Fig 1.
Knock-out of cellular genes leading to the production of IP6.
(A) Inositol phosphate pathway in H. sapiens. Inositol-pentakisphosphate 2-kinase (IPPK) adds the sixth phosphate to position 2 of IP5 (yellow box). IP5 synthesis from I(1,3,4,5)P4 and I(1,3,4,6)P4 has not been fully resolved. Other abbreviations: phospholipase C (PLC), IP33K (inositol-triphosphate 3-kinase), IPMK (inositol-polyphosphate multikinase), INPP5 (inositol-polyphosphate 5-phosphatase), and ITPK1 (inositol-tetrakisphosphate 1-kinase). (B-C) Chromatograms showing insertion-deletions of inositol-phosphate pathway KOs in HEK293FTs. Red bars delineate the 20-base pair guide RNA sequence used for CRISPR/Cas9 targeting. (B) KO of IPPK has a 10-base pair (bp) deletion. (C) KO of IPMK has three copies with 1- and 10-bp deletions and a 1-bp insertion.
Fig 2.
IP pathway KOs have reduced IP6 and IP5 levels and have a loss of infectious particle release.
(A) 33% PAGE gel separating inositol phosphates. Two dilutions of purified 1 M IP6 were used as a standard and had IP5 breakdown products. The number of cells in each sample is indicated. (B) IP6 quantification of panel A in ng per million cells and μM. (C) Relative IP5 quantification normalized to the HEK293FT control. (D) Experimental timeline. (E) Percent infectious particle release normalized to HEK293FT cells. Student’s t-test was used for pair-wise comparison (n = 4, *** p < 0.001, error bars = mean + SD). (F) Representative western blot of experiments from panel D. Full-length HIV-Gag (pr55) and GAPDH loading control are presented on the left panels. Virus released into media is presented in the middle panel. A longer exposure of the virus release blot was also taken and presented on the right panel.
Fig 3.
Addition of multiple inositol polyphosphate phosphatase 1 (MINPP1) removes endogenous IP6 and relevant IP5 species from cells.
(A) Inositol phosphate pathway showing MINPP1 removal of the 3-position phosphate from IP6, IP5, and IP4. Removal of 3-phosphate from IP6 and I(1,3,4,5,6)P5 results in an equatorial hydroxyl group. (B) Experimental timeline. (C) 33% PAGE gel separating inositol phosphates. Dilution of purified 1 M IP6 was used as a standard and had an IP5 breakdown product. The number of cells in each sample is indicated. (D) IP6 quantification of panel C in ng per million cells and μM. (E) Relative IP5 quantification of panel C normalized to the HEK293FT control.
Fig 4.
Exogenous expression of MINPP1 is toxic in IPPK-KO cells.
(A) Plasmid map of expression vectors. (B) Experimental timeline. (C) Line plot of a representative experiment. The percentage of cells expressing EGFP over time are normalized to the starting population of EGFP positive cells for each cell line. (D) Bar chart of percent EGFP positive cells on the last day of collection from panel C (day 21). Student’s t-test was used for pair-wise comparison (n = 4, ** p < 0.01, *** p < 0.001, error bars = mean ± SD).
Fig 5.
IPPK-KO cells expressing MINPP1 have substantial loss in infectious particle production due to a block in viral release.
(A) Experimental timeline. (B) Bar chart of percent infectious particle release normalized to virus from HEK293FT cells expressing the empty vector. Student’s t-test was used for pair-wise comparison (n = 5, *** p < 0.001, error bars = mean ± SD). (C) Representative western blot of two experiments from panel B. The rows are full-length HIV-Gag (top), GAPDH loading control (middle), and virus released into media (bottom). A longer exposure was also taken for the blot of released virus. (D) Relative quantification of uncleaved pr55 Gag normalized to GAPDH levels in cell lysate. (E) Relative quantification of p24 Gag in virus released into media.
Fig 6.
IP6 and IP5 levels in target cells do not affect susceptibility to HIV-1 infection.
(A) Bar chart of percent infectious particle release normalized to HEK293FT cells. Student’s t-test was used for pair-wise comparison (n = 4, * p < 0.05). (B) Experimental timeline of the assay. (C) Plasmid map of HIV-1ΔEnv-CD4. (D) Example flow plots show output from the assay. (E) Bar chart of the ratio of the actual percent double positive cells to the expected double positive cells. The expected percent of double positive cells was calculated from the total percent of red cells and green cells. Student’s t-test was used for pair-wise comparison (n = 4, * p < 0.05, error bars = mean ± SD).
Fig 7.
Gammaretroviruses and Betaretroviruses do not require IP6 or IP5 as assembly co-factors.
Bar charts of percent infectious particle release of other retroviral genera normalized to virus from HEK293FT cells expressing the empty vector. (A) The Gammaretrovirus Murine leukemia virus (MLV, n = 4). (B) The Betaretrovirus Mason-Pfizer monkey virus (MPMV, n = 5). (C) Multiple sequence alignment of CA proteins of HIV-1, MLV, and MPMV. Note the lack of K290 and K359 homology in MLV and MPMV.
Fig 8.
IP6 and IP5 are required assembly co-factors for primate lentiviruses.
Bar charts of percent infectious particle release of lentiviruses normalized to virus from HEK293FT cells expressing the empty vector. (A) Simian immunodeficiency virus from macaques (SIV, n = 8). (B) Feline immunodeficiency virus (FIV, n = 7). (C) Equine infectious anemia virus (EIAV, n = 5). (D) Multiple sequence alignment of CA proteins of HIV-1, SIV, FIV, and EIAV. Note the homology of K290 and K359 in HIV-1 to lysines in SIV, FIV, and EIAV.
Fig 9.
Addition of IP6 stimulates in vitro immature assembly of lentiviruses.
Representative images at 36000x and 110000x of virus like particles (VLPs) from in vitro assembly reactions at pH8. Assembly reactions were performed with 0, 5, or 50 μM of IP6. (A) VLPs from HIV-1 sCASPNC (ectopic Serine preceding CASPNC) assemblies. (B) VLPs from SIV sCASPNC assemblies. (C) VLPs from FIV sCACSPNC assemblies. (D) VLPs from EIAV sCASPNC assemblies. (E) Quantification of spherical VLPs from each virus assembly reaction (n = 4–6). (F) Quantification of tubular VLPs from each virus assembly reaction (n = 5–6). Should include a definition of the box plot.
Table 1.
Primers used for cloning.