Essential functions of inositol hexakisphosphate (IP6) in murine leukemia virus replication

ABSTRACT We have investigated the function of inositol hexakisphosphate (IP6) and inositol pentakisphosphate (IP5) in the replication of murine leukemia virus (MLV). While IP6 is known to be critical for the life cycle of HIV-1, its significance in MLV remains unexplored. We find that IP6 is indeed important for MLV replication. It significantly enhances endogenous reverse transcription (ERT) in MLV. Additionally, a pelleting-based assay reveals that IP6 can stabilize MLV cores, thereby facilitating ERT. We find that IP5 and IP6 are packaged in MLV particles. However, unlike HIV-1, MLV depends upon the presence of IP6 and IP5 in target cells for successful infection. This IP6/5 requirement for infection is reflected in impaired reverse transcription observed in IP6/5-deficient cell lines. In summary, our findings demonstrate the importance of capsid stabilization by IP6/5 in the replication of diverse retroviruses; we suggest possible reasons for the differences from HIV-1 that we observed in MLV. IMPORTANCE Inositol hexakisphosphate (IP6) is crucial for the assembly and replication of HIV-1. IP6 is packaged in HIV-1 particles and stabilizes the viral core enabling it to synthesize viral DNA early in viral infection. While its importance for HIV-1 is well established, its significance for other retroviruses is unknown. Here we report the role of IP6 in the gammaretrovirus, murine leukemia virus (MLV). We found that like HIV-1, MLV packages IP6, and as in HIV-1, IP6 stabilizes the MLV core thus promoting reverse transcription. Interestingly, we discovered a key difference in the role of IP6 in MLV versus HIV-1: while HIV-1 is not dependent upon IP6 levels in target cells, MLV replication is significantly reduced in IP6-deficient cell lines. We suggest that this difference in IP6 requirements reflects key differences between HIV-1 and MLV replication.


Figure S1: Optimization of ERT reaction:
A) The bar graph indicates ERT product formation in the presence of different concentrations of melittin (x-axis).B) ERT product formation was also observed when Triton-X was used instead of melittin.IP6 concentration in these assays was 40µM.C) Early, intermediate, and late ERT product formation measured during different time intervals (x-axis).D) The bar graph shows the formation of ERT products in different reaction conditions indicated below the graph.Concertation of the rNTP mixture is the same as in the ERT buffer (described in materials methods) unless mentioned otherwise in the X-axis.The label "6.7mM" above three bars refers to the concentration of each rNTP.E) IP6 titration with and without rNTPs in the ERT reaction buffer.The graphs represent the mean ± SD of three replicates in the qPCR measurement of a single experiment.F) The bar graph shows MLV RT activity measured using SG-PERT assay.Viruses were lysed in PERT lysis buffer containing 0.125% of triton-X for near-complete lysis.Lysed samples were incubated with an external template (MS2 RNA) and primers.Reverse transcribed products were measured using qPCR.The graphs represent the mean ± SD of three replicates in the qPCR measurement of a single experiment.Except for Figure S1E, all other figures are representative of a single experiment selected from two independent experiments.

Figure S2 :
Figure S2: Pelleting-based stability assays: A) Immunoblot showing recovery of MLV capsid protein with increasing concentration of IP6.Inositol is used as a negative control at a concentration of 80µM.B) Quantification of the p30 CA recovery from the immunoblot.C) Immunoblot showing MLV capsid protein (p30 CA ) recovery after the addition of rNTPs (6.7mM each).IP6 was used as a positive control and inositol was used as a negative control, both at 80µM.D) Quantification of percent p30 CA recovery from immunoblots of three independent experiments.The graphs represent the mean ± SD of three independent experiments.Statistical significance is analyzed using one-way ANOVA.P values are indicated by *, ***P<0.001,** P<0.01, *P<0.05,ns: not significant.

Figure S3 :
Figure S3: R3 residue in MLV capsid is conserved and MLV-WT and R3 capsid mutant particles have similar morphology: A) Sequence alignment of various gammaretrovirus capsid regions shows complete conservation of the R3 residue, which is highlighted in red.The following gammaretroviruses are included in the alignment: Moloney murine leukemia virus (MMLV, Acc No. P03355 ), Friend virus (FV, Acc No. P26808.2 ), Amphotropic murine leukemia virus (A-MLV, Acc No. AAO61195.1),Xenotropic murine leukemia virus (XMRV, Acc No. A1Z651.1 ), Moloney murine leukemia virus neuropathogenic variant (MLVMN, Acc No. Q8UN02.2),endogenous ecotropic MLV in AKR mice (AKV, Acc No. P03356.3),Feline leukemia virus (FeLV, Acc No. NP_047255.1),Koala retrovirus (KoRV, Acc No. Q9TTC1.2),Porcine endogenous retrovirus (PERV, Acc No. CAB65339.1),Gibbon ape leukemia virus (GaLV, Acc No. P21414.2), and RD114 retrovirus (a cat endogenous virus) from a human tumor cell line RD, Acc No. BAM17305.1.Conservation annotation is as follows: an asterisk (*) denotes positions with a single, fully conserved residue, a colon (:) indicates conservation among groups of strongly similar properties, and a period (.) signifies conservation among groups of weakly similar properties.B) Transmission electron microscopy (TEM) analysis of WT and R3 capsid mutants of MLV.HEK293T cells were transfected with full-length MLV-WT or R3A and R3K mutants for virus production.Two days later they were processed for thin-section TEM.The green arrowhead indicates MLV particles displaying electron density underlying the virion membrane, while the red arrowhead points to MLV particles exhibiting electron density in the center of the virion.The scale bar is 200nm.

Figure S4 :
Figure S4: IP6/5 analysis in IPMK-KO and IPPK-KO cells: A) TiO2 PAGE showing levels of IP6 in control, IPMK-KO, and IPPK-KO cells.B) The bar graph shows the amount of IP6 and IP5 quantitated from TLC in control, IPMK-KO, and IPPK-KO cells.

Figure S5 :
Figure S5: IP6 is required for MLV replication: Quantitation of MLV p30 CA in the supernatant (A); MLV p30 CA (B), and Pr65 (C) in the cell lysates of control cells and KO cells shown in the representative immunoblot of Figure 5A.Infectivity (RLU/Actin) in control cells vs KO cells with viruses produced

Figure S6 :
Figure S6: MINPP1 expression in control and IP-KO cells: Immunoblot showing MINPP1 expression in the control and the KO cell lines transfected with 200 ng or 600 ng of MINPP1-expressing plasmid or of pcDNA3.1.