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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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Related in: MedlinePlus

QT-PCR analysis of late reverse transcripts generated in HeLa/CD4 and NP2/CD4/CXCR4 cells challenged with restricted and non-restricted MLV-B and MLV-NB viral vectors. MLV-B and MLV-NB cored viral vectors pseudotyped with the different Envs described in Figure 1 were used to challenge confluent monolayers of HeLa/CD4 and NP2/CD4/CX/CR4 cells in 12 well trays with MOIs of 0.25-1. Controls for infection included incubation of infected cells for 5 minutes at 4°C or trypsinising target cells (to strip cell surface receptors off) just prior to infection. Normal infection involved incubating cell cultures O/N (12-16 hrs) to 48 hrs at 37°C (for the FACS equivalent). Total extracted DNA was normalised to 250 or 500 ng/PCR reaction. Primers and probes were designed to amplify the eGFP target, with an internal genomic GAPDH control. The data shown have been normalised to the GAPDH amplification and for the background level of transcripts seen in uninfected cell controls for each experiment. (a) (i) MLV-B viral vectors pseudotyped with VSV-G Env, MLV-NB viral vectors pseudotyped with HIV-2 MCN Env, cells were incubated 5 minutes at 4°C and overnight 37°C incubation, data shown are for RT transcripts detected after overnight 37°C incubation minus the number detected for the 5 minutes at 4°C incubation for each Env, MOI of 1 (ii) Corresponding infection data (cells incubated for 48 hrs) for the QT-PCR shown in (i); (b) (i and ii) HIV-1 and HIV-2 MCN pseudotyped MLV-NB CA, titrated at MOIs 0.25-1 (iii and iv) Corresponding infection data (cells incubated for 48 hrs) for the QT-PCR shown in (i and ii); (c) MLV-NB viral vectors pseudotyped with GALV and Ampho Envs, cells were incubated O/N, MOI of 0.3. Data shown for the QT-PCRs are representative of 3 or more independent experiments. Error bars represent +/- STDEV within 1 experiment.
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Figure 3: QT-PCR analysis of late reverse transcripts generated in HeLa/CD4 and NP2/CD4/CXCR4 cells challenged with restricted and non-restricted MLV-B and MLV-NB viral vectors. MLV-B and MLV-NB cored viral vectors pseudotyped with the different Envs described in Figure 1 were used to challenge confluent monolayers of HeLa/CD4 and NP2/CD4/CX/CR4 cells in 12 well trays with MOIs of 0.25-1. Controls for infection included incubation of infected cells for 5 minutes at 4°C or trypsinising target cells (to strip cell surface receptors off) just prior to infection. Normal infection involved incubating cell cultures O/N (12-16 hrs) to 48 hrs at 37°C (for the FACS equivalent). Total extracted DNA was normalised to 250 or 500 ng/PCR reaction. Primers and probes were designed to amplify the eGFP target, with an internal genomic GAPDH control. The data shown have been normalised to the GAPDH amplification and for the background level of transcripts seen in uninfected cell controls for each experiment. (a) (i) MLV-B viral vectors pseudotyped with VSV-G Env, MLV-NB viral vectors pseudotyped with HIV-2 MCN Env, cells were incubated 5 minutes at 4°C and overnight 37°C incubation, data shown are for RT transcripts detected after overnight 37°C incubation minus the number detected for the 5 minutes at 4°C incubation for each Env, MOI of 1 (ii) Corresponding infection data (cells incubated for 48 hrs) for the QT-PCR shown in (i); (b) (i and ii) HIV-1 and HIV-2 MCN pseudotyped MLV-NB CA, titrated at MOIs 0.25-1 (iii and iv) Corresponding infection data (cells incubated for 48 hrs) for the QT-PCR shown in (i and ii); (c) MLV-NB viral vectors pseudotyped with GALV and Ampho Envs, cells were incubated O/N, MOI of 0.3. Data shown for the QT-PCRs are representative of 3 or more independent experiments. Error bars represent +/- STDEV within 1 experiment.

Mentions: Figure 3a (i) shows that the level of newly transcribed DNA transcripts after O/N incubation 37°C on HeLa/CD4 cells were equivalent (MOI 1). The corresponding infectivity data (Figure 3a (ii)) show that the MCN pseudotyped virus is, however, much less infectious compared to VSV-G Env pseudotyped virus. Figure 3b (i and ii) shows that regardless of viral Env, pseudotype infection of NP2 cells always resulted in greater numbers of RT transcripts compared to infection of HeLa cells. Importantly, however, a comparison of transcripts produced after challenge of HeLa cells resulted in the same number of transcripts regardless of whether the CA was pseudotyped with restrictive or non-restrictive Envs. The levels of RT transcripts detected correspond to the different MOIs used to challenge cells. The corresponding infectivity data (Figure 3b (iii and iv)) confirm that in NP2 cells the transcripts result in permissive titratable infection for both Envs, but in HeLa cells, while the HIV-1 Env virus results in efficient infection, the HIV-2 MCN virus is restricted.


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)

QT-PCR analysis of late reverse transcripts generated in HeLa/CD4 and NP2/CD4/CXCR4 cells challenged with restricted and non-restricted MLV-B and MLV-NB viral vectors. MLV-B and MLV-NB cored viral vectors pseudotyped with the different Envs described in Figure 1 were used to challenge confluent monolayers of HeLa/CD4 and NP2/CD4/CX/CR4 cells in 12 well trays with MOIs of 0.25-1. Controls for infection included incubation of infected cells for 5 minutes at 4°C or trypsinising target cells (to strip cell surface receptors off) just prior to infection. Normal infection involved incubating cell cultures O/N (12-16 hrs) to 48 hrs at 37°C (for the FACS equivalent). Total extracted DNA was normalised to 250 or 500 ng/PCR reaction. Primers and probes were designed to amplify the eGFP target, with an internal genomic GAPDH control. The data shown have been normalised to the GAPDH amplification and for the background level of transcripts seen in uninfected cell controls for each experiment. (a) (i) MLV-B viral vectors pseudotyped with VSV-G Env, MLV-NB viral vectors pseudotyped with HIV-2 MCN Env, cells were incubated 5 minutes at 4°C and overnight 37°C incubation, data shown are for RT transcripts detected after overnight 37°C incubation minus the number detected for the 5 minutes at 4°C incubation for each Env, MOI of 1 (ii) Corresponding infection data (cells incubated for 48 hrs) for the QT-PCR shown in (i); (b) (i and ii) HIV-1 and HIV-2 MCN pseudotyped MLV-NB CA, titrated at MOIs 0.25-1 (iii and iv) Corresponding infection data (cells incubated for 48 hrs) for the QT-PCR shown in (i and ii); (c) MLV-NB viral vectors pseudotyped with GALV and Ampho Envs, cells were incubated O/N, MOI of 0.3. Data shown for the QT-PCRs are representative of 3 or more independent experiments. Error bars represent +/- STDEV within 1 experiment.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: QT-PCR analysis of late reverse transcripts generated in HeLa/CD4 and NP2/CD4/CXCR4 cells challenged with restricted and non-restricted MLV-B and MLV-NB viral vectors. MLV-B and MLV-NB cored viral vectors pseudotyped with the different Envs described in Figure 1 were used to challenge confluent monolayers of HeLa/CD4 and NP2/CD4/CX/CR4 cells in 12 well trays with MOIs of 0.25-1. Controls for infection included incubation of infected cells for 5 minutes at 4°C or trypsinising target cells (to strip cell surface receptors off) just prior to infection. Normal infection involved incubating cell cultures O/N (12-16 hrs) to 48 hrs at 37°C (for the FACS equivalent). Total extracted DNA was normalised to 250 or 500 ng/PCR reaction. Primers and probes were designed to amplify the eGFP target, with an internal genomic GAPDH control. The data shown have been normalised to the GAPDH amplification and for the background level of transcripts seen in uninfected cell controls for each experiment. (a) (i) MLV-B viral vectors pseudotyped with VSV-G Env, MLV-NB viral vectors pseudotyped with HIV-2 MCN Env, cells were incubated 5 minutes at 4°C and overnight 37°C incubation, data shown are for RT transcripts detected after overnight 37°C incubation minus the number detected for the 5 minutes at 4°C incubation for each Env, MOI of 1 (ii) Corresponding infection data (cells incubated for 48 hrs) for the QT-PCR shown in (i); (b) (i and ii) HIV-1 and HIV-2 MCN pseudotyped MLV-NB CA, titrated at MOIs 0.25-1 (iii and iv) Corresponding infection data (cells incubated for 48 hrs) for the QT-PCR shown in (i and ii); (c) MLV-NB viral vectors pseudotyped with GALV and Ampho Envs, cells were incubated O/N, MOI of 0.3. Data shown for the QT-PCRs are representative of 3 or more independent experiments. Error bars represent +/- STDEV within 1 experiment.
Mentions: Figure 3a (i) shows that the level of newly transcribed DNA transcripts after O/N incubation 37°C on HeLa/CD4 cells were equivalent (MOI 1). The corresponding infectivity data (Figure 3a (ii)) show that the MCN pseudotyped virus is, however, much less infectious compared to VSV-G Env pseudotyped virus. Figure 3b (i and ii) shows that regardless of viral Env, pseudotype infection of NP2 cells always resulted in greater numbers of RT transcripts compared to infection of HeLa cells. Importantly, however, a comparison of transcripts produced after challenge of HeLa cells resulted in the same number of transcripts regardless of whether the CA was pseudotyped with restrictive or non-restrictive Envs. The levels of RT transcripts detected correspond to the different MOIs used to challenge cells. The corresponding infectivity data (Figure 3b (iii and iv)) confirm that in NP2 cells the transcripts result in permissive titratable infection for both Envs, but in HeLa cells, while the HIV-1 Env virus results in efficient infection, the HIV-2 MCN virus is restricted.

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