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The antiviral spectra of TRIM5α orthologues and human TRIM family proteins against lentiviral production.

Ohmine S, Sakuma R, Sakuma T, Thatava T, Takeuchi H, Ikeda Y - PLoS ONE (2011)

Bottom Line: TRIM5αhu did not show notable late restriction activities against these lentiviruses.The antiviral activities and encapsidation efficiencies did not correlate with their relative expression levels in the producer cells.Our results demonstrated the variations in the late restriction activities among closely related TRIM5α orthologues and a subset of human TRIM family proteins, providing further insights into the late restriction activities of TRIM proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine, Mayo Clinic, Rochester, Minnesota, United States of America.

ABSTRACT

Background: Rhesus monkey TRIM5α (TRIM5αrh) recognizes the incoming HIV-1 core through its C-terminal B30.2(PRYSPRY) domain and promotes its premature disassembly or degradation before reverse transcription. Previously, we have shown that TRIM5αrh blocks HIV-1 production through the N-terminal RBCC domain by the recognition of Gag polyproteins. Although all TRIM family proteins have RBCC domains, it remains elusive whether they possess similar late-restriction activities.

Methodology/principal findings: We examined the antiviral spectra of TRIM5α orthologues and human TRIM family members which have a genetic locus proximal to human TRIM5α (TRIM5αhu), against primate lentiviral production. When HIV-1 virus-like particles (VLPs) were generated in the presence of TRIM5α proteins, rhesus, African green and cynomolgus monkey TRIM5α (TRIM5αag and TRIM5αcy), but not TRIM5αhu, were efficiently incorporated into VLPs, suggesting an interaction between HIV-1 Gag and TRIM5α proteins. TRIM5αrh potently restricted the viral production of HIV-1 groups M and O and HIV-2, but not simian lentiviruses including SIV(MAC)1A11, SIV(AGM)Tan-1 or SIV(AGM)SAB-1. TRIM5αhu did not show notable late restriction activities against these lentiviruses. TRIM5αag and TRIM5αcy showed intermediate restriction phenotypes against HIV-1 and HIV-2, but showed no restriction activity against SIV production. A series of chimeric TRIM5α constructs indicated that the N-terminal region of TRIM5αag and TRIM5αcy are essential for the late restriction activity, while the C-terminal region of TRIM5αcy negatively regulates the late restriction activity against HIV-1. When select human TRIM family proteins were examined, TRIM21 and 22 were efficiently incorporated into HIV-1 VLPs, while only TRIM22 reduced HIV-1 titers up to 5-fold. The antiviral activities and encapsidation efficiencies did not correlate with their relative expression levels in the producer cells.

Conclusions/significance: Our results demonstrated the variations in the late restriction activities among closely related TRIM5α orthologues and a subset of human TRIM family proteins, providing further insights into the late restriction activities of TRIM proteins.

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Related in: MedlinePlus

TRIM5α orthologue expression in 293T cells and antiviral spectrum of TRIM5α orthologues against lentiviral production.(A) Verification of the proper expression of TRIM5α orthologues. Control plasmid (−), TRIM5αhu (Hu), TRIM5αrh (Rh), TRIM5αag (Ag) and TRIM5αcy (Cy) expression in transfected 293T cells were verified via immunoblot analysis. Arrow depicts approximate TRIM5α band size. (B) 293T cells were co-transfected with a primate lentivirus proviral plasmid and increasing amounts of human (Hu), rhesus monkey (Rh), African green monkey (Ag) or cynomolgus monkey (Cy) TRIM5α-expressing plasmids. As infectious proviral plasmids, HIV-1 Group M (pNL4-3 and p89.6), HIV-1 Group O (pCMO2.41, pCMO2.5), HIV-2 (pROD10), and SIV (pSIVMAC1A11, pSIVAGMTan-1 and pSIVAGMSAB-1) were used. Viral titers were determined in GHOST(3)R3X4R5 indicator cells and described as infectious units per ml (IU/ml). Error bars indicate one standard deviation.
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pone-0016121-g002: TRIM5α orthologue expression in 293T cells and antiviral spectrum of TRIM5α orthologues against lentiviral production.(A) Verification of the proper expression of TRIM5α orthologues. Control plasmid (−), TRIM5αhu (Hu), TRIM5αrh (Rh), TRIM5αag (Ag) and TRIM5αcy (Cy) expression in transfected 293T cells were verified via immunoblot analysis. Arrow depicts approximate TRIM5α band size. (B) 293T cells were co-transfected with a primate lentivirus proviral plasmid and increasing amounts of human (Hu), rhesus monkey (Rh), African green monkey (Ag) or cynomolgus monkey (Cy) TRIM5α-expressing plasmids. As infectious proviral plasmids, HIV-1 Group M (pNL4-3 and p89.6), HIV-1 Group O (pCMO2.41, pCMO2.5), HIV-2 (pROD10), and SIV (pSIVMAC1A11, pSIVAGMTan-1 and pSIVAGMSAB-1) were used. Viral titers were determined in GHOST(3)R3X4R5 indicator cells and described as infectious units per ml (IU/ml). Error bars indicate one standard deviation.

Mentions: Immunoblot analysis was performed to verify the proper expression of TRIM5αag and TRIM5αcy proteins following transfection into 293T cells (Fig. 2A). TRIM5αrh reduced the titers of two HIV-1 group M clones (NL4-3, 89.6) and HIV-1 group O clones (CMO2.41 and CMO2.5) by up to 60-fold in a dose-dependent manner (Fig. 2B). TRIM5αrh also reduced the titers of HIV-2 up to 40-fold. Although SIVMAC and SIVAGMSAB-1 titers were largely unaffected by TRIM5αrh, we observed a 10-fold decrease in the titers of SIVAGMTan-1 in the presence of TRIM5αrh (Fig. 2B). TRIM5αag and TRIM5αcy showed similar patterns of late restriction activity to its rhesus monkey orthologue, although the reductions in viral titers were modest. NL4-3 titers were reduced by 7-fold in cells expressing TRIM5αag or TRIM5αcy, while 89.6 titers were only reduced by 2-fold. Although group O isolate CMO2.41 virus titers were reduced by up to 7-fold when producer cells expressed TRIM5αag or TRIM5αcy, the titers of another Group O clone CMO2.5, which is based on the CMO2.41 isolate but containing the 5′LTR to vpr sequences from the MVP2171 O-type isolate [40], were strongly affected by TRIM5αag but not TRIM5αcy. TRIM5αag and TRIM5αcy showed little late restriction activity on SIVMAC1A11, SIVAGMTan-1, SIVAGMSAB-1 and HIV-2 production. Intriguingly, TRIM5αag reduced SIVAGMTan-1 titers by 6-fold (Fig. 2B), which may be explained by the difference in the host species of SIVAGM and TRIM5αag: SIVAGMTan-1 is isolated from the Chlorocebus tantalus [31] but the TRIM5αag protein used in these experiments were derived from Chlorocebus aethiops-derived CV1 cells [41]. TRIM5αhu marginally reduced HIV titers, while it did not affect SIV titers (Fig. 2B).


The antiviral spectra of TRIM5α orthologues and human TRIM family proteins against lentiviral production.

Ohmine S, Sakuma R, Sakuma T, Thatava T, Takeuchi H, Ikeda Y - PLoS ONE (2011)

TRIM5α orthologue expression in 293T cells and antiviral spectrum of TRIM5α orthologues against lentiviral production.(A) Verification of the proper expression of TRIM5α orthologues. Control plasmid (−), TRIM5αhu (Hu), TRIM5αrh (Rh), TRIM5αag (Ag) and TRIM5αcy (Cy) expression in transfected 293T cells were verified via immunoblot analysis. Arrow depicts approximate TRIM5α band size. (B) 293T cells were co-transfected with a primate lentivirus proviral plasmid and increasing amounts of human (Hu), rhesus monkey (Rh), African green monkey (Ag) or cynomolgus monkey (Cy) TRIM5α-expressing plasmids. As infectious proviral plasmids, HIV-1 Group M (pNL4-3 and p89.6), HIV-1 Group O (pCMO2.41, pCMO2.5), HIV-2 (pROD10), and SIV (pSIVMAC1A11, pSIVAGMTan-1 and pSIVAGMSAB-1) were used. Viral titers were determined in GHOST(3)R3X4R5 indicator cells and described as infectious units per ml (IU/ml). Error bars indicate one standard deviation.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3021539&req=5

pone-0016121-g002: TRIM5α orthologue expression in 293T cells and antiviral spectrum of TRIM5α orthologues against lentiviral production.(A) Verification of the proper expression of TRIM5α orthologues. Control plasmid (−), TRIM5αhu (Hu), TRIM5αrh (Rh), TRIM5αag (Ag) and TRIM5αcy (Cy) expression in transfected 293T cells were verified via immunoblot analysis. Arrow depicts approximate TRIM5α band size. (B) 293T cells were co-transfected with a primate lentivirus proviral plasmid and increasing amounts of human (Hu), rhesus monkey (Rh), African green monkey (Ag) or cynomolgus monkey (Cy) TRIM5α-expressing plasmids. As infectious proviral plasmids, HIV-1 Group M (pNL4-3 and p89.6), HIV-1 Group O (pCMO2.41, pCMO2.5), HIV-2 (pROD10), and SIV (pSIVMAC1A11, pSIVAGMTan-1 and pSIVAGMSAB-1) were used. Viral titers were determined in GHOST(3)R3X4R5 indicator cells and described as infectious units per ml (IU/ml). Error bars indicate one standard deviation.
Mentions: Immunoblot analysis was performed to verify the proper expression of TRIM5αag and TRIM5αcy proteins following transfection into 293T cells (Fig. 2A). TRIM5αrh reduced the titers of two HIV-1 group M clones (NL4-3, 89.6) and HIV-1 group O clones (CMO2.41 and CMO2.5) by up to 60-fold in a dose-dependent manner (Fig. 2B). TRIM5αrh also reduced the titers of HIV-2 up to 40-fold. Although SIVMAC and SIVAGMSAB-1 titers were largely unaffected by TRIM5αrh, we observed a 10-fold decrease in the titers of SIVAGMTan-1 in the presence of TRIM5αrh (Fig. 2B). TRIM5αag and TRIM5αcy showed similar patterns of late restriction activity to its rhesus monkey orthologue, although the reductions in viral titers were modest. NL4-3 titers were reduced by 7-fold in cells expressing TRIM5αag or TRIM5αcy, while 89.6 titers were only reduced by 2-fold. Although group O isolate CMO2.41 virus titers were reduced by up to 7-fold when producer cells expressed TRIM5αag or TRIM5αcy, the titers of another Group O clone CMO2.5, which is based on the CMO2.41 isolate but containing the 5′LTR to vpr sequences from the MVP2171 O-type isolate [40], were strongly affected by TRIM5αag but not TRIM5αcy. TRIM5αag and TRIM5αcy showed little late restriction activity on SIVMAC1A11, SIVAGMTan-1, SIVAGMSAB-1 and HIV-2 production. Intriguingly, TRIM5αag reduced SIVAGMTan-1 titers by 6-fold (Fig. 2B), which may be explained by the difference in the host species of SIVAGM and TRIM5αag: SIVAGMTan-1 is isolated from the Chlorocebus tantalus [31] but the TRIM5αag protein used in these experiments were derived from Chlorocebus aethiops-derived CV1 cells [41]. TRIM5αhu marginally reduced HIV titers, while it did not affect SIV titers (Fig. 2B).

Bottom Line: TRIM5αhu did not show notable late restriction activities against these lentiviruses.The antiviral activities and encapsidation efficiencies did not correlate with their relative expression levels in the producer cells.Our results demonstrated the variations in the late restriction activities among closely related TRIM5α orthologues and a subset of human TRIM family proteins, providing further insights into the late restriction activities of TRIM proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine, Mayo Clinic, Rochester, Minnesota, United States of America.

ABSTRACT

Background: Rhesus monkey TRIM5α (TRIM5αrh) recognizes the incoming HIV-1 core through its C-terminal B30.2(PRYSPRY) domain and promotes its premature disassembly or degradation before reverse transcription. Previously, we have shown that TRIM5αrh blocks HIV-1 production through the N-terminal RBCC domain by the recognition of Gag polyproteins. Although all TRIM family proteins have RBCC domains, it remains elusive whether they possess similar late-restriction activities.

Methodology/principal findings: We examined the antiviral spectra of TRIM5α orthologues and human TRIM family members which have a genetic locus proximal to human TRIM5α (TRIM5αhu), against primate lentiviral production. When HIV-1 virus-like particles (VLPs) were generated in the presence of TRIM5α proteins, rhesus, African green and cynomolgus monkey TRIM5α (TRIM5αag and TRIM5αcy), but not TRIM5αhu, were efficiently incorporated into VLPs, suggesting an interaction between HIV-1 Gag and TRIM5α proteins. TRIM5αrh potently restricted the viral production of HIV-1 groups M and O and HIV-2, but not simian lentiviruses including SIV(MAC)1A11, SIV(AGM)Tan-1 or SIV(AGM)SAB-1. TRIM5αhu did not show notable late restriction activities against these lentiviruses. TRIM5αag and TRIM5αcy showed intermediate restriction phenotypes against HIV-1 and HIV-2, but showed no restriction activity against SIV production. A series of chimeric TRIM5α constructs indicated that the N-terminal region of TRIM5αag and TRIM5αcy are essential for the late restriction activity, while the C-terminal region of TRIM5αcy negatively regulates the late restriction activity against HIV-1. When select human TRIM family proteins were examined, TRIM21 and 22 were efficiently incorporated into HIV-1 VLPs, while only TRIM22 reduced HIV-1 titers up to 5-fold. The antiviral activities and encapsidation efficiencies did not correlate with their relative expression levels in the producer cells.

Conclusions/significance: Our results demonstrated the variations in the late restriction activities among closely related TRIM5α orthologues and a subset of human TRIM family proteins, providing further insights into the late restriction activities of TRIM proteins.

Show MeSH
Related in: MedlinePlus