypically with TEM micrographs of negatively-stained samples, HIV-1 Gag particle assembly at the
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