CEM-T4 Cells Do Not Lack an APOBEC3G Cofactor

Human APOBEC3G inhibits the replication of Vif-deficient HIV-1 by hypermutating nascent viral cDNA (reviewed in [1]–[3]). Zheng and colleagues recently reported that the HIV-1 restriction activity of APOBEC3G requires a cellular co-factor [4]. Their conclusion depended on three critical observations: i) CEM-T4 cells support the replication of Vif-deficient HIV-1, ii) CEM-T4 cells express restrictive levels of APOBEC3G, and iii) CEM-T4 cells engineered to express more APOBEC3G still permitted Vif-deficient HIV-1 replication [4]. These observations suggested that APOBEC3G alone is insufficient for restriction, and together with subsequent cell fusion experiments, that a recessive cellular co-factor is required. However, the fact that APOBEC3G is capable of restricting a broad number of retroelements, including yeast Ty elements, strongly suggests that other human cellular proteins are not absolutely required for restriction (e.g., [5],[6] and reviewed in [1]–[3]). 
 
An alternative explanation that could account for the observed permissive phenotype of the CEM-T4 line is that it is mixed, composed of a population of cells expressing low and/or variable levels of APOBEC3G. If this were the case, then the permissive phenotype could simply be due to virus replication in the subset of cells expressing low levels of APOBEC3G. To address this hypothesis, we generated subclones of the CEM-T4 line by serial dilution and determined the level of APOBEC3G expression by immunoblotting (Figure 1A). First, we observed that CEM-T4 cells expressed levels of APOBEC3G that were considerably lower than those in the non-permissive line CEM, regardless of whether they were obtained from the AIDS Research and Reference Reagent Program (CEM-T4-A) or directly from the Zheng laboratory (CEM-T4-Z). Second, we found that representative CEM-T4 subclones, regardless of source, expressed both low and variable APOBEC3G levels. It is further notable that none of the subclones expressed fully non-permissive, CEM-like APOBEC3G levels. This heterogeneity is reflected by the kinetics of Vif-deficient virus replication, with some subclones being fully permissive and others being semi-permissive (Figure 1B and 1C). Nevertheless, these data demonstrated that APOBEC3G levels are both low and variable within individual cells of the CEM-T4 line, thus providing a reasonable molecular explanation for the permissive phenotype of this cell line. 
 
 
 
Figure 1 
 
CEM-T4 cells express low APOBEC3G levels and subclones show additional heterogeneity. 
 
 
 
Curiously, Zheng and coworkers [4] also showed that CEM-T4 cells retrovirally transduced with APOBEC3G were still permissive for Vif-deficient HIV-1 replication. This result may be due to the distinct possibility that APOBEC3G itself inactivated some of the transducing viral cDNAs. Such an attempt at complementation would probably result in a CEM-T4 line that is mixed for APOBEC3G expression. To clarify this important point, we used electroporation to generate panels of HA-tagged and untagged APOBEC3G-expressing CEM-T4 clones and used them for virus replication experiments (Figures 2 and ​and3,3, respectively). 
 
 
 
Figure 2 
 
Expression of exogenous APOBEC3G-HA is sufficient to render CEM-T4 cells non-permissive for Vif-deficient HIV-1. 
 
 
 
 
 
Figure 3 
 
Increasing levels of untagged APOBEC3G is sufficient to render CEM-T4 cells non-permissive for Vif-deficient HIV-1 replication. 
 
 
 
Contrary to the findings of Zheng and colleagues [4], our APOBEC3G-expressing CEM-T4 clones were fully restrictive for Vif-deficient HIV-1 replication. The observed non-permissive phenotype was not due to gross overexpression of APOBEC3G because many of the restrictive CEM-T4 clones stably expressed APOBEC3G to levels slightly lower to those of CEM, the original parent of CEM-T4 (e.g., clone G4 in Figure 3). It is also notable that some vector control clones such as V9, like the subclones in Figure 1, expressed low and non-restrictive levels of APOBEC3G. These data suggest that cells must pass an APOBEC3G expression “threshold” before they become fully non-permissive for Vif-deficient HIV-1. Nevertheless, together these virus replication data clearly demonstrated that expression of exogenous APOBEC3G is sufficient to render CEM-T4 cells non-permissive for Vif-deficient HIV-1 replication. We conclude that this cell line does not require a specific endogenous APOBEC3G “co-factor” to restrict Vif-deficient HIV-1. 
 
In this report, we question the obligate APOBEC3G co-factor hypothesis put forth by Zheng and colleagues [4]. Our data clearly provide an alternative explanation that—rather than lacking a co-factor for APOBEC3G—the CEM-T4 line is mixed with individual cells expressing heterogeneous and less-than-restrictive levels of APOBEC3G. Importantly, when transfections were used to generate stable APOBEC3G-expressing CEM-T4 clones, the resulting lines were non-permissive for Vif-deficient HIV-1 replication. Based on these results, we conclude that CEM-T4 cells do not lack an APOBEC3G co-factor and, more generally, that APOBEC3G may not strictly require another cellular protein for HIV-1 restriction. However, we would like to emphasize that neither study rules out the attractive possibility that APOBEC3G, like most other proteins, is likely to be regulated by a variety of cellular processes.

Human APOBEC3G inhibits the replication of Vif-deficient HIV-1 by hypermutating nascent viral cDNA (reviewed in [1][2][3]). Zheng and colleagues recently reported that the HIV-1 restriction activity of APOBEC3G requires a cellular co-factor [4]. Their conclusion depended on three critical observations: i) CEM-T4 cells support the replication of Vif-deficient HIV-1, ii) CEM-T4 cells express restrictive levels of APOBEC3G, and iii) CEM-T4 cells engineered to express more APOBEC3G still permitted Vif-deficient HIV-1 replication [4]. These observations suggested that APOBEC3G alone is insufficient for restriction, and together with subsequent cell fusion experiments, that a recessive cellular co-factor is required. However, the fact that APOBEC3G is capable of restricting a broad number of retroelements, including yeast Ty elements, strongly suggests that other human cellular proteins are not absolutely required for restriction (e.g., [5,6] and reviewed in [1][2][3]).
An alternative explanation that could account for the observed permissive phenotype of the CEM-T4 line is that it is mixed, composed of a population of cells expressing low and/or variable levels of APOBEC3G. If this were the case, then the permissive phenotype could simply be due to virus replication in the subset of cells expressing low levels of APOBEC3G. To address this hypothesis, we generated subclones of the CEM-T4 line by serial dilution and determined the level of APOBEC3G expression by immunoblotting ( Figure 1A). First, we observed that CEM-T4 cells expressed levels of APO-BEC3G that were considerably lower than those in the non-permissive line CEM, regardless of whether they were obtained from the AIDS Research and Reference Reagent Program (CEM-T4-A) or directly from the Zheng laboratory (CEM-T4-Z). Second, we found that representative CEM-T4 subclones, regardless of source, expressed both low and variable APO-BEC3G levels. It is further notable that none of the subclones expressed fully nonpermissive, CEM-like APOBEC3G levels. This heterogeneity is reflected by the kinetics of Vif-deficient virus replication, with some subclones being fully permissive and others being semi-permissive (Figure 1B  [8,9]) on the indicated lines or subclones. Virus replication was monitored by mixing cell-free culture supernatant with the reporter cells CEM-GFP, and infectivity was determined by determining the percentage of GFP-positive CEM-GFP cells by flow cytometry [7,8]. doi:10.1371/journal.ppat.1000528.g001 and 1C). Nevertheless, these data demonstrated that APOBEC3G levels are both low and variable within individual cells of the CEM-T4 line, thus providing a reasonable molecular explanation for the permissive phenotype of this cell line.
Curiously, Zheng and coworkers [4] also showed that CEM-T4 cells retrovirally transduced with APOBEC3G were still permissive for Vif-deficient HIV-1 replication. This result may be due to the distinct possibility that APOBEC3G itself inactivated some of the transducing viral cDNAs. Such an attempt at complementation would probably result in a CEM-T4 line that is mixed for APOBEC3G expres-sion. To clarify this important point, we used electroporation to generate panels of HA-tagged and untagged APOBEC3Gexpressing CEM-T4 clones and used them for virus replication experiments (Figures 2  and 3, respectively).
Contrary to the findings of Zheng and colleagues [4], our APOBEC3G-expressing CEM-T4 clones were fully restrictive for Vifdeficient HIV-1 replication. The observed non-permissive phenotype was not due to gross overexpression of APOBEC3G because many of the restrictive CEM-T4 clones stably expressed APOBEC3G to levels slightly lower to those of CEM, the original parent of CEM-T4 (e.g., clone G4 in Figure 3). It is also notable that some vector control clones such as V9, like the subclones in Figure 1, expressed low and non-restrictive levels of APOBEC3G. These data suggest that cells must pass an APOBEC3G expression ''threshold'' before they become fully non-permissive for Vif-deficient HIV-1. Nevertheless, together these virus replication data clearly demonstrated that expression of exogenous APOBEC3G is sufficient to render CEM-T4 cells non-permissive for Vif-deficient HIV-1 replication. We conclude that this cell line does not require a specific endogenous APOBEC3G ''co-factor'' to restrict Vif-deficient HIV-1.
In this report, we question the obligate APOBEC3G co-factor hypothesis put forth by Zheng and colleagues [4]. Our data clearly provide an alternative explanation that-rather than lacking a co-factor for APOBEC3G-the CEM-T4 line is mixed with individual cells expressing heterogeneous and less-than-restrictive levels of APOBEC3G. Importantly, when transfections were used to generate stable APO-BEC3G-expressing CEM-T4 clones, the resulting lines were non-permissive for Vifdeficient HIV-1 replication. Based on these results, we conclude that CEM-T4 cells do not lack an APOBEC3G co-factor and, more generally, that APOBEC3G may not strictly require another cellular protein for HIV-1 restriction. However, we would like to emphasize that neither study rules out the attractive possibility that APOBEC3G, like most other proteins, is likely to be regulated by a variety of cellular processes.