Protease-dependent uncoating of a complex retrovirus

Abstract

Although retrovirus egress and budding have been partly unraveled, little is known about early stages of the replication cycle. In particular, retroviral uncoating, a process during which incoming retroviral cores are altered to allow the integration of the viral genome into host chromosomes, is poorly understood. To get insights into these early events of the retroviral cycle, we have used foamy complex retroviruses as a model. In this report, we show that a protease-defective foamy retrovirus is noninfectious, although it is still able to bud and enter target cells efficiently. Similarly, a retrovirus mutated in an essential viral protease-dependent cleavage site in the central part of Gag is noninfectious. Following entry, wild-type and mutant retroviruses are able to traffic along microtubules towards the microtubule-organizing center (MTOC). However, whereas nuclear import of Gag and of the viral genome was observed for the wild-type virus as early as 8 hours postinfection, incoming capsids and genome from mutant viruses remained at the MTOC. Interestingly, a specific viral protease-dependent Gag cleavage product was detected only for the wild-type retrovirus early after infection, demonstrating that cleavage of Gag by the viral protease at this stage of the virus life cycle is absolutely required for productive infection, an unprecedented observation among retroviruses.

FIG. 1.

Schematic representation of pcPFV proviral constructs. In all constructs, the U3 region of the 5′ LTR is replaced by the CMV immediate-early promoter. The I-to-E amino acid substitution at the protease cleavage site ₃₁₀IR↓SV₃₁₃ in the Gag open reading frame and the D-to-A substitution in the viral protease active site DSGA result in the pcPFV-Gag_I310E and pcPFV-PR_D/A proviral mutants, respectively. The viral protease-dependent cleavage sites along the p71 Gag protein (648 amino acids) are depicted by arrows. The major ₆₂₁VN↓TV₆₂₄ cleavage site, generating the p68/p3 Gag proteins, and the three internal secondary sites, ₃₁₀IR↓SV₃₁₃, ₃₃₈VF↓PV₃₄₁, and ₃₅₁IN↓AI₃₅₄, are represented. Gly-Arg (GR)-rich boxes are depicted as I, II, and III.

FIG. 2.

Characterization of protease and Gag mutants. A) Western blot analysis of protein extracts from provirus-transfected 293T cells using anti-PFV antibodies. Analysis of intracellular protein lysates performed 48 h posttransfection reveals the presence of the characteristic Gag doublet in cells transfected with wild-type and pcPFV-Gag_I310E proviruses and confirms the absence of protease activity in the pcPFV-PR_D/A. In that case, trans-complementation with a p68-expressing vector (lane pcPFV-PR_D/A + p68) leads to detection of the Gag doublet. Western blot analysis performed on cell-free virus supernatant of transfected cells reveals efficient extracellular virion production from wild-type, pcPFV-PR_D/A, and pcPFV-Gag_I310E proviruses, whereas an Env-defective clone, used as a control, remains intracellular. B) Electron micrographs of intracellular capsids. Normally shaped intracellular capsids are observed in 293T cells transfected with pcPFV, pcPFV-PR_D/A71/68, and pcPFV-Gag_I310E corresponding to PFV, PR_D/A71/68, and Gag_I310E viruses. Magnification, ×12,750. A high magnification of viral particles is presented in the left corner. C) Relative virus entry efficacy. Two hours postinfection, virus entry was evaluated by the ratio of input viral RNA content to intracellular viral RNA content assessed by qRT-PCR in pronase-treated cells. The results represent the means of three independent experiments, and the wild-type virus is reported as 100%.

FIG. 3.

Intracellular trafficking of incoming Gag. Shown is subcellular localization of incoming Gag proteins studied by confocal microscopy following indirect immunofluorescence at 4, 6, and 8 h postinfection using anti-Gag antisera (green). As early as 4 h postinfection of U373 MG cells, Gag from wild-type virus (A) reaches the MTOC, stained with γ-tubulin antibodies (red), and is detected in the nucleus as early as 6 h postinfection. While Gag from the wild-type virus is mainly nuclear at 8 h postinfection, Gag from PFV-PR_D/A71/68 and PFV-Gag_I310E viruses (B) remains localized at the MTOC. Nuclei are stained with DAPI (blue).

FIG. 4.

Nuclear import of the viral genome. Shown is subcellular localization of the DNA genome from wild-type and mutant viruses studied by confocal microscopy following in situ hybridization at either 4 or 8 h postinfection using a biotin-labeled PFV full-length probe (green). As shown on these confocal slices, whereas the viral DNA genome of the wild-type virus reaches the MTOC 4 h postinfection, it is detected in discrete interchromatin domains in the nucleus at 8 h postinfection. In contrast, the viral DNA genome of mutant viruses remains at the MTOC at 8 h postinfection and was never detected within the nucleus at this stage of the viral replication cycle. Nuclei are stained with DAPI (blue).

FIG. 5.

Early Gag processing. Fifteen minutes or 7 h postinfection, the fate of incoming Gag proteins was studied by Western blot in protein extracts from pronase-treated U373 MG cells using anti-Gag antisera. The characteristic Gag doublet is clearly detected as early as 15 min postinfection for all viruses (wild type, PFV-PR_D/A71/68, and PFV-Gag_I310E viruses). In contrast, 7 h postinfection, smaller Gag products migrating at 60, 28, and 22 kDa are detected in all protein extracts, whereas a 38-kDa product is detected only in extracts from cells infected with the wild-type virus and totally absent in extracellular virions. The mock lane represents noninfected cells.

FIG. 6.

Model for FV disassembly and nuclear import. After viral entry, apparently intact FV cores migrate along the MT network in a dynein/dynactin-dependent manner to reach the MTOC. During this trafficking, cellular cues trigger viral core-associated protease activation. This enzyme will process the structural Gag protein, weakening the viral core. This disassembly process might be initiated and/or enhanced by cellular proteases and interactions with cellular cofactors. Consequently, Gag processing may uncover NLSs present on Pol and Gag GRII, the viral genome being linked with this protein complex through the nucleic acid binding (NAB) domain in the GRI box. This complex could actively import the viral genome into the nucleus or await at the MTOC for nuclear membrane breakdown during mitosis.