Report. Evolution of HIV-1 Isolates that Use a Novel Vif-Independent Mechanism to Resist Restriction by Human APOBEC3G

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1 Current Biology 18, , June 3, 2008 ª2008 Elsevier Ltd All rights reserved DOI /j.cub Evolution of HIV-1 Isolates that Use a Novel Vif-Independent Mechanism to Resist Restriction by Human APOBEC3G Report Guylaine Haché, 1 Keisuke Shindo, 1 John S. Albin, 1 and Reuben S. Harris 1, * 1 Department of Biochemistry, Molecular Biology, and Biophysics Institute for Molecular Virology Beckman Center for Genome Engineering University of Minnesota Minneapolis, Minnesota Summary The human APOBEC3G protein restricts the replication of Vif-deficient HIV-1 by deaminating nascent viral cdna cytosines to uracils, leading to viral genomic strand G-to-A hypermutations [1 4]. However, the HIV-1 Vif protein triggers APOBEC3G degradation, which helps to explain why this innate defense does not protect patients [5]. The APOBEC3G- Vif interaction is a promising therapeutic target, but the benefit of the enabling of HIV-1 restriction in patients is unlikely to be known until Vif antagonists are developed. As a necessary prelude to such studies, cell-based HIV-1 evolution experiments were done to find out whether APOBEC3G can provide a long-term block to Vif-deficient virus replication and, if so, whether HIV-1 variants that resist restriction would emerge. APOBEC3G-expressing T cells were infected with Vif-deficient HIV-1. Virus infectivity was suppressed in 45/48 cultures for more than five weeks, but replication was eventually detected in three cultures. Virus-growth characteristics and sequencing demonstrated that these isolates were still Vif-deficient and that in fact, these viruses had acquired a promoter mutation and a Vpr null mutation. Resistance occurred by a novel tolerance mechanism in which the resistant viruses packaged less APOBEC3G and accumulated fewer hypermutations. These data support the development of antiretrovirals that antagonize Vif and thereby enable endogenous APOBEC3G to suppress HIV-1 replication. Results APOBEC3G has been shown to decrease the infectivity of Vifdeficient HIV-1 by 10- to several-100-fold (e.g., [2 4, 6 9]). However, in almost all reported experiments there was clearly a population of viruses that appeared unrestricted. These apparent escapees could be attributable to low levels of virus replication in the presence of APOBEC3G, to a failure of APOBEC3G to express in some cells, and/or to assays operating near resolution limits. Regardless of the explanations, the overall and long-term potency of this important antiviral protein is unknown. To address these questions and gain further insights into the mechanism of HIV-1 restriction, we used a long-term continuous cell-culture system to ask whether Vif-deficient viruses could evolve resistance to APOBEC3G. The human T cell line *Correspondence: rsh@umn.edu CEM-SS was transfected stably with an APOBEC3G-expression construct or a vector control. APOBEC3G-expressing clones restricted the short-term spreading infection of Vif-deficient HIV-1 IIIB but not that of the Vif-proficient virus (Figure 1A and Table S1 available online; originally shown in [3]). In contrast, vector-control clones supported the replication of both viruses. It is noteworthy that the CEM-SS clones selected for these experiments expressed near-physiologic APOBEC3G levels, similar to those in the parental line CEM (nonpermissive for Vif-deficient virus replication), the unrelated T cell line H9, or activated CD4-positive primary T lymphocytes (Figure 1B and Figure S1). Two independent APOBEC3G-expressing CEM-SS clones were each split into 24 wells, infected with Vif-deficient HIV- 1 IIIB, and maintained continuously for six weeks. Cell-free supernatants were used in the periodic assaying for the presence of viable virus with the use of reporter cells, in which a Tat-activated HIV-1 LTR promoter drives GFP expression and therefore provides a quantitative measure of virus infectivity (see Supplemental Experimental Procedures). Although most cultures showed no signs of virus replication, one culture from experiment 1 and two cultures from experiment 2 began to produce virus around the five-week time point (Figure 1C). These virus isolates were called A3G-R1, A3G-R2, and A3G- R3. To eliminate cell-based explanations for the apparent resistance phenotypes (such as a loss of APOBEC3G expression), supernatants containing A3G-R1, -R2, and -R3 were purified and used to infect fresh APOBEC3G-expressing cells. All three of the virus isolates were now clearly capable of growth on APOBEC3G-expressing cells, indicating that they had evolved a strong, heritable resistance to APOBEC3G (Figure 1D). These virus-replication kinetics were similar to those of the Vif-proficient virus on APOBEC3G-expressing cells, and they were stable over a minimum of five passages, each using fresh APOBEC3G-expressing cells (Table S1). These data demonstrated that APOBEC3G is capable of providing a long-term block to Vif-deficient HIV-1 replication, which is only rarely alleviated by resistance mutation(s). To find out whether the APOBEC3G-resistant viral isolates had restored Vif function, we assessed whether the resistant isolates could replicate in two human T cell lines, CEM and H9, in which Vif is required to overcome APOBEC3G, the related restriction factor APOBEC3F, and potentially other cellular proteins (e.g., Figure 1, Figure 2, Figure S1, and refs. [3, 9]). Curiously, none of the APOBEC3G-resistant isolates replicated in CEM or H9 cells, indicating that Vif was still nonfunctional and that something other than APOBEC3G was still inhibiting virus replication (Figure 2A and Figure S1). Proviral DNA sequencing confirmed that the resistant isolates retained the original vif nonsense mutations. APOBEC3F and APOBEC3G are thought to function similarly, by gaining access to assembling HIV-1 particles and deaminating nascent cdna (e.g., [6, 9 11]). APOBEC3B and APOBEC3DE have also been implicated in HIV-1 restriction (e.g., [10, 12, 13]). To test whether any of these APOBEC3 proteins could mediate restriction of the APOBEC3G-resistant isolates, viral spreading-infection experiments were done with

2 Current Biology Vol 18 No Figure 1. Vif-Deficient HIV-1 Isolates that Resist APOBEC3G (A) Short-term spreading-infection data for Vif-deficient (circles) or Vif-proficient (inverted triangles) viruses. Throughout the manuscript, a unique symbol represents each virus, open symbols and dashed lines represent virus growth on vector-control cells (e.g., V2), and filled symbols and solid lines represent virus growth on APOBEC3-expressing cells (e.g., G1, G2). (B) An immunoblot showing APOBEC3G levels in CEM, vector-control CEM-SS clones (V1 and V2), CEM-SS ( ), and three independently derived APOBE3G-expressing CEM-SS clones (G1, G2, and G3). The same membrane was stripped and probed for Tubulin (TUB). Complementary immunoblot data comparing model cell lines to primary cells are shown in Figure S1. (C) Highlights of the long-term spreading-infection experiments for Vif-deficient viruses on vector-control- or APOBEC3G-expressing CEM-SS cell lines. Of the cultures, 45/48 showed no virus replication on APOBEC3G-expressing cells (flat lines not graphed). The three APOBEC3G-resistant isolates that eventually emerged were A3G-R1 (squares), A3G-R2 (triangles), and A3G-R3 (diamonds). (D) Spreading infections of A3G-R1, -R2, or -R3 on CEM-SS clones expressing APOBEC3G (G1, G2) or a vector control (V2). Parental viruses were also analyzed in parallel, and these data resembled those in Figure 1A (not shown). See the APOBEC3G panels in Figure S2 for complementary data and Table S1 for additional information. CEM-SS clones that expressed CEM-like levels of APOBEC3F and analogous experiments were independently done with clones that expressed HA-tagged APOBEC3G, APOBEC3F, APOBEC3B, or APOBEC3DE (Figure 2 and Figure S2). APOBEC3B or APOBEC3DE did not inhibit the replication of the Vif-deficient parental virus, let alone the APOBEC3G-resistant derivatives (Figure S2). These data suggested that neither of these is relevant to HIV-1 restriction in the CEM-SS system, but additional experiments will be necessary for determining whether these proteins influence HIV-1 in other cell types. In contrast, APOBEC3F inhibited the replication of both the Vif-deficient virus and the APOBEC3G-resistant isolates but not the Vif-proficient parent (Figure 2 and Figure S2). Inhibition was not attributable to differences in protein levels (Figure S2). Figure 2. APOBEC3G-Resistant Isolates Are Still Susceptible to APOBEC3F (A and B) Replication kinetics of the parental viruses and A3G-R1, -R2, and -R3 on CEM cells or APOBEC3F-expressing CEM-SS cells (F1 or F2). These experiments were done in parallel, so the vector-control data (V2) shown in the CEM panels are also applicable to the APOBEC3F panels. The A3G-R2 and -R3 data on APOBEC3F lines are presented in one panel to make space for Figure 2C. A3G-R1, -R2, and -R3 also spread with rapid kinetics on APOBEC3G-expressing cells (G2; not shown). The higher overall titers of the APOBEC3G-resistant viruses warranted expanded y axes. Symbol and line designations are identical to those in Figure 1. See Figure S1 and Figure S2 for complementary data. (C) An immunoblot of APOBEC3F levels in two APOBEC3F-expressing CEM-SS clones (F1 and F2), parental CEM-SS cells ( ), and two nonpermissive cell lines (H9 and CEM). The same membrane was stripped and probed for Tubulin (TUB).

3 Proof that APOBEC3G Lethally Restricts HIV Figure 3. HIV-1 Mutations that Confer Vif-Independent Resistance to APOBEC3G (A) A schematic of HIV-1 IIIB indicating the coding regions and relevant restriction sites (GenBank Accession EU541617). Asterisks denote the tandem stop mutations in vif. (B) Summaries of the types of base substitutions found in A3G-R1, -R2, and -R3 proviral DNA. Each horizontal line is a composite of four independent proviral DNA sequences from large or small PCR amplicons (solid and dashed lines, respectively). G-to-A substitutions are depicted by vertical bars, and other substitutions are depicted by closed circles. Vertical arrows highlight the common A-to-T/C and vpr-inactivating mutations. See Figure S4 for an overview of 800 kb of sequencing data. (C) Replication kinetics of molecular clone-derived viruses with the indicated genotypes. Symbol and line designations are nearly identical to those in Figure 1, the exception being that the double mutants share the same symbols as the APOBEC3G-resistant triple mutant (diamonds for A3G-R3M). See Figure S3 for supporting data, including experiments with A3G-R1M, A3G-R2M, and their double-mutant derivatives. These data therefore (1) indicated that resistance is specific to APOBEC3G and that the mechanisms of restriction by APO- BEC3F and APOBEC3G differ at some fundamental level, (2) supported the hypothesis that APOBEC3F is at least partly responsible for the inability of Vif-deficient viruses to replicate on CEM cells, and (3) discouraged most trivial explanations for APOBEC3G resistance, such as improved fitness or growth rate (given that crossresistance to APOBEC3F would probably have been observed in such scenarios). The APOBEC3G-resistance mutations were identified by sequencing of proviral DNA. Remarkably, the APOBEC3Gresistant isolates only had two types of mutations in common: an A200T or A200C (A200T[C]) noncoding transversion and a Vpr truncation mutation (Figure 3 and Table S2). It is likely that these two mutations arose independently, because the nature of these and several associated mutations suggested that both APOBEC3G-dependent and -independent mechanisms contributed to the genesis of the resistance-conferring mutations (Table S2). For inquiring whether a Vpr-truncation and an A200 transversion combined to confer resistance, these mutations were incorporated into Vif-deficient molecular clones and used for spreading-infection experiments (Figure 3C and Figure S3; an M suffix label distinguishes molecular-clone-derived viruses from original isolates). All of the molecular clones with both mutations, A3G-R1M, A3G-R2M, and A3G-R3M, spread efficiently on APOBEC3G-expressing cells, whereas viruses with either mutation alone were unable to replicate. Thus, both mutations were required for Vif-deficient viruses to overcome APOBEC3G-dependent restriction. Mammalian retroviruses use multiple strategies to escape the APOBEC3 proteins of their hosts. HIV-1 and SIV use Vif to degrade APOBEC3s, foamy viruses use Bet to inhibit APOBEC3s, and others such as HTLV, MuLV, and MPMV simply appear to exclude APOBEC3s from encapsidation (e.g., [5, 14 19]). However, the frequent G-to-A hypermutations observed during identification of the APOBEC3G-resistance mutations strongly suggested that the resistance mechanism described here would be unique. Indeed, extensive proviral DNA-sequence analyses showed that the resistant viruses were being hypermutated at a significant frequency, 2-fold lower than that of the Vif-deficient parental virus (1.5 G-to-A versus 3.3 G-to-A per kb, respectively) but still much higher than the Vif-proficient virus (0.03 G-to-A per kb) (Figure S4). These hypermutation data implied that significant but perhaps lower levels of APOBEC3G were being encapsidated. For addressing this directly, APOBEC3G encapsidation was quantified in spreading-infection experiments with CEM-SS cells expressing a catalytically defective but otherwise fully intact APOBEC3G protein that does not inhibit Vif-deficient HIV-1 (A3G-E259Q; e.g., [2, 8, 20]). Immunoblots of virionassociated proteins from the spreading-infection peaks demonstrated that the resistant molecular clones packaged 2- to 3-fold less APOBEC3G than did the Vif-deficient parent (Figure 4A and Figure S5). Moreover, the APOBEC3G-resistance phenotype and the partial-encapsidation defect were apparent in a single round of virus replication in APOBEC3Gexpressing CEM-SS cells (Figure S6 and Supplemental Discussion). Taken together, the hypermutation and packaging data demonstrated that the resistant viruses had evolved a novel tolerance mechanism for escaping APOBEC3Gdependent restriction. This mechanism may be particularly applicable to a population of viruses, given that the population clearly expands while many individual viruses are undoubtedly being restricted. Interestingly, the APOBEC3G packaging defect was also apparent in Vif-deficient A200T(C) viruses (Figure 4A). This correlated inversely with data showing that the resistant isolates and A200T(C) mutants yielded higher virus titers (Figure 4B). One way that these two results may be linked is for the A200T(C) mutation to upregulate HIV-1 transcription, which would produce more substrate for particle production and in turn cause less APOBEC3G to be packaged (i.e., more particle

4 Current Biology Vol 18 No a T200A compromised the activity of the HIV-1 LAI promoter (Figure 4C). Taken together, these data indicated that transcriptional upregulation underlies both the observed titer increases and the APOBEC3G encapsidation defect, thereby constituting an integral part of the APOBEC3G-resistance mechanism. Discussion Figure 4. A200T(C) Contributes to APOBEC3G Resistance by Enhancing HIV-1 Transcription, Increasing Virus Titers, and Diminishing APOBEC3G Encapsidation (A) Immunoblots showing the level of virus-particle-associated APOBEC3G- E259Q or p24 Gag from a spreading-infection experiment with the indicated viruses. Relative to the Vif (-) virus with an APOBEC3G/p24 Gag ratio normalized to 1 (n = 3), the Vif (-) /Vpr (-), A200T(C)/Vif (2) and A200T(C)/Vif (2) /Vpr (2) viruses had mean ratios of (n = 9), (n = 9), and (n = 8), respectively. See Figure S5 for additional quantification and Figure S6 for complementary single-cycle data. (B) Relative titers of the indicated viruses produced after a single round of replication on CEM-SS cells expressing APOBEC3G-E259Q. Each histogram bar shows the mean and SD of three independently derived supernatants (some error bars were too small to graph). The titer for the Vif-proficient virus was normalized to 1 to facilitate comparisons. (C) Expression levels of the indicated LTR-GFP reporter constructs in HEK293 cells. Each histogram bar shows the mean and SD of three independent replicas. The GFP-expression level of the A200 HIV-1 IIIB construct was normalized to 1 to facilitate comparisons. The inset depicts the LTR-GFPexpression construct, with position of nucleotide 200 marked by an asterisk. assembly effectively dilutes available cellular APOBEC3G). The A200T(C) mutation is located on the promoter proximal end of a demonstrated transcriptional-activator-binding site (DBF [21]), which supports such a mechanism. Therefore, to test whether the A200T(C) mutation triggers higher levels of transcription, we made a series of LTR-promoter-driven GFP-expression constructs containing A200, T200, or C200, transfected HEK293 cells, and monitored GFP expression with flow cytometry. The T200 and C200 constructs each caused an 8- to 10-fold increase in GFP expression (Figure 4C). It is notable that most HIV-1 isolates already have a pyrimidine at position 200 ( content/hiv-db). We therefore tested whether the reciprocal mutation would diminish promoter activity and found that Our studies demonstrate that APOBEC3G is capable of blocking the replication of Vif-deficient HIV-1 over an extended duration. Virus replication was only detectable after two mutations appeared: a noncoding A200T(C) transversion and a Vpr null mutation. The fact that significant virus evolution was required for Vif-deficient viruses to bypass restriction by APOBEC3G demonstrates that this arm of the innate immune response is capable of imposing a lethal block to virus replication. Our studies therefore provide an important proof of principle that can be appreciated with analogies drawn to HIV-1 drug resistance. The potency of APOBEC3G can now be compared to that of bona fide antiretroviral drugs such as reverse transcriptase inhibitors, given that both impose blocks to virus replication such that resistance mutations provide the only means of escape. The combination of APOBEC3G and APOBEC3F may be further likened to two antiretroviral drugs that block replication through distinct paths (e.g., nucleoside and nonnucleodside reverse transcriptase inhibitors), because resistance to APOBEC3G did not confer crossresistance to APOBEC3F. Our studies therefore provide strong additional justifications for efforts to develop Vif-neutralizing therapeutics. The novel combination of resistance mutations reported here also provides a unique opportunity to better understand the mechanism of APOBEC3G-dependent HIV-1 restriction. It is remarkable that APOBEC3G did not select for a restoration of Vif function. This strongly indicates that Vif has other, potentially more important, roles in the biology of HIV-1 and related lentiviruses. Together with prior work [6, 9 11], our studies suggest that at least one of those roles will be APOBEC3F neutralization. Our data indicate that the A200T(C) mutation contributes to APOBEC3G resistance by elevating LTR transcription, increasing virus production, and ultimately causing less APO- BEC3G encapsidation (Figure S7). Diminished APOBEC3G levels thereby contribute to an overall shift from lethal to sublethal levels of restriction (i.e., a shift to tolerable APOBEC3G levels). This suggests that anything the virus does to minimize APOBEC3G packaging is likely to prove beneficial. Some viruses, like HTLV, MuLV, and MTMV appear to have taken this to the extreme by simply excluding APOBEC3G from particles [14 17]. A Vpr-inactivating mutation was also required for Vif-deficient HIV-1 to evolve resistance to APOBEC3G. Vpr-inactivation was not observed in Vif-deficient viruses that replicated on non-apobec3g-expressing CEM-SS cells (not shown). These unexpected results suggested that Vpr might actually facilitate APOBEC3G-dependent restriction by, for instance, packaging a cellular factor (Figure S7). Such a model is appealing because Vpr is abundant in virions and it has been shown to interact with many proteins (e.g., uracil DNA glycosylase UNG2, Vpr-binding protein 1, and others; reviewed in [22]). However, the most obvious Vpr-binding candidate, UNG2, is not involved in APOBEC3G-dependent restriction [7, 8, 23, 24]. Much additional work will therefore be necessary for the

5 Proof that APOBEC3G Lethally Restricts HIV identification of this putative restriction cofactor and for the testing of other tenable models. In addition to its clear relevance to HIV-1, the novel tolerance mechanism described here has several broad implications. It highlights the distinct possibility that some retroviruses might have evolved to coexist with and possibly even benefit from the mutagenic activity of the APOBEC3 proteins. For example, the majority of reverse-transcribing viruses and transposons lack obvious APOBEC3-neutralizing factors such as Vif or Bet (e.g., [5, 18, 19]). Moreover, it is unlikely that all remaining retroelements escape restriction by HTLV-, MuLV-, or MPMVlike avoidance mechanisms (e.g., [14 17]). Thus, on the basis of current data, the APOBEC3G resistance by tolerance mechanism described here might well apply to many viruses. Indeed, G-to-A hypermutations are evident in multiple viruses, such as other lentiviruses, the hepadnavirus HBV, the gammaretrovirus SNV, the endogenous murine retrovirus Pmv, and the DNA tumor virus HPV (e.g., [25 29]). Therefore, the ability to tolerate and perhaps even regulate APOBEC3-dependent restriction has clear implications for virus evolution, immune escape, and drug resistance. Supplemental Data Experimental procedures, a supplemental discussion, two tables, and seven figures are included with this article online at com/cgi/content/full/18/11/819/dc1/. Acknowledgments We thank P. Bieniasz, J. Coffin, S. Goff, A. Haase, P. Hackett, L. Mansky, V. Planelles, and P. Southern for valuable feedback; M. Malim, J. Lingappa, and M. Stevenson for key reagents; and M. Hertzberg and L. Mansky for the generous provision of facilities. These studies were supported by grants from the National Institutes of Health (AI064046), the Campbell Foundation, and the University of Minnesota (Grant-In-Aid and Academic Health Center Faculty Research Development Awards). G.H., J.S.A., and R.S.H., respectively, were supported in part by a Canadian Institutes of Health Research predoctoral studentship, a Medical Scientist Training Program grant (T32 GM008244, with supplemental support from the Mayo Foundation), and a Searle Scholarship. The University of Minnesota Advanced Genetic Analysis Center assisted with DNA sequencing, and the Minnesota Supercomputing Institute provided computational support. Received: December 7, 2007 Revised: April 18, 2008 Accepted: April 25, 2008 Published online: May 22, 2008 References 1. Harris, R.S., Bishop, K.N., Sheehy, A.M., Craig, H.M., Petersen-Mahrt, S.K., Watt, I.N., Neuberger, M.S., and Malim, M.H. (2003). DNA deamination mediates innate immunity to retroviral infection. 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