Mutations in the HIV-1 envelope glycoprotein can broadly rescue blocks at multiple steps in the virus replication cycle Rachel Van Duynea, Lillian S. Kuoa,1, Phuong Phama, Ken Fujiia,2, and Eric O. Freeda,3 aVirus–Cell Interaction Section, HIV Dynamics and Replication Program, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702 Edited by Joseph G. Sodroski, Dana-Farber Cancer Institute, Boston, MA, and accepted by Editorial Board Member Stephen P. Goff March 19, 2019 (received for review November 29, 2018) The p6 domain of HIV-1 Gag contains highly conserved peptide efficient mode of viral propagation than cell-free infection, is motifs that recruit host machinery to sites of virus assembly, thereby initiated by interactions between Env expressed on the surface of promoting particle release from the infected cell. We previously the infected cell and CD4 on the surface of the target cell, in the – reported that mutations in the YPXnL motif of p6, which binds the absence of cell cell fusion, inducing the formation of a virological host protein Alix, severely impair HIV-1 replication. Propagation of synapse (VS) (27). Alternatively, when cell-surface HIV-1 Env en- the p6–Alix binding site mutants in the Jurkat T cell line led to the gages CD4 on target cells, cell fusion can occur, resulting in the emergence of viral revertants containing compensatory mutations formation of multinucleated cells, or syncytia. Several studies have not in Gag but in Vpu and the envelope (Env) glycoprotein subunits demonstrated the importance of cell-to-cell transmission in vitro in gp120 and gp41. The Env compensatory mutants replicate in Jurkat overcoming barriers to cell-free infection, including target cell T cells and primary human peripheral blood mononuclear cells, de- infectability, virus stability, and defects in virus production (28–30). spite exhibiting severe defects in cell-free particle infectivity and Env- Additionally, cell-to-cell transmission can allow HIV-1 spread in the mediated fusogenicity. Remarkably, the Env compensatory mutants presence of broadly neutralizing antibodies (bNabs) (31). Finally, can also rescue a replication-delayed integrase (IN) mutant, and ex- cell-to-cell transmission of HIV-1 has been shown to be less sensitive hibit reduced sensitivity to the IN inhibitor Dolutegravir (DTG), dem- to antiretrovirals (ARVs) compared with cell-free transmission (29, onstrating that they confer a global replication advantage. In 32–35). The ability of the virus to evade blocks to infection may in addition, confirming the ability of Env mutants to confer escape from part be attributed to a higher multiplicity of infection (MOI) during DTG, we performed de novo selection for DTG resistance and ob- cell-to-cell vs. cell-free infection, allowing for a higher percentage of served resistance mutations in Env. These results identify amino acid cells to be infected with more than one virus (36). These findings substitutions in Env that confer broad escape from defects in virus raise the intriguing possibility that HIV-1 could potentially escape replication imposed by either mutations in the HIV-1 genome or by an the inhibitory activity of antiviral agents through the acquisition of – antiretroviral inhibitor. We attribute this phenotype to the ability of mutations in Env that promote highly efficient cell cell transmission. the Env mutants to mediate highly efficient cell-to-cell transmission, We have previously shown that mutations in the Alix binding resulting in an increase in the multiplicity of infection. These findings site of p6 induce relatively minor defects in Gag processing, virus have broad implications for our understanding of Env function and release, and cell-free particle infectivity, but impose significant the evolution of HIV-1 drug resistance. delays in replication kinetics in physiologically relevant cell types drug resistance | cell–cell transmission | Dolutegravir | virological synapse Significance he assembly of HIV type 1 (HIV-1) particles is driven by the HIV-1 adapts over time to bypass blocks imposed by genetic Texpression of the viral Gag polyprotein precursor, Pr55Gag, lesions in the viral genome, typically by acquiring compensa- which contains several major structural domains required for tory mutations in the defective gene itself. Here we report that virus-like particle production, including the p6 domain that HIV-1 can evade replication blocks by acquiring mutations in promotes membrane scission to release budding virions (1–4). the envelope (Env) glycoprotein that enhance cell-to-cell HIV-1 p6 encodes two highly conserved peptide motifs, known as transmission. We identified mutations in Env that arose in “late domains,” that recruit components of the cellular endosomal the presence of the antiretroviral inhibitor Dolutegravir, thereby circumventing restriction. These data, which demon- sorting complexes required for transport (ESCRT) machinery to – sites of virus assembly (5–7). The physiological function of the strate that mutations in Env can provide escape from an anti HIV-1 drug in vitro, could have broad implications for HIV-1 drug ESCRT apparatus is to drive membrane-scission reactions that resistance and viral transmission. occur in a variety of cellular contexts, including the biogenesis of – multivesicular bodies and cytokinesis (5 7). The Pro-Thr/Ser-Ala- Author contributions: R.V.D., L.S.K., K.F., and E.O.F. designed research; R.V.D., L.S.K., P.P., Pro (PT/SAP) motif of p6 interacts directly with the ESCRT-I and K.F. performed research; R.V.D., L.S.K., P.P., and K.F. analyzed data; and R.V.D. and subunit Tsg101 (8–14); the Tyr-Pro-Xn-Leu (YPXnL, where X is E.O.F. wrote the paper. any residue and n = 1–3 amino acids) motif of p6 binds the ESCRT- The authors declare no conflict of interest. associated protein Alix (15–21). While the requirement for the p6– This article is a PNAS Direct Submission. J.G.S. is a guest editor invited by the Tsg101 interaction in HIV-1 release is well established, the physi- Editorial Board. ological role of p6–Alix binding is less well defined. Published under the PNAS license. Expression of Gag alone is sufficient for the formation of 1Present address: Division of AIDS, National Institute of Allergy and Infectious Diseases, virus-like particles, but the incorporation of the HIV-1 envelope Bethesda, MD 20892. (Env) glycoprotein complex is required for the generation of 2Present address: Neurovirology Project, Tokyo Metropolitan Institute of Medical Science, infectious particles. Env expression on the membranes of both 156-8506 Tokyo, Japan. free virions and infected cells promotes viral spread. Productive 3To whom correspondence should be addressed. Email: [email protected]. viral transmission from infected to uninfected cells can occur via This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. two pathways: cell-free infection or cell-to-cell transmission (22– 1073/pnas.1820333116/-/DCSupplemental. 26). The latter pathway, which is thought to be a more rapid and Published online April 11, 2019. 9040–9049 | PNAS | April 30, 2019 | vol. 116 | no. 18 www.pnas.org/cgi/doi/10.1073/pnas.1820333116 Downloaded by guest on October 1, 2021 (37). To further characterize the significance of p6–Alix interac- structed pNL4-3 p6/Env and p6/Vpu mutant clones and evalu- tions, we selected for viral revertants that alleviate the replication ated their replication kinetics, in parallel with WT and the defects imposed by a panel of mutations in the p6 YPXnLmotif. original p6-mutant clones, in Jurkat cells. The Vpu-inactivating We identified second-site compensatory changes in both Vpu and mutations partially rescued the replication-defective p6-Y36S/L44R Env that rescue replication defects imposed by the mutations in p6. and p6-L41A mutants (SI Appendix,Fig.S1C and D). The p6-Y36A The three Env compensatory mutations that arose can rescue virus replication defect was largely rescued by both Env-P81S and Env- replication despite exhibiting severe defects in cell-free particle in- A556T (Fig. 2A). Similarly, the replication defects of both p6-Y36S/ fectivity. Strikingly, these Env mutations also provide a replication L44H and p6-L41R were rescued by Env-A556T, with reverse- advantage in the context of an integrase (IN) mutant and in the transcriptase (RT) peaks occurringatornearthedayofpeakRT presence of the IN strand-transfer inhibitor (INSTI) Dolutegravir for the WT (Fig. 2 B and D, respectively). In contrast, the replication (DTG). De novo selection in the presence of DTG led to the defective p6-Y36A substitution was not rescued by Env-A327T (SI acquisition of at least one additional Env mutation that confers Appendix,Fig.S1B) and the p6-Y36S/L44R mutations were not cell-line–independent resistance to DTG in vitro. We attribute rescued by Env-I744V or Env-R786K (SI Appendix,Fig.S1C). The the decreased DTG sensititivity of the Env mutants to their ability delay in replication exhibited by p6-L41A was rescued by Env-Y61H, to efficiently transmit viral material in a cell-associated manner, but not by Env-R166I (Fig. 2C and SI Appendix,Fig.S1D). Because resulting in an increased MOI during spreading infections. the Env mutations R166I, A327T, I744V, and R786K did not con- tribute to rescue of the p6 mutants, they were not analyzed further. Results Similarly, considering that Vpu mutations often arise during propa- p6–Alix-Binding Site Mutants Acquire Second-Site Mutations in Vpu gation of replication-defective HIV-1 mutants in Jurkat cells, we and Env. To further characterize the role of the p6–Alix in- elected to focus on the Env compensatory mutations. teraction in HIV-1 replication, we propagated the p6 mutants (Fig. 1A, Left) in culture to select for viral revertants. The Jurkat T cell Env Compensatory Mutants Display Highly Efficient Replication line was transfected with pNL4-3 WT and p6 mutant proviral clones Kinetics in Jurkat T Cells and Peripheral Blood Mononuclear Cells and virus replication was monitored over time.