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A novel envelope mediated post entry restriction of murine leukaemia virus in human cells is Ref1/TRIM5α independent.

Oliveira NM, Trikha R, McKnight Á - Retrovirology (2010)

Bottom Line: For example rhesus TRIM5α (rhTRIM5α) can potently restrict HIV-1 infection while human TRIM5α (huTRIM5α) only has a mild effect on SIVmac and HIV-1 infectivity (Lv1).The HeLa/CD4 cell line restricted both MLV CAs in an Env dependent manner, compared to NP2/CD4/CXCR4 cells.We discuss the relevance of these findings in light of the growing evidence supporting the complexities involved in innate host immunity to retroviral infection.

View Article: PubMed Central - HTML - PubMed

Affiliation: HIV/AIDS Group, Centre for Immunology and Infectious Disease, Blizard Institute of Cell and Molecular Science, Barts and the London School of Medicine and Dentistry, 4 Newark Street, Whitechapel, London E1 2AT, UK.

ABSTRACT

Background: 'Intrinsic' resistance to retroviral infection was first recognised with the Friend virus susceptibility gene (Fv1), which determines susceptibility to murine leukaemia virus (MLV) infection in different murine species. Similarly, the tripartite motif (TRIM) family of proteins determine lentiviral restriction in a primate host-species specific manner. For example rhesus TRIM5α (rhTRIM5α) can potently restrict HIV-1 infection while human TRIM5α (huTRIM5α) only has a mild effect on SIVmac and HIV-1 infectivity (Lv1). Human TRIM5α is able to restrict MLV-N virus replication, but is ineffective against MLV-B or MLV-NB virus infection. Lv2 restriction of some HIV-2 viruses is seen in human cells. Like Lv1, Lv2 is a post-entry restriction factor, whose viral determinants have been mapped to the viral capsid (CA). Unlike Lv1, however, Lv2 is determined by envelope (Env) in addition to CA. Here we present evidence of a novel Env determined post entry restriction to infection in human cells of pseudotyped MLV-B and MLV-NB cores.

Results: We generated retroviral vectors pseudotyped with various gamma and lentiviral Envs on MLV-B and -NB CAs containing a green fluorescent protein (GFP) reporter. Flow cytometry was used to determine transduction efficiencies in NP2/CD4/CXCR4 (glioma cell line stably transduced with the HIV receptors) and HeLa/CD4 cell lines. The HeLa/CD4 cell line restricted both MLV CAs in an Env dependent manner, compared to NP2/CD4/CXCR4 cells. Quantitative polymerase chain reaction (QT-PCR) analysis of reverse transcription (RT) transcripts demonstrates that this restriction occurs at a post entry and RT level. siRNA knockdown of huTRIM5α ruled out a direct role for this cellular component in mediating this restriction. We describe a previously unobserved Env determined restriction of MLV-B and MLV-NB CAs in HeLa/CD4 cells when pseudotyped with HIV-2 and RD114 Envs, but not gibbon ape leukaemia virus (GALV), HIV-1 or Amphotrophic (Ampho) Envs.

Conclusions: Our data further demonstrate the variability of Env and CA mediated susceptibility to post entry host cell restriction. We discuss the relevance of these findings in light of the growing evidence supporting the complexities involved in innate host immunity to retroviral infection.

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siRNA knockdown of huTRIM5α reduces VSV-G MLV-N restriction in HeLa/CD4 cells but has no effect on MLV-B and MLV-NB. (a) VSV-G pseudotyped MLV-N viral vectors were used to challenge HeLa/CD4 cells that were pre-treated with 50 pmol siRNA huTRIM5α, control siRNA and untreated cells. As expected, treatment with siRNA huTRIM5α resulted in a relief of the restriction of MLV-N cores, while treatment of cells with the control siRNA had no effect on the transduction. MLV-B viral vectors pseudotyped with restricted (MCN, MCR, RD114) and non restricted (VSV-G, Ampho and GALV) Envs were used to infect HeLa/CD4 cells that were pre-treated with the optimal siRNA huTRIM5α concentration determined for MLV-N (50 pmol), control siRNA or non-siRNA treated. Untreated NP2/CD4/CXCR4 cells were also infected as a control. The siRNA huTRIM5α treated cells showed no relief of restriction of the MLV-B viral vectors. (b) As for Figure 4a, but with MLV-NB virus. Data represent the average of three or more independent experiments +/- S.E.M.
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Figure 4: siRNA knockdown of huTRIM5α reduces VSV-G MLV-N restriction in HeLa/CD4 cells but has no effect on MLV-B and MLV-NB. (a) VSV-G pseudotyped MLV-N viral vectors were used to challenge HeLa/CD4 cells that were pre-treated with 50 pmol siRNA huTRIM5α, control siRNA and untreated cells. As expected, treatment with siRNA huTRIM5α resulted in a relief of the restriction of MLV-N cores, while treatment of cells with the control siRNA had no effect on the transduction. MLV-B viral vectors pseudotyped with restricted (MCN, MCR, RD114) and non restricted (VSV-G, Ampho and GALV) Envs were used to infect HeLa/CD4 cells that were pre-treated with the optimal siRNA huTRIM5α concentration determined for MLV-N (50 pmol), control siRNA or non-siRNA treated. Untreated NP2/CD4/CXCR4 cells were also infected as a control. The siRNA huTRIM5α treated cells showed no relief of restriction of the MLV-B viral vectors. (b) As for Figure 4a, but with MLV-NB virus. Data represent the average of three or more independent experiments +/- S.E.M.

Mentions: HuTRIM5α has been well documented as a restriction to MLV-N, but not MLV-B and MLV-NB pseudotyped with a VSV-G Env. We down regulated TRIM5α production using specific siRNA knockdown to determine whether or not it had a role in the restricted phenotype described here. As shown by others, siRNA knockdown of huTRIM5α relieved the restriction in HeLa/CD4 (with an increase from 18% to 38% in transduction) of VSV-G pseudotyped MLV-N viral vectors, but not with control siRNA (Figure 4a). Similar results were obtained on NP2/CD4/CXCR4 cells (7.6% to 26.4% (data not shown). By comparison, siRNA knockdown of huTRIM5α did not affect the HIV-2 (MCN, MCR) and RD114 Env mediated restriction of MLV-B CAs in the HeLa/CD4 cells (Figure 4a). The results were similar for MLV-NB cored viruses (Figure 4b). Therefore, the restriction of MLV-B and MLV-NB cores, pseudotyped by HIV-2 and RD114 Envs, is not due to the direct activity of huTRIM5α on incoming CAs.


A novel envelope mediated post entry restriction of murine leukaemia virus in human cells is Ref1/TRIM5α independent.

Oliveira NM, Trikha R, McKnight Á - Retrovirology (2010)

siRNA knockdown of huTRIM5α reduces VSV-G MLV-N restriction in HeLa/CD4 cells but has no effect on MLV-B and MLV-NB. (a) VSV-G pseudotyped MLV-N viral vectors were used to challenge HeLa/CD4 cells that were pre-treated with 50 pmol siRNA huTRIM5α, control siRNA and untreated cells. As expected, treatment with siRNA huTRIM5α resulted in a relief of the restriction of MLV-N cores, while treatment of cells with the control siRNA had no effect on the transduction. MLV-B viral vectors pseudotyped with restricted (MCN, MCR, RD114) and non restricted (VSV-G, Ampho and GALV) Envs were used to infect HeLa/CD4 cells that were pre-treated with the optimal siRNA huTRIM5α concentration determined for MLV-N (50 pmol), control siRNA or non-siRNA treated. Untreated NP2/CD4/CXCR4 cells were also infected as a control. The siRNA huTRIM5α treated cells showed no relief of restriction of the MLV-B viral vectors. (b) As for Figure 4a, but with MLV-NB virus. Data represent the average of three or more independent experiments +/- S.E.M.
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Figure 4: siRNA knockdown of huTRIM5α reduces VSV-G MLV-N restriction in HeLa/CD4 cells but has no effect on MLV-B and MLV-NB. (a) VSV-G pseudotyped MLV-N viral vectors were used to challenge HeLa/CD4 cells that were pre-treated with 50 pmol siRNA huTRIM5α, control siRNA and untreated cells. As expected, treatment with siRNA huTRIM5α resulted in a relief of the restriction of MLV-N cores, while treatment of cells with the control siRNA had no effect on the transduction. MLV-B viral vectors pseudotyped with restricted (MCN, MCR, RD114) and non restricted (VSV-G, Ampho and GALV) Envs were used to infect HeLa/CD4 cells that were pre-treated with the optimal siRNA huTRIM5α concentration determined for MLV-N (50 pmol), control siRNA or non-siRNA treated. Untreated NP2/CD4/CXCR4 cells were also infected as a control. The siRNA huTRIM5α treated cells showed no relief of restriction of the MLV-B viral vectors. (b) As for Figure 4a, but with MLV-NB virus. Data represent the average of three or more independent experiments +/- S.E.M.
Mentions: HuTRIM5α has been well documented as a restriction to MLV-N, but not MLV-B and MLV-NB pseudotyped with a VSV-G Env. We down regulated TRIM5α production using specific siRNA knockdown to determine whether or not it had a role in the restricted phenotype described here. As shown by others, siRNA knockdown of huTRIM5α relieved the restriction in HeLa/CD4 (with an increase from 18% to 38% in transduction) of VSV-G pseudotyped MLV-N viral vectors, but not with control siRNA (Figure 4a). Similar results were obtained on NP2/CD4/CXCR4 cells (7.6% to 26.4% (data not shown). By comparison, siRNA knockdown of huTRIM5α did not affect the HIV-2 (MCN, MCR) and RD114 Env mediated restriction of MLV-B CAs in the HeLa/CD4 cells (Figure 4a). The results were similar for MLV-NB cored viruses (Figure 4b). Therefore, the restriction of MLV-B and MLV-NB cores, pseudotyped by HIV-2 and RD114 Envs, is not due to the direct activity of huTRIM5α on incoming CAs.

Bottom Line: For example rhesus TRIM5α (rhTRIM5α) can potently restrict HIV-1 infection while human TRIM5α (huTRIM5α) only has a mild effect on SIVmac and HIV-1 infectivity (Lv1).The HeLa/CD4 cell line restricted both MLV CAs in an Env dependent manner, compared to NP2/CD4/CXCR4 cells.We discuss the relevance of these findings in light of the growing evidence supporting the complexities involved in innate host immunity to retroviral infection.

View Article: PubMed Central - HTML - PubMed

Affiliation: HIV/AIDS Group, Centre for Immunology and Infectious Disease, Blizard Institute of Cell and Molecular Science, Barts and the London School of Medicine and Dentistry, 4 Newark Street, Whitechapel, London E1 2AT, UK.

ABSTRACT

Background: 'Intrinsic' resistance to retroviral infection was first recognised with the Friend virus susceptibility gene (Fv1), which determines susceptibility to murine leukaemia virus (MLV) infection in different murine species. Similarly, the tripartite motif (TRIM) family of proteins determine lentiviral restriction in a primate host-species specific manner. For example rhesus TRIM5α (rhTRIM5α) can potently restrict HIV-1 infection while human TRIM5α (huTRIM5α) only has a mild effect on SIVmac and HIV-1 infectivity (Lv1). Human TRIM5α is able to restrict MLV-N virus replication, but is ineffective against MLV-B or MLV-NB virus infection. Lv2 restriction of some HIV-2 viruses is seen in human cells. Like Lv1, Lv2 is a post-entry restriction factor, whose viral determinants have been mapped to the viral capsid (CA). Unlike Lv1, however, Lv2 is determined by envelope (Env) in addition to CA. Here we present evidence of a novel Env determined post entry restriction to infection in human cells of pseudotyped MLV-B and MLV-NB cores.

Results: We generated retroviral vectors pseudotyped with various gamma and lentiviral Envs on MLV-B and -NB CAs containing a green fluorescent protein (GFP) reporter. Flow cytometry was used to determine transduction efficiencies in NP2/CD4/CXCR4 (glioma cell line stably transduced with the HIV receptors) and HeLa/CD4 cell lines. The HeLa/CD4 cell line restricted both MLV CAs in an Env dependent manner, compared to NP2/CD4/CXCR4 cells. Quantitative polymerase chain reaction (QT-PCR) analysis of reverse transcription (RT) transcripts demonstrates that this restriction occurs at a post entry and RT level. siRNA knockdown of huTRIM5α ruled out a direct role for this cellular component in mediating this restriction. We describe a previously unobserved Env determined restriction of MLV-B and MLV-NB CAs in HeLa/CD4 cells when pseudotyped with HIV-2 and RD114 Envs, but not gibbon ape leukaemia virus (GALV), HIV-1 or Amphotrophic (Ampho) Envs.

Conclusions: Our data further demonstrate the variability of Env and CA mediated susceptibility to post entry host cell restriction. We discuss the relevance of these findings in light of the growing evidence supporting the complexities involved in innate host immunity to retroviral infection.

Show MeSH
Related in: MedlinePlus