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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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Flow cytometry analysis shows equivalent numbers of CD4 and CXCR4 molecules on the surface of HeLa/CD4 and NP2/CD4/CXCR4 cells. Cells were stained with Pacific blue conjugated mouse anti human CD4 and PE conjugated mouse anti human CXCR4, with appropriate isotype and unlabelled cell controls. (a) NP2/CD4/CXCR4 and HeLa/CD4 cells showed similar log shifts in CD4 fluorescent intensity, compared to isotype controls. (b) NP2/CD4/CXCR4 and HeLa/CD4 cells show similar log shifts in CXCR4 fluorescent intensity, compared to isotype controls.
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Figure 2: Flow cytometry analysis shows equivalent numbers of CD4 and CXCR4 molecules on the surface of HeLa/CD4 and NP2/CD4/CXCR4 cells. Cells were stained with Pacific blue conjugated mouse anti human CD4 and PE conjugated mouse anti human CXCR4, with appropriate isotype and unlabelled cell controls. (a) NP2/CD4/CXCR4 and HeLa/CD4 cells showed similar log shifts in CD4 fluorescent intensity, compared to isotype controls. (b) NP2/CD4/CXCR4 and HeLa/CD4 cells show similar log shifts in CXCR4 fluorescent intensity, compared to isotype controls.

Mentions: The results above demonstrate that Env is a strong determinant of the ability of viral core to complete early events in replication. We next determined the HIV receptor levels of NP2 and HeLa cells to further support the notion that the restriction was not due to a difference in the expression of these receptors. We immunostained the cells with fluorescently labelled antibodies to CD4 and CXCR4 and used flow cytometry for their detection. These experiments show similar log shifts in fluorescent intensity for CD4 staining (Figure 2a) and CXCR4 staining (Figure 2b) on both HeLa/CD4 and NP2/CD4/CXCR4 cells. Hence, the restriction in HeLa/CD4 cells is not due to the reduced level of receptors on the surface of these cells in comparison to those seen on the unrestricted NP2/CD4/CXCR4 cells.


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)

Flow cytometry analysis shows equivalent numbers of CD4 and CXCR4 molecules on the surface of HeLa/CD4 and NP2/CD4/CXCR4 cells. Cells were stained with Pacific blue conjugated mouse anti human CD4 and PE conjugated mouse anti human CXCR4, with appropriate isotype and unlabelled cell controls. (a) NP2/CD4/CXCR4 and HeLa/CD4 cells showed similar log shifts in CD4 fluorescent intensity, compared to isotype controls. (b) NP2/CD4/CXCR4 and HeLa/CD4 cells show similar log shifts in CXCR4 fluorescent intensity, compared to isotype controls.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2959036&req=5

Figure 2: Flow cytometry analysis shows equivalent numbers of CD4 and CXCR4 molecules on the surface of HeLa/CD4 and NP2/CD4/CXCR4 cells. Cells were stained with Pacific blue conjugated mouse anti human CD4 and PE conjugated mouse anti human CXCR4, with appropriate isotype and unlabelled cell controls. (a) NP2/CD4/CXCR4 and HeLa/CD4 cells showed similar log shifts in CD4 fluorescent intensity, compared to isotype controls. (b) NP2/CD4/CXCR4 and HeLa/CD4 cells show similar log shifts in CXCR4 fluorescent intensity, compared to isotype controls.
Mentions: The results above demonstrate that Env is a strong determinant of the ability of viral core to complete early events in replication. We next determined the HIV receptor levels of NP2 and HeLa cells to further support the notion that the restriction was not due to a difference in the expression of these receptors. We immunostained the cells with fluorescently labelled antibodies to CD4 and CXCR4 and used flow cytometry for their detection. These experiments show similar log shifts in fluorescent intensity for CD4 staining (Figure 2a) and CXCR4 staining (Figure 2b) on both HeLa/CD4 and NP2/CD4/CXCR4 cells. Hence, the restriction in HeLa/CD4 cells is not due to the reduced level of receptors on the surface of these cells in comparison to those seen on the unrestricted NP2/CD4/CXCR4 cells.

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