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Molecular mechanisms by which HERV-K Gag interferes with HIV-1 Gag assembly and particle infectivity
© The Author(s) 2017
Received: 30 November 2016
Accepted: 18 April 2017
Published: 26 April 2017
Human endogenous retroviruses (HERVs), the remnants of ancient retroviral infections, constitute approximately 8% of human genomic DNA. Since HERV-K Gag expression is induced by HIV-1 Tat in T cells, induced HERV-K proteins could affect HIV-1 replication. Indeed, previously we showed that HERV-K Gag and HIV-1 Gag coassemble and that this appears to correlate with the effect of HERV-K Gag expression on HIV-1 particle release and its infectivity. We further showed that coassembly requires both MA and NC domains, which presumably serve as scaffolding for Gag via their abilities to bind membrane and RNA, respectively. Notably, however, despite possessing these abilities, MLV Gag failed to coassemble with HIV-1 Gag and did not affect assembly and infectivity of HIV-1 particles. It is unclear how the specificity of coassembly is determined.
Here, we showed that coexpression of HERV-K Gag with HIV-1 Gag changed size and morphology of progeny HIV-1 particles and severely diminished infectivity of such progeny viruses. We further compared HERV-K-MLV chimeric constructs to identify molecular determinants for coassembly specificity and for inhibition of HIV-1 release efficiency and infectivity. We found that the CA N-terminal domain (NTD) of HERV-K Gag is important for the reduction of the HIV-1 release efficiency, whereas both CA-NTD and major homology region of HERV-K Gag contribute to colocalization with HIV-1 Gag. Interestingly, these regions of HERV-K Gag were not required for reduction of progeny HIV-1 infectivity.
Our results showed that HERV-K Gag CA is important for reduction of HIV-1 release and infectivity but the different regions within CA are involved in the effects on the HIV-1 release and infectivity. Altogether, these findings revealed that HERV-K Gag interferes the HIV-1 replication by two distinct molecular mechanisms.
Long terminal repeat (LTR)-bounded elements, which are called human endogenous retroviruses (HERVs), comprise about 8% of the human genome [1–3]. HERVs have infected germ lineage cells, and therefore their proviruses are transmitted vertically from ancestors to progeny in human genomic DNA . During a period exceeding a million years, they have acquired numerous mutations or deletions and therefore no longer encode infectious retrovirus . HERV-K, which is relatively new endogenous retrovirus among HERV families, apparently contains a set of intact open reading frames . However, all known HERV-K proviruses are replication incompetent [7–9]. Two groups reconstructed infectious HERV-K sequences by aligning full-length HERV-K proviruses [9, 10]. The infectious HERV-K clones have become a widely used tool for biological research of HERV-K.
Virion assembly of HIV-1 as well as HERV-K occurs at the plasma membrane (PM) [9, 11]. HIV-1 Gag consists of four major domains, matrix (MA), capsid (CA), nucleocapsid (NC) and p6 . These domains mediate each step of the virion assembly events. The MA domain promotes Gag targeting and binding to the PM. The CA domain mediates the Gag–Gag interactions for assembly of the immature virion and formation of conical shell of the mature viral core. The NC domain binds the viral genome through the two zinc finger motifs and facilitates Gag multimerization during viral assembly. p6, which contains late domain motifs, binds TSG101 and ALIX, recruits the ESCRT machinery and facilitates release of nascent virus particles from the PM [13–15]. Similar to HIV-1 Gag, HERV-K Gag consists of 4 major domains, MA, CA, NC and p15 [16, 17]. The N-terminus of the MA domain is likely myristoylated and essential for Gag binding to the PM . The CA domain contains the major homology region (MHR) that is conserved among retroviruses . The NC domain encodes two zinc finger motifs for RNA binding [16, 17]. The late domain in p15 is essential for the pinch-off of virus from the PM . Unlike the p6 domain of HIV-1 Gag, p15 is located between MA and CA in HERV-K Gag. The functions of each domain of HERV-K Gag are likely to be similar to those of HIV-1 Gag [16–20].
All human cells harbor HERV-K genomes. HERV-K is expressed in germ cells and under some pathological conditions [21–25]. In HIV-1-infected patients, antibodies and T cell responses against HERV-K are detected [26–30]. Furthermore, HERV-K RNA and Gag protein are upregulated in plasma samples of HIV-1-infected patients [28, 31–36]. It has been also shown that HIV-1 Tat changes the state of heterochromatin and induces the HERV-K expression in somatic cells [37–39]. Thus, it is possible that HERV-K Gag exists simultaneously with HIV-1 Gag in same host cells. In our previous study, we observed that HERV-K Gag of the reconstructed clone coassembles with HIV-1 Gag at the PM when overexpressed . For the coassembly of HERV-K Gag with HIV-1 Gag, membrane binding via MA domains and RNA binding via NC domains are essential. Importantly, the virus release efficiency and infectivity of HIV-1 were substantially reduced when coassembly of HIV-1 Gag with HERV-K Gag was observed.
Previously, we showed that HIV-1 Gag coassembles with HERV-K Gag but not MLV Gag . However, MLV Gag binds the PM and RNA like HERV-K Gag. Therefore, it is likely that there is an unknown mechanism that determines the specificity of interaction between HERV-K Gag and HIV-1 Gag. In this study, we determined the domains responsible for the specific interaction between HERV-K Gag and HIV-1 Gag. We found that HERV-K Gag CA MHR promotes specific colocalization with HIV-1 Gag most efficiently, whereas HERV-K Gag CA N-terminal domain (NTD) is needed for HERV-K-Gag-mediated interference of HIV-1 release. However, both HERV-K Gag CA-MHR and CA-NTD were not required for reduction of HIV-1 infectivity, which coincided with a change in the size of HIV-1 particles. Together, these data provide the two distinct molecular mechanisms for the HERV-K Gag interference against HIV-1 replication.
For expression of HERV-K Gag, pCRVI/HERV-K/Gag, a kind gift from P. Bieniasz, was used in this study . This plasmid encodes the HERV-KCON Gag sequence following a CMV promoter and a sequence corresponding to the HIV-1 5′ untranslated region (nt 428–785 in pNL4-3), along with ORFs encoding HIV-1 Rev, Tat, and Vpu. pCRVI/HERV-K/Gag-Flag, pCRVI/HERV-K/Gag-Venus, pCRVI/HIV-1/Gag-Flag, pCRVI/HIV-1/Gag-Venus, pCRVI/HIV-1/Gag-mRFP, pCRVI/MLV/Gag-Flag, and pCRVI/MLV/Gag-Venus were described previously . Chimeric HERV-K Gag constructs were derived from the pCRVI/HERV-K Gag-Flag and pCRVI/MLV Gag-Flag. TSG101-DN was constructed in the same design as Tsg-5’ described previously . HIV-1/YP(−) was created by PCR mutagenesis and contains amino acid substitutions in the ALIX binding motif (YP).
HeLa cells and TZM-bl cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma) supplemented with 5% FBS (DMEM-10). TZM-bl (also called JC.53.bl-13) is a HeLa cell derivative that stably expresses large amounts of CD4 and CCR5 . TZM-bl cells that harbor Tat-responsive reporter genes for firefly luciferase (Luc) and Escherichia coli B-galactosidase were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH from Dr. John C. Kappes, Dr. Xiaoyun Wu and Tranzyme Inc .
HeLa cells were cotransfected with pNL4-3 and indicated pCRVI plasmids using lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. At 16 h post-transfection, the supernatants were filtered through 0.45-μm filters, and virions in the supernatants were pelleted down by ultracentrifugation (83,500×g, 4 °C, 45 min). Gag proteins in the virion lysates were quantified by p24 ELISA according to the manufacturer’s instruction (MBL).
Virus release assay
HeLa cells were cotransfected with pNL4-3 and indicated pCRVI plasmids. At 16 h post-transfection, virions in the supernatants were collected and pelleted down by ultracentrifugation (83,500×g, 4 °C, 45 min). Cells and virions were lysed with 0.5% TritonX lysis buffer [50 mM Tris–HCl pH7.5 containing 0.5% TritonX-100, 300 mM NaCl, 10 mM Iodoacetamide with protease inhibitor cocktail (Roche)]. Gag proteins in the cell and virion lysates were detected by immunoblotting using HIV-Ig (NIH AIDS Research and Preference Reagent Program), mouse monoclonal anti-Flag antibody (Wako), mouse monoclonal anti-HERV-K Gag antibody (HERM-1831-5) (Austral Biologicals) as primary antibodies. HRP-conjugated anti-human Ig antibodies (Jackson ImmunoResearch) and anti-mouse Ig (Amersham) were used as a secondary antibody. Detection using a HRP-conjugated secondary antibody was performed using the Chemi-Lumi One L (Nacalai tesque).
Rate-zonal gradient analysis
Rate-zonal gradient analysis was performed as previously described . Virions in cell-free supernatants were collected and centrifuged at a low speed (8000×g, 5 min) to remove cellular debris. Virions were pelleted by ultracentrifugation and re-suspended in 1 ml of RPMI-10. Each concentrated sample was layered onto 10–30% sucrose and ultracentrifuged (83,500×g, 4 °C, 45 min) in a Beckman SW41Ti. Then 1 ml fractions were collected from each gradient. Amounts of HIV-1 Gag proteins in each fraction were measured by p24 ELISA.
Infectivity analysis of virions in each sucrose fraction
Each sucrose fraction was two-fold diluted with RPMI-10. For removing sucrose from each fraction, virions in each fraction were pelleted down by ultracentrifugation and resuspended with RPMI-10. Amounts of virions were determined by p24 ELISA.
For analysis of virus infectivity using TZM-bl cells, 3 × 104 cells were inoculated with virus stocks normalized by the amount of p24 Gag (2 ng of p24 Gag) for 2 h. Two days post-infection, Luc activities in TZM-bl cells were measured according to the manufacturer’s instruction (Promega).
HeLa cells were plated in 8-well chamber slides (Nunc) 1 day before transfection at 3.0 × 104 cells/well. At 16 h post-transfection, HeLa cells cotransfected with plasmids encoding YFP- and mRFP-tagged Gag proteins were fixed with 4% paraformaldehyde (Wako) in PBS for 30 min at room temperature, washed once with PBS, and mounted in Fluoromount-G (Dako). The images of 20–50 fields were recorded using a Zeiss LSM 700 laser-scanning confocal microscopy. Colocalization between YFP- and mRFP-tagged Gag was quantified using the ZEN software (Zeiss) with which we calculate the Pearson correlation coefficient (R-value). We set the entire cell body of each YFP- and mRFP-coexpressing cell as the region of interest for this analysis. R = 1 represents perfect co-localization, and R = 0 represents random distributions of fluorescence intensities.
Transmission electron microscopy analysis
HeLa cells were transfected with indicated plasmids. Cells were fixed 16 h post-transfection with 2% glutaraldehyde in PBS. Cells were analyzed on a Hitachi H7600 transmission electron microscope as previously described .
Coassembly of HERV-K Gag alters HIV-1 particle properties
HERV-K Gag CA-NTD is required for reduction of HIV-1 release
HERV-K Gag reduces the HIV-1 assembly at the early steps
HERV-K CA-MHR plays a key role in robust colocalization between HERV-K Gag and HIV-1 Gag at the plasma membrane
HERV-K CA-NTD and MHR are not required for reduction of HIV-1 infectivity
To examine the infectivity of HIV-1 particles with the larger size, we purified the fractionated viruses from sucrose gradients. The purified virus in each fraction was normalized by amounts of p24 antigens. TZM-bl cells, which encodes Tat-driven luciferase gene, were infected with these normalized viruses. The infectivity of HIV-1 in the fraction #04 was not severely reduced by HERV-K Gag (Fig. 9b). However, the infectivity of HIV-1 in the fractions #07-09, which are likely to be enriched in coassembled particles, was drastically reduced (Fig. 9b). Chimeric HERV-K Gags encoding MLV Gag CA-NTD reduced the infectivity of HIV-1 similarly. These results suggest that HERV-K CA-NTD is not necessary for inhibition of HIV-1 infectivity (Fig. 9b). In addition, the presence of HERV-K CA MHR, which is important for extensive colocalization with HIV-1 Gag (Fig. 8), did not exacerbate the infectivity defect of HIV-1 virions, suggesting that HERV-K CA MHR and the high-level colocalization dependent on this region are dispensable for inhibition of HIV-1 infectivity.
In this study, we found that HERV-K Gag CA-NTD is important for efficient inhibition of HIV-1 release, whereas CA-MHR is required for higher colocalization between HIV-1 Gag and HERV-K Gag, and yet both CA-NTD and CA-MHR are not essential for inhibition of infectivity. These findings suggest that HERV-K Gag inhibits HIV-1 release and impairs infectivity of released progeny virions in genetically separable mechanisms.
HERV-K Gag consists of 4 major domains, MA, p15, CA and NC domains. Previously, we suggested that HERV-K Gag coassembles with HIV-1 Gag at the PM in a manner dependent on MA-mediated membrane binding and NC-mediated RNA binding . However, MLV Gag, which binds to the PM and RNA just like HERV-K Gag, did not colocalize or coassemble with HIV-1 Gag and failed to inhibit HIV-1 release. It was unclear how HERV-K Gag, but not MLV Gag, targets to the assembly sites of HIV-1 Gag at the PM. In this study, we found that HERV-K CA, when replaced with MLV CA, can promote colocalization of MLV Gag with HIV-1 Gag at the PM in most cases (Fig. 7b). Among the tested HERV-K CA regions, MHR promotes the colocalization most efficiently (Fig. 8b). Nonetheless, it appears that any of the tested HERV-K CA regions is capable of promoting colocalization with HIV-1 Gag.
We observed that a single chimera, HeHeM, showed minimal correlation of distribution with HIV-1 Gag despite containing the entire HERV-K CA. It is possible that this chimera suppresses the HIV-1 assembly process at the step of membrane binding of Gag multimer. We speculate that heteromultimerization between HIV-1 Gag and HeHeM Gag might destabilize PM binding of the Gag multimer at an early step of HIV-1 assembly (see below), prevent formation of prominent coassemblies at the PM, and thereby reduce colocalization between HIV-1 Gag and the chimera. In this regard, it is of note that the colocalization efficiency represented by the R value may underestimate the ability of Gag constructs to heteromultimerize with HIV-1 Gag if a large fraction of cells display only cytosolic HIV-1 Gag as observed in cultures coexpressing WT HERV-K Gag or chimeras containing the entire HERV-K CA.
Based on TSG101 experiments, coassembly of HERV-K Gag is likely to take place at the early stage of HIV-1 Gag assembly (Fig. 5). The Lingappa group reported that three classes of HIV-1 CA residues are involved in distinct steps of virus assembly [52, 56]. Six residues in CA-CTD, VK181/182, WM184/185 and LL189/190, are involved in the dimerization of CA-CTD. Three residues in CA-CTD, K158, D197 and P224, are involved in the low order multimerization. Finally, eight residues, EE75/76, RS100/102, TT107/108 and TQ110/112 in CA-NTD are important for the completion of particle formation. In this study, we found that release efficiency of 5 HIV-1 mutants, VK181/182AA, WM184/185AA, LL189/190AA, K158A and P224A is not reduced by HERV-K Gag (Fig. 6). Therefore, the early assembly steps, which require Gag dimerization or low-order multimerization, are likely to be the target of HERV-K Gag or prerequisite for the process inhibited by HERV-K Gag. It is unclear why the virus release efficiency of D197A mutant was reduced by HERV-K Gag unlike that of K158A or P224A mutants. However, we observed that the D197A mutant releases 10-fold more p24 than K158A and P224A mutants in our experiments (Fig. 6). Therefore, it is possible that D197A might impose less severe suppression than K158A or P224A on the stage susceptible to the inhibition by HERV-K. We also do not rule out an alternative possibility that VK181/182AA, WM184/185AA, LL189/190AA, K158A and P224A may directly or indirectly disrupt the interface for HERV-K Gag.
We observed that coexpression of HERV-K Gag reduces the number of HIV-1 particles with mature core formation (Fig. 1c, e) although it did not cause accumulation of HIV-1 Pr55 Gag in virions (Fig. 3b). In this regard, the effect of HERV-K Gag is reminiscent of the effect of bevirimat, a maturation inhibitor, except that the CA-SP1 fragment was not detected in either cells or viruses unlike with the case with bevirimat treatment. Indeed, the morphology of HIV-1 virions produced from cells coexpressing HERV-K Gag (frame 2 of Fig. 1c) appears similar to that of HIV-1 particles produced by cells treated with bevirimat [57–59]. Notably, within HERV-K CA regions, both CA-NTD and MHR are not required for impairing the infectivity of HIV-1 (Fig. 9b). Therefore, it is possible that HERV-K Gag impairs HIV-1 core formation or stability after the processing of HIV-1 Gag and does so by interactions with HIV CA via its CA C-terminal region outside of MHR. Further studies focused on the morphology and biochemical properties of cores of co-assembled particles containing HERV-K CA chimeras should help establish the relationship between HERV-K Gag-induced changes in HIV-1 maturation and impairment of its infectivity.
Previous studies observed that HERV-K expression is increased upon HIV-1 infection in T cells [33, 39]. Consistent with this, we observed HERV-K induction by HIV-1 infection in an in vitro experiment (data not shown). It is tempting to suggest that HERV-K sequences have existed in the human genome under several selection pressures and might have protected the host cells from the threat of exogenous viruses as are the case with other endogenized viruses. Fv1, which is a remnant of mouse endogenous retrovirus Gag, interferes with the post-entry process of MLV infection [60–63]. Endogenous retroelements Fv4, enJSRV Env and Refrex-1 Env prevent the entry of exogenous retroviruses MLV , JSRV  and FeLV-2 [66, 67], respectively, through receptor masking. Similar to HERV-K , enJSRV also blocks the JSRV particle formation via its Gag . Recently, Wysocka group reported that HERV-K element (Rec), which is expressed in early embryogenesis, appears to induce an innate immune response (IFITM1) and protect the host cells from exogenous viral infection . In the current study, we showed that HERV-K Gag can suppress the HIV-1 assembly at an early step and alter the properties of HIV-1 particles, via distinct molecular mechanisms. Altogether, endogenous retroviral elements are likely to have been contributing survival of the hosts in the evolutionary time scale via a wide variety of mechanisms.
HERV-K is principally expressed in germ cells, but eventually silenced through the development process. Interestingly, however, accumulating evidence suggests that HERV-K reappears in HIV-1-infected patients. We previously reported that HERV-K Gag coassembles with HIV-1 Gag and interferes with the HIV-1 release. Moreover, HERV-K Gag reduces the infectivity of HIV-1. However, the molecular mechanisms by which HERV-K Gag interferes with HIV-1 replication remain poorly understood. In this study, we found that HERV-K CA domain is important for specific incorporation into HIV-1 virions and reduction of HIV-1 release. HERV-K Gag interfered with HIV-1 Gag assembly at an early step(s) and changed HIV-1 particle properties including core formation and infectivity. The effects on HIV-1 release and infectivity were genetically separable.
KM, YM and AO conceived and coordinated the study. KM, HT and YN performed experiments. FS and KN performed transmission electron microscopy analysis. KM and AO prepared the manuscript. All authors read and approved the final manuscript.
We would like to thank Dr. Shinji Harada for helpful discussions and critical review of the manuscript. We would also like to thank Dr. Paul D. Bieniasz for providing plasmids.
The authors declare that they have no competing interests.
The following reagents were obtained through AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: HIV-Ig from NABI and NHLBI. This work was supported by MEXT KAKENHI Grant Number 24790445 to K.M., Takeda Science Foundation to K.M., Kumamoto University AIDS Global COE Program International Research Scientist Development Awards Young Investigator Grant to K.M., the JSPS Institutional Program for Young Researcher Overseas Visits to K.M., Okukubo Memorial Fund for medical Research in Kumamoto University School of medicine to K.M., as well as by the National Institutes of Health grant R01 AI071727 to A.O. and contract HHSN26120080001E from the National Cancer Institute, National Institutes of Health, to K.N.
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