Limits...
Variability in a dominant block to SIV early reverse transcription in rhesus monkey cells predicts in vivo viral replication and time to death.

Rogers TF, Lim SY, Sundsvold TJ, Chan T, Hsu A, Letvin NL - Virol. J. (2010)

Bottom Line: While it has long been appreciated that there is considerable variability in host containment of HIV/SIV replication, the determinants of that variability are not fully understood.Previous studies demonstrated that the degree of permissivity of a macaque's peripheral blood mononuclear cells (PBMC) for infection with simian immunodeficiency virus (SIV) in vitro predicted that animal's peak plasma virus RNA levels following SIV infection in vivo.In a cohort of 15 unrelated Indian-origin rhesus monkeys, infectability of PBMC of individual animals with SIVsmE660, as defined by tissue culture infectious dose (TCID50), varied by more than 3 logs and was a stable phenotype over time.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Viral Pathogenesis, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02115, USA. thomas_rogers@hms.harvard.edu

ABSTRACT
While it has long been appreciated that there is considerable variability in host containment of HIV/SIV replication, the determinants of that variability are not fully understood. Previous studies demonstrated that the degree of permissivity of a macaque's peripheral blood mononuclear cells (PBMC) for infection with simian immunodeficiency virus (SIV) in vitro predicted that animal's peak plasma virus RNA levels following SIV infection in vivo. The present study was conducted to define the mechanisms underlying the variable intrinsic susceptibility of rhesus monkey PBMC to SIVsmE660 infection. In a cohort of 15 unrelated Indian-origin rhesus monkeys, infectability of PBMC of individual animals with SIVsmE660, as defined by tissue culture infectious dose (TCID50), varied by more than 3 logs and was a stable phenotype over time. Susceptibility of a monkey's PBMC to wild type SIVsmE660 infection correlated with the susceptibility of that monkey's PBMC to infection with VSV-G pseudotyped SIVsm543-GFP. Moreover, the permissivity of an individual monkey's PBMC for infection with this construct correlated with the permissivity of a B-lymphoblastoid cell line (B-LCL) generated from PBMC of the same animal. We found that the degree of intrinsic resistance of monkey B-LCL correlated with the copy number of early reverse transcription (ERT) SIV DNA. The resistance of monkey B-LCL to SIVsmE660 replication could be abrogated by preincubation of cells with the SIV virus-like particles (VLPs) and SIV resistance phenotype could be transferred to a SIV susceptible B-LCL through cell fusion. Finally, we observed a positive correlation between susceptibility of monkey B-LCL to SIV infection with a VSV-G pseudotyped SIV-GFP construct in vitro and both the peak plasma virus RNA levels in vivo and time to death following wild type SIV infection. These findings suggest that a dominant early RT restricting factor that can be saturated by SIV capsid may contribute to the variable resistance to SIV infection in rhesus monkey B-LCL and that this differential intrinsic susceptibility contributes to the clinical outcome of an SIV infection.

Show MeSH

Related in: MedlinePlus

Fusion of SIV resistant and susceptible rhesus monkey B-LCLs demonstrates a dominant SIV resistance phenotype. SIV susceptible and resistant B-LCLs were labeled with either Vybrant DID (Invitrogen) or Oregon Green (Invitrogen). Susceptible and resistant cell lines were fused using PEG incubation and double positive fusion events were sorted by flow cytometry. Fused cells were subsequently infected with SIVsmE543-GFP and quantified for % GFP+ by flow cytometry.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2874537&req=5

Figure 6: Fusion of SIV resistant and susceptible rhesus monkey B-LCLs demonstrates a dominant SIV resistance phenotype. SIV susceptible and resistant B-LCLs were labeled with either Vybrant DID (Invitrogen) or Oregon Green (Invitrogen). Susceptible and resistant cell lines were fused using PEG incubation and double positive fusion events were sorted by flow cytometry. Fused cells were subsequently infected with SIVsmE543-GFP and quantified for % GFP+ by flow cytometry.

Mentions: On the basis of these data, we hypothesized that rhesus monkey B-LCL express a protein or variable forms of a protein that inhibit early reverse transcription by interacting with incoming lentiviral capsid. To test this hypothesis, we first examined the dominance of the SIV resistance phenotype in B-LCL. To determine if the resistance of B-LCL derived from some rhesus monkeys to SIV replication is a dominant or non-dominant phenotype, we performed SIV infection assays on cells that were fusions of SIV-resistant and SIV-sensitive B-LCL. By examining the SIV susceptibility phenotype in a cell that is a fusion between a resistant and a susceptible B-LCL, we could determine if resistance to SIV replication in B-LCL is mediated by a factor that is present in resistant cells or reflects the absence of a factor necessary for early reverse transcription. Since nonfused cells and homokaryotypic fusions would impact the MOI of the VSV-G pseudotyped SIVsm543-GFP infection, we generated PEG-mediated fusions of resistant and susceptible cell lines that were stained with intracellular dyes. Heterokaryotypic fusion products could then be isolated through detect of the intermixing of the dyes by flow cytometry. Using this strategy, we showed that a sensitive/sensitive cell fusion maintained a sensitive phenotype, while a resistant/sensitive cell fusion was resistant to SIV infection (Fig. 6). These data suggest that a dominant factor exists in resistant B-LCL that can inhibit SIV replication.


Variability in a dominant block to SIV early reverse transcription in rhesus monkey cells predicts in vivo viral replication and time to death.

Rogers TF, Lim SY, Sundsvold TJ, Chan T, Hsu A, Letvin NL - Virol. J. (2010)

Fusion of SIV resistant and susceptible rhesus monkey B-LCLs demonstrates a dominant SIV resistance phenotype. SIV susceptible and resistant B-LCLs were labeled with either Vybrant DID (Invitrogen) or Oregon Green (Invitrogen). Susceptible and resistant cell lines were fused using PEG incubation and double positive fusion events were sorted by flow cytometry. Fused cells were subsequently infected with SIVsmE543-GFP and quantified for % GFP+ by flow cytometry.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2874537&req=5

Figure 6: Fusion of SIV resistant and susceptible rhesus monkey B-LCLs demonstrates a dominant SIV resistance phenotype. SIV susceptible and resistant B-LCLs were labeled with either Vybrant DID (Invitrogen) or Oregon Green (Invitrogen). Susceptible and resistant cell lines were fused using PEG incubation and double positive fusion events were sorted by flow cytometry. Fused cells were subsequently infected with SIVsmE543-GFP and quantified for % GFP+ by flow cytometry.
Mentions: On the basis of these data, we hypothesized that rhesus monkey B-LCL express a protein or variable forms of a protein that inhibit early reverse transcription by interacting with incoming lentiviral capsid. To test this hypothesis, we first examined the dominance of the SIV resistance phenotype in B-LCL. To determine if the resistance of B-LCL derived from some rhesus monkeys to SIV replication is a dominant or non-dominant phenotype, we performed SIV infection assays on cells that were fusions of SIV-resistant and SIV-sensitive B-LCL. By examining the SIV susceptibility phenotype in a cell that is a fusion between a resistant and a susceptible B-LCL, we could determine if resistance to SIV replication in B-LCL is mediated by a factor that is present in resistant cells or reflects the absence of a factor necessary for early reverse transcription. Since nonfused cells and homokaryotypic fusions would impact the MOI of the VSV-G pseudotyped SIVsm543-GFP infection, we generated PEG-mediated fusions of resistant and susceptible cell lines that were stained with intracellular dyes. Heterokaryotypic fusion products could then be isolated through detect of the intermixing of the dyes by flow cytometry. Using this strategy, we showed that a sensitive/sensitive cell fusion maintained a sensitive phenotype, while a resistant/sensitive cell fusion was resistant to SIV infection (Fig. 6). These data suggest that a dominant factor exists in resistant B-LCL that can inhibit SIV replication.

Bottom Line: While it has long been appreciated that there is considerable variability in host containment of HIV/SIV replication, the determinants of that variability are not fully understood.Previous studies demonstrated that the degree of permissivity of a macaque's peripheral blood mononuclear cells (PBMC) for infection with simian immunodeficiency virus (SIV) in vitro predicted that animal's peak plasma virus RNA levels following SIV infection in vivo.In a cohort of 15 unrelated Indian-origin rhesus monkeys, infectability of PBMC of individual animals with SIVsmE660, as defined by tissue culture infectious dose (TCID50), varied by more than 3 logs and was a stable phenotype over time.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Viral Pathogenesis, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02115, USA. thomas_rogers@hms.harvard.edu

ABSTRACT
While it has long been appreciated that there is considerable variability in host containment of HIV/SIV replication, the determinants of that variability are not fully understood. Previous studies demonstrated that the degree of permissivity of a macaque's peripheral blood mononuclear cells (PBMC) for infection with simian immunodeficiency virus (SIV) in vitro predicted that animal's peak plasma virus RNA levels following SIV infection in vivo. The present study was conducted to define the mechanisms underlying the variable intrinsic susceptibility of rhesus monkey PBMC to SIVsmE660 infection. In a cohort of 15 unrelated Indian-origin rhesus monkeys, infectability of PBMC of individual animals with SIVsmE660, as defined by tissue culture infectious dose (TCID50), varied by more than 3 logs and was a stable phenotype over time. Susceptibility of a monkey's PBMC to wild type SIVsmE660 infection correlated with the susceptibility of that monkey's PBMC to infection with VSV-G pseudotyped SIVsm543-GFP. Moreover, the permissivity of an individual monkey's PBMC for infection with this construct correlated with the permissivity of a B-lymphoblastoid cell line (B-LCL) generated from PBMC of the same animal. We found that the degree of intrinsic resistance of monkey B-LCL correlated with the copy number of early reverse transcription (ERT) SIV DNA. The resistance of monkey B-LCL to SIVsmE660 replication could be abrogated by preincubation of cells with the SIV virus-like particles (VLPs) and SIV resistance phenotype could be transferred to a SIV susceptible B-LCL through cell fusion. Finally, we observed a positive correlation between susceptibility of monkey B-LCL to SIV infection with a VSV-G pseudotyped SIV-GFP construct in vitro and both the peak plasma virus RNA levels in vivo and time to death following wild type SIV infection. These findings suggest that a dominant early RT restricting factor that can be saturated by SIV capsid may contribute to the variable resistance to SIV infection in rhesus monkey B-LCL and that this differential intrinsic susceptibility contributes to the clinical outcome of an SIV infection.

Show MeSH
Related in: MedlinePlus