Clathrin Facilitates the Morphogenesis of Retrovirus Particles

The morphogenesis of retroviral particles is driven by Gag and GagPol proteins that provide the major structural component and enzymatic activities required for particle assembly and maturation. In addition, a number of cellular proteins are found in retrovirus particles; some of these are important for viral replication, but many lack a known functional role. One such protein is clathrin, which is assumed to be passively incorporated into virions due to its abundance at the plasma membrane. We found that clathrin is not only exceptionally abundant in highly purified HIV-1 particles but is recruited with high specificity. In particular, the HIV-1 Pol protein was absolutely required for clathrin incorporation and point mutations in reverse transcriptase or integrase domains of Pol could abolish incorporation. Clathrin was also specifically incorporated into other retrovirus particles, including members of the lentivirus (simian immunodeficiency virus, SIVmac), gammaretrovirus (murine leukemia virus, MLV) and betaretrovirus (Mason-Pfizer monkey virus, M-PMV) genera. However, unlike HIV-1, these other retroviruses recruited clathrin primarily using peptide motifs in their respective Gag proteins that mimicked motifs found in cellular clathrin adaptors. Perturbation of clathrin incorporation into these retroviruses, via mutagenesis of viral proteins, siRNA based clathrin depletion or adaptor protein (AP180) induced clathrin sequestration, had a range of effects on the accuracy of particle morphogenesis. These effects varied according to which retrovirus was examined, and included Gag and/or Pol protein destabilization, inhibition of particle assembly and reduction in virion infectivity. For each retrovirus examined, clathrin incorporation appeared to be important for optimal replication. These data indicate that a number of retroviruses employ clathrin to facilitate the accurate morphogenesis of infectious particles. We propose a model in which clathrin contributes to the spatial organization of Gag and Pol proteins, and thereby regulates proteolytic processing of virion components during particle assembly

The Gag Cleavage Product, p12, is a Functional Constituent of the Murine Leukemia Virus Pre-Integration Complex

The p12 protein is a cleavage product of the Gag precursor of the murine leukemia virus (MLV). Specific mutations in p12 have been described that affect early stages of infection, rendering the virus replication-defective. Such mutants showed normal generation of genomic DNA but no formation of circular forms, which are markers of nuclear entry by the viral DNA. This suggested that p12 may function in early stages of infection but the precise mechanism of p12 action is not known. To address the function and follow the intracellular localization of the wt p12 protein, we generated tagged p12 proteins in the context of a replication-competent virus, which allowed for the detection of p12 at early stages of infection by immunofluorescence. p12 was found to be distributed to discrete puncta, indicative of macromolecular complexes. These complexes were localized to the cytoplasm early after infection, and thereafter accumulated adjacent to mitotic chromosomes. This chromosomal accumulation was impaired for p12 proteins with a mutation that rendered the virus integration-defective. Immunofluorescence demonstrated that intracellular p12 complexes co-localized with capsid, a known constituent of the MLV pre-integration complex (PIC), and immunofluorescence combined with fluorescent in situ hybridization (FISH) revealed co-localization of the p12 proteins with the incoming reverse transcribed viral DNA. Interactions of p12 with the capsid and with the viral DNA were also demonstrated by co-immunoprecipitation. These results imply that p12 proteins are components of the MLV PIC. Furthermore, a large excess of wt PICs did not rescue the defect in integration of PICs derived from mutant p12 particles, demonstrating that p12 exerts its function as part of this complex. Altogether, these results imply that p12 proteins are constituent of the MLV PIC and function in directing the PIC from the cytoplasm towards integration

p12 Tethers the Murine Leukemia Virus Pre-integration Complex to Mitotic Chromosomes

<div><p>The p12 protein of the murine leukemia virus (MLV) is a constituent of the pre-integration complex (PIC) but its function in this complex remains unknown. We developed an imaging system to monitor MLV PIC trafficking in live cells. This allowed the visualization of PIC docking to mitotic chromosomes and its release upon exit from mitosis. Docking occurred concomitantly with nuclear envelope breakdown and was impaired for PICs of viruses with lethal p12 mutations. Insertion of a heterologous chromatin binding module into p12 of one of these mutants restored PICs attachment to the chromosomes and partially rescued virus replication. Capsid dissociated from wild type PICs in mitotic cells but remained associated with PICs harboring tethering-negative p12 mutants. Altogether, these results explain, in part, MLV restriction to dividing cells and reveal a role for p12 as a factor that tethers MLV PIC to mitotic chromosomes.</p> </div

Characteristics of real time imaging system for MLV PICs.

<p>(A) Schematics of MLV genome, Gag, modified Gags, p12 and mutations. Mutated residues are aligned with the cognate wt residues (in bold). Arrowhead marks LANA31 and Myc epitope insertion site. Arrows and ✗ represent protease-cleavage sites and protease-resistant linker (GGSI), respectively. (B) Western blot of chimeric virions. Anti-GFP antibody was used to detect the processing of GFP fusion proteins in virion pellets, purified from supernatants of cultures, transfected with the indicated modified Gags and wt MLV. Mock represents transfection with no plasmid DNA. (C) Infectivity of chimeric virions. The indicated molar ratios of MA-GFP/p12-CA-NC and wt MLV plasmids were co-transfected into 293T cells, together with pQCXIP-GFP-C1 vector. Virions in culture supernatants, normalized by RT activity, were used to infect NIH3T3 cells, which were analyzed by FACS for GFP fluorescence, 2 days post-infection. Average infectivity from 3 independent experiments is presented as percentage of infectivity of wt particles with no modified Gag (wt). Error bars indicate SEM. (D) Confocal fluorescence microscopy of wt GFP-infected U/R cell. Serial optical sections were reconstituted into a 3D image, with a 10 µm grid. PICs are in green and Hoechst-stained chromosomes in blue.</p

Docking onto mitotic chromosomes coincides with NE breakdown.

<p>wt GFP-infected U/R/RFP-H2A (A) and U/R/RFP-laminA (B, C) cells, were imaged upon entry to mitosis. Shown are frames from the resulting movies (Movie S3; parts A and B). Full arrowheads (A) mark PICs (green) anchored to the mitotic chromosomes (red). Empty arrowheads (B) mark gaps in the NE (red). (C) Visualization of PICs' movement during NE breakdown. Three pairs of frames were chosen from the start, middle and end of Movie S3, part B. For each pair, the PICs in the second frame were superimposed on the first frame and pseudo-colored with red. Lamin A signal was pseudo-colored with white. PICs with a relative large shift in their position appear in red or green while PICs with a minimal shift appear in yellow. Time (minutes and seconds) from start of imaging is shown for each frame. Bars represent 10 µm.</p

CA-p12 dissociation in mitotic cells: discrepancy between wt and p12 mutants.

<p>Immunofluorescence of interphase (A) and mitotic (B–E) U/R cells, infected with 1xMycR (A, B), or with 1xMycR clones carrying PM14 (C), S(61,65)A (D) and S(61,65)A/M63I (E) mutations. 12 hpi cells were stained with anti-CA (green) and anti-Myc (red) antibodies. Chromosomes were stained with DAPI (blue). Extracellular virions are to the right of the dashed lines (A, B). An asterisk (C) marks non-typical p12 staining. (F) Quantification of the percentage of p12 signal that overlaps CA signal in interphase and mitotic cells (∼400 p12 dots/cell were analyzed in 4 and 3 1xMycR-infected mitotic and interphase cells, respectively; ∼40–130 dots/cell in 5 cells were analyzed for the rest of the viruses). (G) Quantification of the percentage of p12 and CA signals that overlap the DAPI-stained chromatin in 1xMycR-infected mitotic cells. Error bars indicate standard error of the mean (SEM). Bars represent 10 µm.</p

MLV PICs dock to mitotic chromosomes independently of IN activity.

<p>U/R/RFP-laminA (A, B) and U/R/RFP-H2A (C–E) cells were infected with wt GFP (A–D), or with D184A GFP (E) virions. Shown are representing kymographs (A–E) of cells imaged in Movie S2. White arrows (shown only in A but apply to all kymographs of all figures) represent the order of the movie frames over time (<i>t</i>); where <i>t</i> = 30, 47, 152, 150 and 26 seconds for A, B, C, D and E, respectively. In kymographs, immobile PICs appear as continues green lines (A, C, E); in contrast, motile PICs change x, y coordinates over time, resulting in the scattered/dotted pattern (B, D). Bars represent 10 µm.</p

LANA31 insertion into p12 of PM14 virus rescues PIC docking to chromosomes and viral replication.

<p>Representing kymographs from Movie S6 of unsynchronized, mitotic U/R/RFP-H2A (A, B) or U/R cells that exit mitosis (F), infected with wt/LANA31 GFP (A), PM14/LANA31 GFP (B), or wt mCherry together with wt/LANA31 GFP (F). Hoechst-stained chromosomes are in blue (F). <i>t</i> = 24, 24 and 72 seconds for A, B and F, respectively. Bars represent 10 µm. (C) Quantification of spatial retention of PICs over time. Retention of PM14/LANA31 GFP was calculated as described for <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003103#ppat-1003103-g005" target="_blank">Fig. 5C</a>. Shown are the average values obtained from four cells, each from an independent movie, and each with approximately 20 PICs. Error bars indicate SEM. (D) Virus spreading. NIH3T3 cells were infected with the indicated viruses, normalized by RT activity. Samples of culture supernatants were harvested at the indicated time points and assayed for RT activity to detect virus spreading. Mock represents uninfected cells. Shown are results of two experiments (Exp. I and II). (E) Single-cycle infection assay. VLPs, harboring the indicated modifications, and normalized by exogenous RT assay, were used to transduce the pQCXIN vector into NIH3T3 cells. 2 dpi the cells were diluted (1∶10 and 1∶100) and selected in G418 medium for additional 10 days. G418-resistant colonies were fixed and stained with crystal violet. Shown is one of three independent experiments.</p