Previously, we showed that HERC5 inhibits an early stage of HIV-1 Gag assembly at the plasma membrane by a mechanism correlating with the modification of Pr55Gag with ISG15 . Here we demonstrate that HERC5 inhibits HIV-1 particle production by a second distinct mechanism targeting Rev/RRE function. A region of the RCC1-like domain of HERC5 was required for this inhibition, which is also evolving under strong positive selection. Although deleting the RCC1-like domain of HERC5 rescued inhibition of Rev/RRE function, HERC5-ΔRLD inhibited HIV-1 particle release at levels comparable to wild type HERC5. HERC5-ΔRLD has been shown to possess some E3 ligase activity for ISG15 conjugation; therefore, it is likely that this E3 ligase activity contributed to inhibition of particle release via ISGylation of HIV-1 Gag [19, 48]. However, the infectivity of HIV-1 particles released from cells expressing HERC5-ΔRLD did not differ from those released from the control cells. By inhibiting the nuclear export of HIV-1 genomic RNA, the RCC1-like domain may also promote the release of non-infectious HIV-1 particles (i.e. particles lacking genomic RNA). We also showed that HERC5-C994A was able to inhibit HIV-1 particle production better than wild type HERC5. This potent inhibition correlated with the loss of ISGylated forms of HERC5. Given that HERC5 modifies itself with ISG15, it is possible that auto-ISGylation negatively regulates HERC5 antiviral activity. This auto-regulation may represent a mechanism by which HERC5 maintains tight control over its E3 ligase-independent activity, particularly during periods of high-expression such as after induction by IFN. A similar finding was previously observed for the E3 ligase TRIM25 where auto-ISGylation negatively regulated its activity for conjugating ISG15 to 14-3-3sigma .
HERC5 expression is significantly up-regulated in cells after exposure to IFN [18, 19]. We showed here that knocking down HERC5 in a background of IFN exposure substantially increased intracellular HIV-1 Gag production after 48 hours, indicating that HERC5 is an important mediator of the IFN response towards HIV-1. Knocking down endogenous levels of HERC5 in primary macrophages, in the absence of exogenous IFN, also resulted in a substantial increase in HIV-1 particle production. This finding suggests that endogenous levels of HERC5 may serve to limit, but not fully restrict, HIV-1 particle production in the absence of IFN. As HERC5 levels increase, such as after exposure to IFN, HERC5 may be more able to restrict HIV-1 particle production. This finding contrasts our previous finding that knockdown of HERC5 did not exhibit a substantial effect on the intracellular production of Pr55Gag from full-length replication-competent HIV-1 . A likely explanation for the difference is that the data in the present study was obtained 48 hours post-transfection, compared to 24 hours in our previous study. Another contributing factor could be that the level of HERC5 knockdown achieved in the present study was higher than our previous study (3.2-fold versus 2.3-fold respectively). In both studies, HERC5 had no substantial effect on intracellular Pr55Gag levels when expressed from a Rev-independent Gag-only construct.
Most eukaryote messenger RNAs undergo splicing to remove introns before they are exported to the cytoplasm via the NXF1/NXT1-dependent nuclear export pathway [50–53]. However, the expression of HIV-1 genes is a notable exception. Unspliced and singly-spliced HIV-1 RNA must be exported to the cytoplasm before they are fully-spliced by host machinery in the nucleus [54–57]. These incompletely-spliced RNAs are essential for steps such as incorporation of full-length genomes into new virions and for expression of Gag, Gag-Pol, Env, Vif, Vpr and Vpu proteins. Nuclear export of incompletely-spliced HIV-1 RNAs occurs when the Rev/RRE complex recruits the dimeric complex of Crm1/RanGTP before translocating through the nuclear pore to the cytoplasm via the Crm1/RanGTP-dependent nuclear export pathway. Once in the cytoplasm, the complex dissociates and RanGTP is converted to RanGDP with the help of RanBP1 and RanGAP1, which then shuttles back into the nucleus for another round of export [35–39, 58].
We showed that HERC5 interacts with Ran and substantially reduces intracellular levels of RanGTP and/or inhibits the association of RanGTP with RanBP1. Cells require a high concentration (>1000-fold) of RanGTP in the nucleus, which is believed to provide directionality for nuclear export to the cytoplasm . Perturbing this nuclear:cytoplasmic RanGTP gradient by either reducing total RanGTP levels or increasing the cytoplasmic RanGTP level by interfering with the interaction between RanGTP and RanBP1, halts nuclear export of Crm1/RanGTP-dependent cargo. Consistent with this idea, we observed altered localization of Rev protein and Rev/RRE-dependent RNA in the presence of HERC5. With the ability of HERC5 to interact with Ran, it is possible that HERC5 binds and sequesters Ran in the cytoplasm. This activity would interfere with the shuttling of RanGDP into the nucleus, thereby interfering with the production of RanGTP in the nucleus. Another possibility is that HERC5 stimulates guanine nucleotide release from Ran in the cytoplasm. It was previously shown that the related RCC1-like domain 1 of human HERC1 stimulates GDP release from the small GTPase proteins ARF1/6 and Rab, but not from Ran [59, 60]. Therefore, it is plausible that the HERC5 RCC1-like domain performs a similar function on Ran, with which it interacts. Further experiments are needed to further dissect this mechanism.
HERC5 orthologs have been identified in a variety of evolutionarily diverse mammals spanning >75 million years of evolution. Genetic conflict arising from the co-evolution of hosts and pathogens can lead to rapid selection of amino acid substitutions that alter amino acid composition of the host factors and their pathogen antagonists, thereby conferring an evolutionary advantage to the host or the pathogen [61, 62]. This process of positive selection is not a common phenomenon and is typically not apparent in most examined datasets [63, 64]. However, recent evolutionary studies on host antiviral factors have shown that they are rapidly evolving genes due to genetic conflict between hosts and pathogens . Several of these host factors such as apolipoprotein B mRNA-editing enzyme catalytic polypeptide 3G (APOBEC3G) ; tripartite motif protein 5 alpha (TRIM5α) ; bone marrow stromal antigen 2 (BST2)/tetherin [67, 68]; sterile alpha motif (SAM) domain and histidine/aspartic acid domain (HD)-containing protein 1 (SAMHD1) ; MxA  contain genetic ‘signatures’ of positive selection. Positively selected residues have been shown to play key functional roles in the antiviral activities of these proteins.
We demonstrated here that HERC5 also contains genetic signatures of strong positive selection. Twenty-seven of 50 codons predicted to be evolving under strong positive selection in HERC5 map to blades 1 and 2 of the predicted β-propeller structure of the RCC1-like domain. This finding identifies blades 1 and 2 as a functionally important region of HERC5 and may represent a highly dynamic interface with viral antagonists. Fifteen of 50 codons predicted to be evolving under strong positive selection map to the spacer region of HERC5. The high proportion of amino acids predicted to be evolving under purifying selection in blades 3–7 of the RCC1-like domain and the majority of the HECT domain indicates that purifying selection is playing an important role in maintaining the long-term stability of these domains. These two domains are highly conserved in evolutionarily diverse mammals, suggesting they play fundamental roles in biology. Indeed, the HECT domain of HERC5 confers its E3 ligase activity and HERC5 has been shown to be the main cellular E3 ligase for host ISGylation.