Figure 1.
Profile of N-MLV CA isolated from pgsA and pgsA-huTRIM5α cells in sucrose gradients.
PgsA cells stably expressing huTRIM5α (huT5α) and control pgsA cells (none) were infected with VSV-G pseudotyped N-MLV carrying a GFP reporter and 3 copies of HA tag in IN (IN-3×HA). (A) Infectivity of N-MLV on pgsA and pgsA-huTRIM5α cells was determined by FACS at 2 days post infection. (B, C) Cells were harvested either immediately after virion binding (T = 0 hr) or after a further incubation at 37°C for 2 hours (T = 2 hr). Post-nuclear supernatants were fractionated on 10–50% (w/v) sucrose gradients and 10 fractions from top of were collected, as explained in Materials and Methods. Western blot analysis of CA (p30) in sucrose fractions collected from T = 0 hr (B) and T = 2 hr (C) post-infection samples. Data is from a representative experiment.
Figure 2.
Profile of N-MLV core components isolated from pgsA and pgsA-huTRIM5α cells in sucrose gradients.
PgsA cells stably expressing huTRIM5α (huT5α) and control pgsA cells (none) were infected with VSV-G pseudotyped N-MLV (IN-3×HA). Infected cells were processed as explained in legend to Fig. 1 and Materials & Methods. (A, B) Sucrose fractions collected from T = 0 hr (A) or T = 2 hr (B) samples were analyzed for the presence of integrase (IN) by western blotting using antibodies against the HA tag. (C, D) viral RNA was quantitated by Q-RT-PCR in sucrose fractions collected from T = 0 hr (C) or T = 2 hr (D) samples. (E) Q-RT-PCR analysis of cellular GAPDH RNA in fractions collected from pgsA cells. (F) Q-PCR analysis of reverse-transcription products at T = 2 hr. Data is from a representative experiment.
Figure 3.
Effect of preventing endosome acidification in N-MLV infected cells.
PgsA-huTRIM5α (huT5α) and control pgsA cells (none) were infected in parallel by VSV-G pseudotyped N-MLV, carrying a single HA tag in IN (IN-HA), in the presence of 50 mM NH4Cl for 2 hours. Samples were processed as in Fig. 1. (A, B) Proteins in sucrose fractions were analyzed by western blotting using antibodies against CA-p30 (A) and HA tag for detection of integrase (B). The non-specific (n.s.) cross-reacting protein band is indicated on the HA western blot. Note that the single HA tag-IN used in this experiments results in different migration relative to the n.s. band that appears in fractions1-3 of several of the anti-HA blots in this study. (C) Viral RNA in the same sucrose fractions was reverse transcribed and analyzed by Q-PCR. Data is from a representative experiment.
Figure 4.
B-MLV cores isolated from both pgsA and pgsA-huTRIM5α cells migrate in dense sucrose fractions.
PgsA-huTRIM5α (huT5α) and control pgsA cells (none) were infected with VSV-G pseudotyped B-MLV, carrying a GFP reporter and IN-3×HA. Infected cells were processed as explained in legend to Fig. 1 and in Materials & Methods. (A) Proteins in fractions collected at T = 0 hr were analyzed by western blotting using antibodies against CA (p30) and HA tag for detection of IN. (B) Q-RT-PCR analysis of viral RNA in fractions collected from T = 0 hr samples. (C, D) Western blot analysis of CA (p30) and IN (C) and Q-RT-PCR analysis of viral RNA (D) in fractions collected from T = 2 hr samples. (E) Reverse transcription products in fractions collected at T = 2 hr was analyzed by Q-PCR. Data is from a representative experiment.
Figure 5.
Inhibition of proteasomes in huTRIM5α expressing cells restores large subviral N-MLV complexes.
PgsA-huTRIM5α (huT5α) and control pgsA cells (none) were infected with VSV-G pseudotyped N-MLV (IN-HA) in the absence (mock) or presence of 2 µM MG132 for 2 hours. Samples were processed as explained in legend to Fig. 1 and in Materials & Methods. (A, B) Protein and RNA samples isolated from mock-treated cells were analyzed by western blotting using antibodies against CA-p30 (A) and by Q-RT-PCR for detection of viral RNA (B), respectively. (C, D) CA and viral RNA in parallel fractions of MG132-treated samples were analyzed by western blotting against CA (C) and Q-RT-PCR (D), respectively. (E) Q-PCR analysis of reverse transcription products isolated from mock-treated and MG132-treated pgsA- huTRIM5α cells. (F) Infectivity of N-MLV on mock-treated and MG132-treated pgsA and pgsA- huTRIM5α cells was determined by FACS. Data is from a representative experiment.
Figure 6.
Effect of reverse transcription and proteasomes in restricting cells on large N-MLV subviral complexes.
PgsA-huTRIM5α (huT5α) and control pgsA cells (none) were infected with VSV-G pseudotyped N-MLV (IN-3×HA) in the presence of either 1 mM AZT alone or 1 mM AZT together with 2 µM MG132 for 2 hours. Samples were processed as explained in legend to Fig. 1 and in Materials & Methods. (A) Protein samples in sucrose fractions were analyzed by western blotting using antibodies against CA (p30) and HA tag for detection of IN. (B, C) Viral RNA in parallel fractions of AZT treated (B) or AZT and MG132-treated (C) samples were reverse-transcribed and analyzed by Q-PCR. Data is from a representative experiment.
Figure 7.
Profile of HIV-1 proteins and RNA isolated from pgsA-huTRIM5α and pgsA-rhTRIM5α cells.
PgsA-huTRIM5α (huT5α) and pgsA-rhTRIM5α (rhT5α) cells were infected with VSV-G pseudotyped HIV-1, carrying a GFP reporter. Infected cells were processed at T = 0 hr and T = 2 hr as explained in legend to Fig. 1. (A) Proteins in fractions collected at T = 0 hr were analyzed by western blotting using antibodies against CA (p24) and integrase (IN). (B) Q-RT-PCR analysis of viral RNA in fractions collected from T = 0 hr samples. (C, D) Western blot analysis of CA and IN (C) and Q-RT-PCR analysis of viral RNA (D) in fractions collected from T = 2 hr samples. (E) Q-PCR analysis of viral reverse transcription products in sucrose fractions of T = 2 hr samples. Data is from a representative experiment.
Figure 8.
Inhibition of proteasomes restores large sub-viral complexes in HIV-1 infected pgsA-rhTRIM5a cells.
PgsA-huTRIM5α (huT5α) and pgsA-rhTRIM5α (rhT5α) cells were infected with VSV-G pseudotyped HIV-1 in the presence of either 25 µM nevirapine alone or together with 2 µM MG132 for 2 hours. Samples were processed as explained in legend to Fig. 1 and in Materials & Methods. (A) Protein samples from fractions 1–10 were analyzed by western blotting using antibodies against CA (p24) and integrase (IN). (B, C) Viral RNA in parallel fractions of nevirapine-treated (B) or nevirapine and MG132-treated (C) samples was reverse-transcribed and quantitated by Q-PCR. (D, E) huT5α and rhT5α cells were infected with VSV-G pseudotyped HIV-1 in the absence (mock) or presence of 2 µM MG132 for 2 hours. Samples were processed as indicated above. (D) Q-PCR analysis of reverse transcription products isolated from mock-treated and MG132-treated pgsA-rhTRIM5α. (E) Infectivity of HIV-1 in mock-treated and MG132-treated huT5α and rhT5α cells was determined by FACS. Data is from a representative experiment.
Figure 9.
Inhibition of proteasomes restores large sub-viral complexes in HIV-1 infected omkTRIMCyp-expressing cells.
(A) Infectivity of HIV-1 in mock-, cyclosporin A (CsA, 5 µM)-, MG132 (2 µM)-, and CsA and nevirapine (25 µM)-treated pgsA-omkTRIMCyp cells was determined by FACS. (B) pgsA-TRIMCyp cells were infected with VSV-G pseudotyped HIV-1 in the absence (mock) or the presence of 5 µM CsA alone, or together with 25 µM nevirapine. Samples were processed and reverse transcription products were analyzed by Q-PCR as explained above. (C–E) PgsA-omkTRIMCyp cells were infected with VSV-G pseudotyped HIV-1 in the presence of either 25 µM nevirapine alone or together with 5 µM cyclosporine A (CsA) or 2 µM MG132 for 2 hours. Samples were processed as explained in legend to Fig. 1 and in Materials & Methods. Protein samples from fractions 1–10 were analyzed by western blotting using antibodies against CA (p24, C) and integrase (IN, D). Viral RNA in parallel fractions were reverse-transcribed and analyzed by Q-PCR (E). Data is from a representative experiment.
Figure 10.
Model for mechanism of restriction by TRIM5 proteins.
Soon after entry into the cytoplasm, retroviral cores are recognized by TRIM5 proteins, which leads to both disassembly and degradation of core components. Inhibition of proteasomes prevents both of these events and restores viral cores where reverse transcription can take place. However, inhibition of proteasomes does not restore virus infectivity indicating that another event (e.g. coating of viral cores by TRIM5 proteins) is crucial for restriction.