Lipin1 is rate limiting for HCV infection

Given the relevance of lipin1 for lipid metabolism and its steatogenic potential, we set out to independently verify data from published differential transcriptomic profile studies that suggested that HCV infection may alter LPIN1 mRNA abundance in cell culture [40, 41]. A specific and statistically significant LPIN1 mRNA induction (Fig 1A) was observed in Huh-7 cells after single cycle infection experiments [multiplicity of infection (MOI) 10] with a cell culture-adapted genotype 2a HCV (D183) variant [42] at the peak of the infection (48 and 72 hours post-infection) and as compared with mock-infected cells (Fig 1B). LPIN1 mRNA induction was prevented when infected cells were treated with 1μM sofosbuvir (Fig 1C), an HCV RNA polymerase inhibitor [43] that reduced viral RNA accumulation by more than two orders of magnitude (Fig 1D), indicating that active HCV replication is required to induce LPIN1 mRNA accumulation.

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Fig 1. HCV infection induces lipin1 expression.

Huh-7 cells were infected at MOI 10 with HCV D183 virus and samples of control (black bars) and infected (gray bars) cells were collected 24, 48 and 72 hours post-infection to determine LPIN1 mRNA levels (A) and intracellular HCV RNA (B). RNAs from mock-infected cells and HCV infected. Impact of sofosbuvir (1μM; DAA) (C, D) or N-acetylcysteine (10mM; NAC) treatment (E, F) on HCV RNA accumulation and LPIN1 mRNA levels. Data are shown as average and standard deviation of two independent infection experiments performed in triplicate (n = 6). G- Protein samples of infected and control cells collected at 48 hours post-infection were subjected to Western-blot analysis to determine lipin1 and NS3 levels, using beta-actin as loading control H-Quantitation of the relative lipin1 protein levels in mock and HCV-infected cells (n = 3). Statistical significance was determined using Student´s t-test (*p<0.05; **p<0.01).

https://doi.org/10.1371/journal.ppat.1007284.g001

LPIN1 mRNA has been shown to be upregulated by mechanisms that involve induction of reactive oxygen species (ROS), as treatment of the cells with ROS-scavenger molecule N-acetylcysteine (NAC) is capable of preventing LPIN1 mRNA induction under glucose deprivation [44] or during H₂O₂ treatment [45]. Given that transcriptional activation of a subset of lipogenic genes during HCV infection is also prevented by addition of antioxidants [46], we sought to determine if LPIN1 mRNA induction by HCV infection is mediated by ROS production and therefore dampened by NAC treatment. LPIN1 mRNA induction was prevented in the presence of the antioxidant (Fig 1E), despite comparable HCV RNA accumulation in mock-treated and NAC-treated HCV-infected cells (Fig 1F), suggesting that virus replication-induced ROS production is required to induce LPIN1 mRNA accumulation. Western-blot analysis confirmed that the observed transcriptional change leads to a correlative protein accumulation (Fig 1G and 1H); reinforcing the notion that acute HCV infection alters lipin1 expression.

In order to determine if lipin1 subcellular localization was altered during HCV infection, we performed confocal microscopy studies in control and HCV-infected Huh-7 cells. Lipin1 staining was observed as cytoplasmic punctated structures both in control and HCV-infected cells. To study if lipin1 signal colocalized with viral antigens, we performed double staining with antibodies against lipin1 and double-stranded RNA (dsRNA) or replicase subunits NS3 and NS5A. None of the viral antigens strictly colocalized with lipin1 (Pearson´s<0.5) (S1A Fig). However, Mander´s coefficients indicate that the majority of lipin1 overlapped with a small fraction of NS3 and NS5A (S1C Fig). In contrast to lipin1, lipin2 signal did not overlap with that of viral proteins NS3 and NS5A (S1B Fig). Our results indicate that no major lipin1 rearrangements are observed after HCV infection and that only a minor fraction of NS proteins colocalize with lipin1.

To study if lipin1 plays any role in HCV infection, lipin1-deficient cells were generated by transducing human hepatoma (Huh-7) cells with lentiviral vectors expressing shRNAs targeting LPIN1 mRNA or a control vector expressing an irrelevant shRNA. Lipin1 expression silencing was verified by Western-Blot, typically 7 days post-transduction (Fig 2A). A partial (50%; shLPIN1-1) and a more profound (>95%; shLPIN1-2) reduction in lipin1 accumulation was observed after transduction with specific shRNAs as compared with the control (Fig 2B). As expected, lipin1 and lipin2 expression is inversely correlated in lipin1 shRNA-expressing cells (Fig 2B). The viability of lipin-deficient cells, as determined by MTT assay [47] was comparable to that of control cells (Fig 2B). These results illustrate that lipin1 shRNA-expressing cells respond homeostatically to functionally compensate partial (shLPIN1-1) and more pronounced (shLPIN1-2) loss of lipin1 (Fig 2B).

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Fig 2. HCV propagation is limited in lipin1-deficient cells.

Huh-7 cells were transduced with lentiviral vectors expressing control or LPIN1-specific shRNAs. Seven days after transduction samples of the cells were collected for Western-blot analysis using antibodies against lipin1 and lipin2 and actin as loading control. Parallel cultures were subjected to an MTT assay to determine their viability as described in the methods section. (A) Representative Western-Blot showing cellular lipin1 and lipin2 protein expression levels and a loading control (actin). (B) Average expression values for lipin1 and lipin2 as determined by Western-Blot and cell viability as determined by an MTT assay. Data are shown as average and SD of six independent transduction experiments (n = 6). (C) Huh-7 cells were transduced with lentiviral vectors expressing control or LPIN1-specific shRNAs. At day 3 post-transduction, cells were infected at MOI 0.01 with HCV D183 virus. Samples of cell supernatants were collected at days 3 and 5 post-infection to determine the extracellular infectivity titer. Average and SD of the infectivity titers at day 3 and 5 of two independent infections performed in triplicate (n = 6). (D) Huh-7.5 cells were transduced with lentiviral vectors expressing control or LPIN1-specific shRNAs. Silenced cells were infected 7 days after transduction at MOI 0.05 with genotype 1a HCV (TNcc) in the presence or absence of 2mAde (10μM; shControl+DAA). Intracellular HCV RNA was determined by RT-qPCR 72 hours post-infection. Data are shown as average and SD of three experiments performed in triplicate (n = 9). Statistical significance was determined using Student´s t-test (*p<0.05; **p<0.01).

https://doi.org/10.1371/journal.ppat.1007284.g002

Control and lipin1-deficient cells were infected with a genotype 2a D183 at MOI 0.01 to study viral spread by determining extracellular infectivity titers at different times post infection. Fig 2C shows limited propagation of this virus in lipin1-deficient cells, with a statistically significant reduction of up to two (shLPIN1-1) and three orders of magnitude (shLPIN1-2) in viral titer between the control and lipin1-deficient cell lines at day 5 post-infection. These results indicate that lipin1 is required for efficient HCV propagation and that homeostatic lipin2 accumulation (Fig 2B) is not sufficient to support efficient HCV infection.

Since important differences in the interaction of different HCV genotypes with cellular lipid metabolism have previously been described [48], we set out to determine if the observations made with a JFH1-derived virus (genotype 2a) were extensive to other HCV genotypes. We performed low multiplicity infections (MOI 0.05) with genotype 1a TNcc virus strain in lipin1-deficient Huh-7.5 cell lines, which are susceptible to infection by this recombinant virus [49]. First, we verified that Huh-7.5.1 also display a significant reduction in genotype 2a infection efficiency when lipin1 is silenced (S2B Fig). In order to determine TNcc infection efficiency, intracellular HCV RNA accumulation was determined 72 hours post-inoculation in control and lipin1-deficient Huh-7.5 cells. Viral RNA detected under these conditions reflects the ability of genotype 1a to infect and replicate viral RNA in the different cell lines, as treatment of control cells with an HCV polymerase inhibitor 2´-C-methyladenosine (2mAde; 10 μM) [50] reduced viral RNA content by two orders of magnitude (Fig 2D). Lipin1 silencing consistently and significantly reduced TNcc replication by approximately 3-fold both in shLPIN1-1 and shLPIN1-2 cell lines (Fig 2D), indicating that lipin1 is also limiting for genotype 1a HCV infection.

To determine the specificity of these observations, identical lipin1-deficient and control Huh-7 cultures were inoculated at MOI 0.01 with a human alpha-coronavirus CoV-229E bearing a GFP reporter gene (hCoV-229E-GFP) [51]. Inoculation of control and lipin1-deficient cells with this virus resulted in comparable progeny virus production, as determined by infectivity titration in cell supernatants 48 hours post-infection (S3 Fig). These results suggest that lipin1 is not rate limiting for CoV-229E-GFP infection and that lipin1 expression is particularly limiting for HCV.

Early aspects of HCV infection are limited in lipin1-deficient cells

To determine which aspects of the HCV replication cycle are limited by lipin1 silencing, single cycle infection experiments were conducted by inoculating control and lipin1-deficient cell cultures at MOI 10 with genotype 2a D183 virus. Infection efficiency was measured by titration of progeny virus infectivity present in the supernatant of infected cells and intracellular HCV RNA accumulation at 48 and 72 hours post-infection. Infection of lipin1-deficient cells resulted in a significant reduction of progeny infectious virus production in shLPIN1-1 and shLPIN1-2 cells as compared with the titers observed in the supernatants of control cells (Fig 3A), reinforcing the notion that lipin1 silencing interferes with HCV infection. Reduced virus production is likely due to parallel reduction of intracellular HCV RNA levels observed in lipin1-deficient cells as compared with the control cell line (Fig 3B). This reduction was observed at all time points, except for that at 5 hours, indicating that the size of the inoculum and initial virus adsorption is comparable among the different cell lines (Fig 3B). Thus, lipin1 silencing suppresses HCV infection by interfering with a step of the HCV lifecycle preceding intracellular HCV RNA accumulation.

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Fig 3. Lipin1 silencing interferes with early steps of HCV infection.

Panels A, B- Huh-7 cells were transduced with lentiviral vectors expressing control or LPIN1-specific shRNAs. At day 7 post-transduction, cells were infected at MOI 10 with HCV D183. Samples of the supernatants and the cells were collected at 24, 48 and 72 hours post-infection. (A) Relative infectivity titers found in infected cell supernatants. (B) Intracellular HCV RNA determined at the indicated time points is shown as genome equivalents per microgram of total RNA GE/μg). Panels C, D-Persistently infected cultures were generated by inoculation with JFH-1 virus at MOI 0.01. Once cultures reached >95% of HCV-positive cells, they were transduced with lentiviral vectors expressing control, HCV RNA-targeting or LPIN1-specific shRNAs. At day 7 post-transduction, cells were split and samples of the cells and supernatants were collected 24 hours later to determine infectious virus production rate by infectivity titration HCV (C) and RNA levels by RT-qPCR (D). All data are shown as mean and SD of 3 independent experiments performed in triplicate (n = 9). Statistical significance was determined using Student´s t-test (*p<0.05; **p<0.01).

https://doi.org/10.1371/journal.ppat.1007284.g003

Next, we set out to determine if lipin1 silencing has any impact on persistent HCV infections to verify if lipin1 is also limiting for late aspects of the virus lifecycle. Persistently infected cells continuously replicate viral RNA, express viral antigens and secrete infectious virions. Thus, it is a valuable system to measure steady-state HCV RNA replication as well as infectious particle assembly and secretion. Persistently infected cultures were transduced with the lentiviral vectors described above to produce persistently infected, lipin1-deficient cells (S4 Fig). Analysis of extracellular infectivity titers revealed that infectivity titers in lipin1-deficient and control cells were comparable, with the exception of a marginal reduction in shLPIN1-2 expressing cells, indicating that lipin1 silencing does not strongly interfere with infectious virus production (Fig 3C). Intracellular HCV RNA levels in lipin1-deficient cells were comparable to that of the control cells (Fig 3D), indicating that lipin1 expression is not rate limiting for HCV RNA replication once infection has been established. Taken together, the results shown above indicate that lipin1 is only limiting at early steps of HCV infection leading to viral RNA accumulation.

As an independent verification of the hypothesis that lipin1 is limiting for early aspects of HCV infection, we used a single cycle surrogate infection model based on the production of HCV virions bearing a defective reporter genome encapsidated by trans-complementation (HCV_tcp). HCV_tcp are capable of producing abortive single-cycle infections, efficiency of which is proportional to the luciferase activity found in the target cells [52]. As expected from the results shown in Fig 3, infection of lipin1-deficient cells with HCV_tcp resulted in a 75% reduction in the reporter luciferase activity in shLPIN1-1 cells and 90% in shLPIN1-2 determined at 48 hours post-infection, proportionally to the degree of silencing in these cells (Fig 4A and 4B). These results underscore the role that lipin1 plays at early steps of HCV infection and indicate that either viral entry or a step leading to efficient HCV RNA replication is impaired in these cells.

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Fig 4. Infection of lipin1-deficient cells with HCV_tcp.

Huh-7 cells were transduced with lentiviral vectors expressing control or LPIN1-specific shRNAs. shLPIN1-1 and shLPIN1-2 target both isoforms, while shLPIN1-3 is specific for lipin1 isoform alpha and shLPIN-4 for isoform beta. Silencing was verified at day 7 post-transduction and control and silenced cells were inoculated with HCV_tcp. Luciferase activity was determined in the different cell lines 48 hours post-inoculation. (A) Total lipin1 protein quantitation by Western-Blot. (B) Relative HCV_tcp infection efficiency. (C) Plot displaying lipin1 expression data versus HCV_tcp infection efficiency and the correlation index adjusted to an exponential curve. Data are shown as average and SD of four independent experiments performed in triplicate (n = 12). Statistical significance was determined using Student´s t-test (*p<0.05; **p<0.01).

https://doi.org/10.1371/journal.ppat.1007284.g004

LPIN-1 mRNA is alternatively spliced in human liver to produce isoforms α and β, which may differ in catalytic activity, subcellular localization and gene expression regulation [53]. To determine the relative contribution of these isoforms to HCV infection, isoform-specific shRNAs were generated and used to transduce Huh-7 cells, as described above. LPIN1α-specific shRNA (shLPIN1-3) reduced total lipin1 protein by 70%, while LPIN1β-specific shRNA (shLPIN1-4) reduced total lipin1 expression by only 30% (Fig 4A). Infection of control and lipin1 isoform-deficient cells with HCV_tcp revealed that lipin1α silencing resulted in a strong (85%) reduction in HCV infection while lipin1β silencing resulted in a milder (55%) but significant reduction in HCV infection efficiency (Fig 4B). These results indicate that both isoforms alpha and beta are limiting for HCV infection and suggest that the total amount of lipin1 present in the cell determines HCV infection efficiency. Taken together, the results obtained with four different shRNAs indicate that total lipin1 expression levels strongly correlate with HCV_tcp infection efficiency (Fig 4C), underscoring a role for this host protein in early aspects of HCV infection.

Lipin1 silencing interferes with initial steps of HCV RNA replication

Reduced HCV RNA accumulation in a single cycle infection (Fig 3) as well as reduced HCV_tcp infection (Fig 4) may be due to a defect in entry of incoming virions. HCV E1E2-pseudotyped retroviral vectors bearing a luciferase gene (HCV_pp) were used to measure viral entry because they constitute a sound model to study viral adsorption, receptor-mediated internalization and E1E2-mediated fusion in endosomes [54]. To assess the specificity of these observations, parallel cultures were inoculated with VSV-G pseudotyped retroviral vectors (VSV_pp). Control and lipin1-deficient cell lines were infected with HCV_pp (genotype 2a; JFH-1 strain) and VSVpp. As a positive control of inhibition of HCV entry, we used hydroxyzine (5 μM), which efficiently blocks HCV infection by interfering with viral entry [55]. As expected, hydroxyzine selectively inhibited HCV_pp infection, as shown by reduced luciferase levels 48 hours post-inoculation only in HCV_pp-infected cells (Fig 5A). Interestingly, lipin1-deficient cells (shLPIN1-1 and shLPIN1-2) were fully susceptible to HCV_pp and VSV_pp infection, as comparable luciferase activity levels were found in all cell lines 48 hours post-inoculation (Fig 5A). These results indicate that lipin1 is not rate limiting for receptor binding, particle internalization or E1E2-mediated endosomal fusion, which are steps recapitulated in this model [54]. Based on the data presented thus far, we hypothesized that lipin1 is limiting for a step in the HCV lifecycle downstream of HCV entry, leading to HCV RNA accumulation. To verify the hypothesis that lipin1 silencing causes strong reduction in initial HCV RNA accumulation by interfering with a step downstream of viral entry, we bypassed this step of the viral replication cycle by transfecting a subgenomic HCV RNA replicon bearing a reporter luciferase gene into control and lipin1-deficient cells. First, we evaluated HCV-IRES driven primary translation of incoming genomes by transfection of a replication-deficient mutant replicon that bears an inactivation mutation in the catalytic site of NS5B RNA polymerase [56]. Luciferase activity measured at 5 hours post-transfection was not reduced in any of the cell lines, indicating that transfection efficiency and HCV IRES-dependent primary translation was not significantly affected by lipin1 silencing (Fig 5B). A significant increase in Renilla luciferase activity was observed in shLPIN1-1 cells both when transfecting a replicon (Fig 5B) or a plasmid expressing Renilla luciferase under a minimal RNA polymerase II promoter (S5A Fig), suggesting that the increase in luciferase activity observed in these cells is not related with HCV. HCV RNA replication was evaluated by measuring accumulation of a reporter luciferase gene at 5 and 48 hours post-transfection of a replication competent HCV subgenomic replicon. Under these experimental conditions luciferase accumulation at 5 hours also represents HCV IRES-driven primary translation of the input RNA, while reporter luciferase activity at 48 hours depends on effective HCV RNA replication. In contrast to primary translation, HCV RNA replication inferred by luciferase activity at 48 hours was strongly reduced in lipin1-deficient cells (90% in shLPIN1-1 and 99% in shLPIN1-2 cells) (Fig 5C), indicating that initiation of HCV replication is dependent on normal lipin1 expression. This reduction was not due to a non-specific defect in luciferase expression, as co-transfection of a plasmid expressing Renilla luciferase lead to comparable luciferase accumulation in all cell lines, discarding the possibility that death of transfected cells or other spurious effects are responsible for the reduced luciferase activity accumulation (S5A Fig). Similar experiments were conducted in ATG4B-deficient cells, as this host factor was shown to be limiting for primary HCV translation [57]. Our studies confirmed that, while partial ATG4B silencing (S5B Fig) significantly interfered with primary translation (S5C Fig) and correlatively with HCV RNA replication efficiency, as expected [57], lipin1-silencing only affected HCV RNA replication (S5D Fig). Overall, these data indicate that HCV RNA replication is not initiated efficiently in lipin1-deficient cells and that blockade occurs at a step downstream translation of incoming HCV genomes.

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Fig 5. Lipin1-silencing interferes with early HCV RNA replication, downstream of viral entry and primary translation.

Huh-7 cells were transduced with lentiviral vectors expressing control or LPIN1-specific shRNAs. (A) Control and silenced cells were inoculated with HCV E1E2 (HCV_pp) or VSV-G-pseudotyped retroviral vectors (VSV_pp) 7 days post-transduction. Control cells treated with hydroxyzine (5μM) were used as positive inhibition control. Luciferase activity was determined in the different cell lines at 48 hours post-inoculation. HDX, hydroxyzine pamoate (5μM). Panels B, C- Control and lipin1-deficient cells were transfected with a replication-deficient mutant (B) or replication competent subgenomic HCV replicon bearing a luciferase gene (C). Luciferase activity was determined in the different cell lines at 5 hours post-transfection for both replicons and 48 hours post-transfection for the replication-competent replicon RNA. Data are expressed as average and SD of three independent experiments performed in triplicate (n = 9). Statistical significance was determined using Student´s t-test (*p<0.05; **p<0.01).

https://doi.org/10.1371/journal.ppat.1007284.g005

Lipin1 phosphatidate phosphatase activity is required to support HCV infection

In order to determine if any of the known functions ascribed to lipin1 is required for HCV infection, we tested the ability to restore HCV infection susceptibility of silencing-resistant wild-type (wt) or mutant lipin1 versions bearing a mutation in the catalytic site responsible for its phosphatase activity (DXDXT) and a mutant in the LXXIL motif, which is inactive both for transcriptional activation as well as for phosphatase activity [31]. Control and lipin1-deficient cells were transfected with wt lipin1 beta cDNA as well as with DXDXT and LXXIL mutants. Comparable overexpression levels of the wt and mutant proteins was obtained in each cell line, although overexpressed lipin1 levels were consistently higher in lipin1-deficient cells (Fig 6A). Cells were subsequently inoculated with HCV D183 at MOI 10 and relative infection efficiency was calculated by determining extracellular infectivity titers 48 hours post-infection. Overexpression of wt lipin1 did not significantly alter susceptibility to HCV infection in control cells, although we observed a small but consistent reduction in extracellular infectivity titers when overexpressing wt and mutant lipin1 constructs (S6A Fig). In contrast to control cells, wt lipin1 overexpression in lipin1-deficient cells consistently increased infectivity titers as compared to mock-transfected cells (S6B Fig) and in clear contrast with the reduction observed in control cells (S6A Fig) or when overexpressing mutant lipin1 in lipin1-deficient cells (S6B Fig). In order to take into account this divergent behavior in control and lipin1-deficient cells, we calculated the relative infection efficiency as the ratio of the infectivity titers found in lipin1-deficient cells and control cells (S6C Fig). Ratios were subsequently normalized to that found in mock-transfected cells in order to average the data from different experiments (S6 Fig). Using relative infection efficiency as readout of this set of experiments, we could clearly observe a statistically significant increase (2-fold) in the relative infection susceptibility in cells overexpressing wt lipin1 cDNA as compared with mock-transfected cells or cells expressing similar or higher levels of the mutants (Fig 6A), suggesting that wt lipin1 modestly, though significantly, rescues HCV infection while phosphatase (DXDXT) and transcriptional coactivation (LXXIL) mutants do not (Fig 6B). These results suggest that lipin1 transcriptional co-activation capacity is not sufficient to support HCV infection while lipin1 phosphatase activity is essential. However, given that LXXIL mutant is deficient both in transcriptional co-activation and PAP activity [31], we cannot determine if transcriptional co-activation by lipin1 is also required to support HCV infection.

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Fig 6. Impact of lipin1 wt and mutant cDNA overexpression in lipin1-deficient cells on HCV infection efficiency.

Huh-7 cells were transduced with lentiviral vectors expressing control or LPIN1-specific shRNAs. At day 3 post-transduction, cells were transfected with plasmids encoding wt, DXDXT or LXXIL lipin1beta cDNA. Forty-eight hours later cells were infected at MOI 10 with HCV D183. Extracellular infectivity titers were determined in the supernatants 48 hours post-infection. (A) Western-blot displaying intracellular lipin1 protein levels in the different cell lines at the time of infection, 48 hours post-transfection. (B) Relative infection efficiency in lipin1-deficient cells transfected with lipin1 cDNAs (see main text and S6 Fig for detailed description). Data are shown as average and SD of the infection efficiency relative to mock-transfected cells in two independent experiments performed in triplicate (n = 6). Statistical significance was determined using Student´s t-test (*p<0.05; **p<0.01).

https://doi.org/10.1371/journal.ppat.1007284.g006

HCV replicase complex formation is reduced in lipin1-deficient cells

The data described above suggest a role for lipin1 phosphatase activity in a step of HCV replication cycle between translation of the viral genome and formation of functional replicase complexes. Data regarding primary translation where inferred from a surrogate model of translation based on a reporter luciferase gene (Fig 5B). To address if indeed viral polyprotein is properly processed and inserted into detergent-resistant microdomains to form the characteristic membranous ultrastructures bearing the viral replicase, we used a replication-independent surrogate model of polyprotein expression. This system is based on a vector encoding the portion of the viral polyprotein corresponding to the replicase (NS3-NS5B) under the transcriptional control of the T7 polymerase and the translational control of encephalomyocarditis virus (EMCV) IRES (pTM-NS3/5B) [58]. Replication-independent polyprotein overexpression systems enable assessment of polyprotein processing as well as studying the formation of virus-derived membranous structures [16, 59].

Control and lipin1-deficient cells were infected with a recombinant vaccinia virus expressing T7 RNA polymerase (VacT7) and subsequently transfected with the plasmid pTM-NS3/5B to enable viral replicase expression. Sixteen hours post-transfection, cells were processed for Western-Blot using anti-NS3. Accumulation of NS3 is comparable in control and both lipin1-deficient cells, underscoring the notion that lipin1 is not limiting for polyprotein translation and processing (Fig 7A). Similarly, NS3 and NS5A expression and subcellular distribution was similar in all cell lines (Fig 7B). These results suggest that there are no major differences in accumulation of viral proteins in lipin1-deficient cells and that a step downstream is affected in these cells.

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Fig 7. HCV-induced vesicular structure formation is reduced in lipin1-deficient cells.

Huh-7 cells were transduced with lentiviral vectors expressing control or LPIN1-specific shRNAs. HCV polyprotein was expressed in the different cells by vaccinia T7 infection and pTM-NS3/5B plasmid transfection at day 7 post-transduction. Samples were collected 16 hours post-infection for Western Blot, immunofluorescence confocal microscopy and TEM. (A) Polyprotein expression efficiency was determined by Western-Blot using an antibody against NS3 and beta actin as loading control. B- Immunofluorescence microscopy showing comparable transfection efficiency and expression of NS3 and NS5A replicase subunits in control and lipin1-deficient cells. Scale bar 50 μm. Panels C, D- Low magnification TEM images from VacT7-infected, mock-transfected (C) and HCV polyprotein-expressing cells (D). LD, lipid droplet. E, F-Higher magnification images of HCV-polyprotein-expressing cells showing DMV, MMV and DMT structures. Scale bars 1μM (panels C, D), 200 nm (panel E), 100 nm (panel F). G- Whisker plot of HCV vesicle diameter distribution in the different cell lines (shControl n = 279; shLPIN1-1 n = 24; shLPIN1-2 n = 61; shControl+DCTV n = 93). Data were collected from three independent experiments. Box plots represent median and quartiles (Q1 and Q3). Whiskers represent 1.5 interquartile range (IQR) above or below the corresponding quartile. (H) Cells were analyzed by immunofluorescence microscopy to determine the percentage of NS3/NS5A-positive cells (shControl n = 84; shLPIN1-1 n = 115; shLPIN1-2 n = 75; shControl+DCTV n = 122). The same samples were analyzed by TEM to determine the presence/absence of vesicular structures (HCV vesicle-positive) in individual to determine the relative incidence of these structures in the different cell lines (shControl n = 31; shLPIN1-1 n = 36; shLPIN1-2 n = 41; shControl+DCTV n = 42 cells). Data are shown as percentage of total cells. DCTV, daclatasvir 100nM. Statistical significance was determined using Fisher´s Exact Test and Chi Square Test (*p<0.05; **p<0.01).

https://doi.org/10.1371/journal.ppat.1007284.g007

Transmission electron microscopy (TEM) of ultrathin cell sections of cells expressing HCV replicase components shows the expected accumulation of a mixture of characteristic double-membrane vesicles (DMV) as well as multiple membrane vesicles (MMV) (Fig 7D, 7E and 7F) that were not found in mock-transfected cells (Fig 7C), as reported in previous studies using similar systems [16]. Individual vesicle diameter displays heterogeneous size distribution in which the predominant population is distributed between 125–150 nm (median 150 nm; average ± SD: 167 ± 81 nm, n = 279) with larger vesicles being less predominant (Fig 7G and S7 Fig). This size distribution is compatible with that observed similar replication-deficient systems and during HCV infection. Treatment of these cells with 100 nM daclatasvir (DCTV), resulted in a strong reduction in the number of vesicles per section area (S7B Fig), without significantly altering the size distribution of the remaining vesicles (Fig 7G), as reported by Berger et al. [59]. Similarly, the diameter distribution of the vesicles found in lipin1-deficient cells was comparable to that in control cells (Fig 7G). However, HCV-induced structures were significantly less abundant 16 hours post-transfection in lipin1-deficient cells than in controls cells (S7C Fig). This reduced abundance is illustrated by a significant reduction in the fraction of cells displaying vesicular structures in lipin1-deficient cell cultures (Fig 7H) despite comparable transfection efficiency and viral protein expression levels, indicating that lipin1 may be required in a critical step leading to formation of the HCV-induced vesicular compartment.

To validate the TEM results independently, we set out to establish a biochemical assay to evaluate replication-independent replicase complex formation. One of the characteristics of the HCV replicase complexes is that they are located in detergent-resistant membranes (DRM) [19, 20]. In this sense, NS proteins are associated with replicase complexes that co-sediment with DRM markers such as caveolin-2 or sigma-1 receptor (SIGMAR1) in low-density fractions in isopycnic gradient ultracentrifugation experiments [19, 21]. Control and lipin1-deficient cells were infected with VacT7 and subsequently transfected with limiting doses of the plasmid pTM-NS3/5B [16]. Parallel samples were treated with DCTV (100 nM). Sixteen hours post-transfection, cell lysates were generated and subjected to equilibrium ultracentrifugation in 10–40% sucrose gradients. Gradient fractions were collected and subjected to Western-Blot analysis to determine the impact of lipin1 silencing on NS3, SIGMAR1 and actin sedimentation profiles. Fig 8A shows how, as previously shown for replicon and JFH1-infected cells, NS3 can be detected in DRM fractions (fractions 3–5), as determined by the presence of SIGMAR1 in those fractions (fractions 3–5; S8A Fig), although in this experimental system, unlike during viral infection [21], most NS3 co-sediments together with solubilized proteins, as shown for actin (fractions 9–12; S8A Fig). DRM-associated NS3 is reduced in DCTV-treated and lipin1-deficient cells, while total NS3 expression remains unchanged (Fig 8A). Four independent experiments were performed in which relative-DRM associated NS3, normalized to that found in solubilized fractions, was calculated for each experimental condition (Fig 8B). Lipin1-deficient cells display a consistent and statistically significant reduction in the DRM-associated, but not total NS3 abundance (Fig 8A and 8B; shLPIN1-1 and shLPIN1-2), similar to that observed in control cells in the presence of DCTV (Figs 8A and 8B; shControl+DCTV), consistent with the TEM data (Figs 7 and S7) and the notion that NS3 in DRM fractions may reflect the abundance of replicase complexes formed in these cells. This reduction is not due to an overall reduction in cellular DRM abundance in lipin1-deficient cells, as lipin1-deficient cells display similar SIGMAR1 distribution pattern as the control cells (S8 Fig). Overall, TEM data and DRM floatation assays strongly suggest that lipin1 is rate limiting for the generation of replicase complexes from fully processed polyprotein subunits.

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Fig 8. Reduced NS3 incorporation into detergent-resistant membranes (DRM) in lipin1-deficient cells.

Huh-7 cells were transduced with lentiviral vectors expressing control or LPIN1-specific shRNAs. HCV polyprotein was expressed in the different cells by vaccinia T7 infection and pTM-NS3/5B plasmid transfection at day 7 post-transduction. Sixteen hours post-transfection, cells were lysed in TNE-0.1% Triton X114 and clear cell lysates were subjected to isopycnic ultracentrifugation in 10–40% sucrose gradients at 120, 000g during 16 hours at 4°C. Gradient fractions were subjected to SDS-PAGE and Western Blot to determine NS3 distribution. (A) Representative Western-Blot of gradient fractions showing the distribution of NS3 in control and lipin1-deficient cells. (B) Quantification of the total and DRM-associated NS3 in the different cell lines, including a control of shControl cells treated with 100nM daclatasvir (shControl+DCTV). Data are expressed as the relative value as compared with the control shRNA. Data are shown as mean and SD of four independent experiments (n = 4). Statistical significance was determined using Student´s t-test (*p<0.05; **p<0.01).

https://doi.org/10.1371/journal.ppat.1007284.g008

Chemicals

HCV antiviral compounds 2´-c-methyladenosine (2mAde), sofosbuvir and daclatasvir were obtained from Boc Sciences (NY, USA), Selleckchem (Texas, USA) and Medchem Express (New Jersey, USA) respectively and dissolved in DMSO to obtain 10mM stock solutions. N-acetylcysteine (NAC) and puromycin were obtained from Sigma-Aldrich (Missouri, USA), dissolved in water to a final concentration of 0.5 M and 50 mg/ml respectively. Hydroxyzine pamoate was purchased from Sigma-Aldrich (Missouri, USA) and dissolved in DMSO to a final 10 mM concentration.

Cells and viruses

Human hepatoma Huh-7 and derived subclones Huh-7.5, Huh-7.5.1 (clone 2) have been described [94–96] and were kindly provided by Dr. Chisari (TSRI-La Jolla, CA). HEK-293T cells [97] were kindly provided by Dr. Ortin (CNB-Madrid, Spain). Cell cultures were maintained subconfluent in Dulbecco´s Modified Eagle´s Medium (Gibco) supplemented with 10 mM HEPES (Gibco), 100U/ml Penicillin/Streptomycin (Gibco), 100μM non-essential amino acids (Gibco) and 10% fetal bovine serum (Sigma-Aldrich).

Production of lipin1-deficient cell lines

Lentiviral vectors expressing control and LPIN1-specific shRNAs were used to inoculate Huh-7 cells. Twenty-four hours later, cells were subjected to selection with 2.5μg/ml of puromycin to assess the lowest lentivirus dose capable of conferring puromycin resistance to 100% of the cell population. Selected cell populations were subsequently cultured in the presence of puromycin until LPIN1 silencing was ascertained by Western-Blot using anti-lipin1 antibodies, typically at day 6–7 post lentiviral transduction, time at which all experiments were performed in the absence of puromycin. Before execution of all the experiments shown in this study, lipin1 expression was assessed by Western-Blot. Cell viability was determined by a thiazolyl blue tetrazolium blue (MTT) formazan formation assay [47].

Single and multiple cycle infections with D183 virus

Control and lipin1-deficient cell lines (5. 10⁴ cells/well) were plated onto 12-well plates and were inoculated with D183 virus at a MOI 10 FFU/cell. Samples of the cells and supernatants were collected 24, 48 and 72 hours post-infection. For multiple cycle infection experiments (MOI 0.01), samples of the supernatants were collected at day 3, 5 and 7 post-inoculation. Cells were split 1:3 in the multiple cycle infection experiments at days 3 and 5 to maintain the cultures subconfluent. Extracellular infectivity titers were determined by endpoint dilution and infection foci counting as previously described [99]. Intracellular HCV RNA was determined by reverse transcription and quantitative PCR (RT-qPCR) as previously described [99].

Western-Blot

Total protein samples were prepared in Laemmli buffer and separated using polyacrylamide denaturing gel electrophoresis (SDS-PAGE). Proteins were subsequently transferred onto PVDF membranes and incubated with 5% milk (lipins) or 3% BSA in PBS-0.25% Tween20 for one hour at room temperature (RT). Primary antibodies against lipin1 (clone B-12; Santa Cruz), lipin2 (H-160; Santa Cruz), NS3 (clone 2E3; Biofront), beta-actin (ab8226; Abcam) and tubulin (clone AA2; Sigma-Aldrich) were diluted in PBS-0.25% Tween20 and incubated for 1 hour (four hours for lipins) at RT. Membranes were subsequently washed for 20 minutes with PBS-0.25% Tween20 three times. Horseradish peroxidase-conjugated secondary antibodies were incubated for 1 hour at room temperature in 5% milk-PBS-0.25% Tween20 and subsequently washed three times for 20 minutes at room temperature. Protein bands were detected using enhanced chemoluminescence reactions and exposure to photographic films. Specific bands were quantitated using the ImageJ Software [100] on non-saturated, scanned films.

Immunofluorescence confocal microscopy

Huh-7 cells were grown on glass coverslips and infected at high multiplicity (MOI 10) with D183 virus. Forty-eight hours post infection cells were fixed for 20 minutes at RT with a 4% formaldehyde solution in PBS, washed twice with PBS and incubated with an incubation buffer (3% BSA; 0.3% Triton X100 in PBS) for 1 hour. Antibodies were diluted in incubation buffer: rabbit anti-lipin1 antibody (1:50; Cell Signaling-Leiden, The Netherlands), rabbit anti-lipin2 antibody (1:100; H-160; Santa Cruz), mouse anti-dsRNA (1:200; J2 clone; Scicons), anti-NS3 (1:500; 2E3 clone; Biofront) or anti-NS5A(1:200; 7E2 clone; Biofront). Primary antibodies were incubated with the cells for 1 hour (4 hours for lipin2 experiments) time after which the cells were washed with PBS and subsequently incubated with a 1:500 dilution of a goat anti-mouse conjugated to Alexa 488 or Alexa 594 (Invitrogen-Carlsbad, CA). Nuclei were stained with DAPI (Life Technologies) during the secondary antibody incubation using the manufacturer´s recommendations. Cells were washed with PBS and mounted on glass slides with Prolong (Invitrogen-Carlsbad, CA).

Confocal microscopy was performed with a Leica TCS SP8 laser scanning system (Leica Microsystems). Images of 1024 × 1024 pixels at eight bit gray scale depth were acquired sequentially every 0.13–0.3 μm through a 63x/1.40 N.A. immersion oil lens, employing LAS AF v 2.6.0 software (Leica Microsystems). Colocalization indexes were calculated using Jacop plugin for Image J [101] from a minimum of 10 regions of interest (ROI). Images were processed using ImageJ, were medians of 1 pixel were obtained for the different channels, only for illustration, not for analysis. Color levels, brightness and contrast were manipulated for illustration using technical and biological controls as reference.

RNA extraction and RT-qPCR

Total RNA extraction was performed using the GTC extraction method [102]. Purified RNA (10–500 ng) was subjected to RT-qPCR using random hexamers and a Reverse Transcription Kit (Applied Biosystems). Quantitative PCR was performed using 2X Reaction Buffer from (Applied Biosystems) and specific oligonucleotides as previously described [99, 103]. Standard curves were prepared by serial dilution of a known copy number of the corresponding amplicon cloned in a plasmid vector.

Infections with genotype 1a TNcc

Control and lipin1-deficient Huh-7.5 cells were inoculated with TNcc virus (MOI 0.05). Due to the relatively low propagation levels of the TNcc virus in this experimental setup, parallel cultures were infected and treated with 2´-C-methyladenosine (2mAde; 10μM) to determine the levels of non-replicative, background HCV RNA. Cells were incubated for 72 hours at 37°C, time after which samples of the cells were collected to determine intracellular HCV RNA levels by RT-qPCR.

Persistently infected cell cultures

To establish persistently infected cell cultures Huh-7 cells were inoculated at MOI 0.01 with JFH-1 HCV strain as previously described [24]. Cell cultures were maintained subconfluent for two weeks, time after which infection rates reach nearly 100% of the cells, as assessed by immunofluorescence microscopy. At this point cells were split and transduced with the corresponding lentiviral vectors in order to generate lipin1-deficient cell cultures as well as control cell lines. Once silencing had been verified by Western-blot, typically at day 6–8 post-transduction, cells were split and samples of the cells and supernatants were collected 24 hours later to determine intracellular HCV RNA levels by RT-qPCR and extracellular infectivity titers by end-point dilution and immunofluorescence microscopy.

Production of trans-complemented infectious HCV virions bearing a reporter gene

Infectious, spread-deficient HCV particles produced by trans-complementation (HCV_tcp) have previously been described [52]. Briefly, Huh-7.5.1 clone 2 cells expressing core-E1 and E2-NS2 regions from JFH-1 by lentiviral transduction, were electroporated with a JFH-1 subgenomic dicistronic replicon bearing a firefly luciferase gene with reagents kindly provided by Dr. Ralf Bartenschlager (U. of Heidelberg). Supernatants containing HCV_tcp were collected 36, 48 and 72 hours post-electroporation, pooled and assayed for viral infectivity. HCV_tcp infection efficiency was determined by inoculating naïve Huh-7 cells with the electroporation supernatants and measuring luciferase activity 48 hours post-infection using a commercially available kit (Promega).

Analysis of viral entry using HCV_pp

Retroviral particle production pseudotyped with different viral envelopes has previously been described [54, 55] with the materials kindly provided by Dr. F. L. Cosset (INSERM, Lyon). Control and lipin-deficient cell lines were inoculated with HCV_pp and VSV_pp and incubated for 48 hours, time at which total cell lysates were assayed for luciferase activity using a commercially available kit (Promega). A selective HCV entry inhibitor, hydroxyzine pamoate (HDX) from Sigma-Aldrich (Missouri, USA), was used as positive control of inhibition [55].

In vitro transcription and HCV RNA transfection

A plasmid containing the sequence corresponding to a subgenomic JFH-1 replicon bearing a firefly luciferase reporter gene was kindly provided by Dr. Ralf Bartenschlager (U. of Heidelberg) [52]. After digestion with the restriction enzyme MluI, the linearized plasmid was transcribed in vitro using a commercial kit (Megascript T7; Ambion-Paisley, UK). The resulting products were digested with DNAse and precipitated with LiCl. Pelleted RNA was washed with 75% and 100% ethanol, and resuspended in nuclease-free water. In vitro transcribed RNA was transfected into the different cell lines together with a plasmid expressing Renilla luciferase under a minimal promoter (pRL-null; Clontech-California, USA) using Lipofectamine 2000 and the manufacturer´s recommendations (Life Technologies- California, USA). Firefly and Renilla luciferase activities were measured in the sample using a commercial kit (Dual Luciferase Assay System; Promega-Wisconsin, USA) at different times post-transfection.

Lipin1 wt and mutant cDNA overexpression in lipin1-deficient cells

Lipin1-deficient cells were generated by lentiviral transduction of shLPIN1-2 shRNA. At day 3 post-transduction, control and lipin1–deficient cell populations (5 X 10⁴ cells/M24 well) were transfected in suspension using lipofectamine 2000 with plasmids (800 ng/M12 well) expressing wt lipin1beta isoform shLPIN1_2-resistant cDNA or DXDXT or LXXIL motif mutants [31]. Transfected cell cultures were incubated for 48 hours and subsequently inoculated at MOI 10 with D183 virus. Infection efficiency was determined by measuring extracellular infectivity titers 48 hours post-infection. Parallel cultures were used to determine relative wt and mutant protein expression efficiency by Western-Blot. Infectivity titers were measured as described above. The relative impact of cDNA expression was estimated by determining the ratio between the infectivity found in lipin1-deficient cells and the control cells transfected with the same plasmid. In order to average experiments with different raw infection efficiency, all the experiments were referenced to the ratio in the mock-transfected cells.

Human coronavirus infection

Control and lipin1-deficient Huh-7 cells were inoculated with CoV-229E (MOI 0.01) for 2 hours at 37°C. Cells were washed twice with warm PBS and replenished with DMEM-10%FCS. Extracellular infectivity titers were determined 48 hours post-infection by end-point dilution and fluorescence microscopy in Huh-7 cells.

T7-driven viral polyprotein overexpression

Huh-7 cells were inoculated at MOI 10 with a recombinant vaccinia virus expressing the T7 phage RNA polymerase (VacT7) [104]. Two hours later, cells were transfected with the plasmid pTM-NS3/5B [16, 58] (kindly provided by Dr. Lohmann; U. of Heidelberg) and Lipofectamine 2000 (ThermoFisher-Massachussets, USA) following the manufacturer´s recommendations in terms of total DNA per well (typically 4μg per 35mm dishes with 7.5 X 10⁵ cells/well) and 50% of the recommended lipofectamine:DNA ratio. Transfected cells were cultured in the presence of the DNA replication inhibitor cytosine β-D-arabinofuranoside (AraC; Sigma-Aldrich) for 16 hours to prevent VacT7 replication [105]. When indicated, media was also supplemented with 100nM daclatasvir (DCTV). Total cell extracts were used to determine viral protein accumulation by Western-blot using anti-NS3 antibody (clone 2E3; Biofront) and β-actin (Abcam; ab8226) as loading control.

Transmission electron microscopy

For ultrastructural electron microscopy studies, control and lipin1-deficient cells expressing NS3-5B polyprotein (see above) were cultured on glass coverslips and fixed in situ after polyprotein expression with a mixture of 2% paraformaldehyde (TAAB) and 2.5% glutaraldehyde (TAAB) (1h at room temperature), post-fixed with 1% osmium tetroxide in PBS (45 min), treated with 1% aqueous uranyl acetate (45 min), dehydrated with increasing quantities of ethanol and embedded in epoxy resin 812 (TAAB). Ultrathin, 70-nm-thick sections were cut in parallel to the monolayer, transferred to formvar-coated EM buttonhole grids and stained with aqueous uranyl acetate (10 min) and lead citrate (3 min). Sections were visualized on a Jeol JEM 1200 EXII electron microscope (operating at 100 kV).

Quantitation of HCV-induced structures was performed as follows. To quantitate the differences in total vesicle abundance, TEM sections were visually inspected under the microscope for the presence/absence of vesicular structures. The number of positive cells and total number of cells were inserted in a 2 X 2 contingency table to determine the statistical significance of the differences between control and lipin1-deficient cells using two-tailed Fisher´s Exact Test or two-tailed Chi Square Test. In addition, we determined the frequency of HCV-induced vesicles in DCTV-treated control cells by dividing the number of structures per inspected area and calculating the average and SD of the frequencies found in the different images. The diameters of individual vesicles were determined manually using size-calibrated images and Image J software.

Detergent-resistant membrane (DRM) ultracentrifugation

Lipin1-deficient and control cells (7.5 X 10⁵ cells) were infected with VacT7 virus (MOI 10) and transfected with limiting doses of pTM-NS3/5B plasmid (typically 800 ng/ well), as higher plasmid doses may difficult observing the reported differences. Cells were lysed by adding 250 μl of TNE (50mM Tris-HCl pH 7.5, NaCl 150 mM and EDTA 2 mM) buffer containing 0.5% Triton X-114 and protease inhibitors (Complete; Roche- Basel, SW). Lysates were incubated for 30 minutes on ice before clearing them by 10-minute centrifugation at 12,000 r.p.m. Clear supernatants were mixed 1:1 with 60% sucrose TNE solution. This mixture was applied on top of a 40% sucrose-TNE cushion and was overlaid with 20% and 10% sucrose-TNE until completing the discontinuous gradient. Gradients were centrifuged for 16 hours at 120,000 X g. Fourteen fractions were collected from the top and analyzed by SDS-PAGE and Western-Blot using antibodies against NS3 (clone 2E3; Biofront), SIGMAR1 (S-18; Santa-Cruz), caveolin-2 (Epitomics; 3643–1) and beta actin as described previously [21]. NS3 signal was quantitated using ImageJ software and the fraction of DRM-associated NS3 was determined as the ratio of NS3 signal in fractions 3, 4 and 5 to the total NS3 signal in the gradient.