Use of a novel assay based on intact recombinant viruses expressing green (EGFP) or red (DsRed2) fluorescent proteins to examine the contribution of pol and env genes to overall HIV-1 replicative fitness

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Abstract

Multiple studies have described a reduction in the replicative fitness of HIV-1 isolates harboring mutations that confer resistance to antiretroviral drugs. Contradictory results, however, have been obtained depending on the methodology used in each study (Quinones-Mateu, M.E., Arts, E.J., 2002. Fitness of drug resistant HIV-I: methodology and clinical implications. Drug Resist. Update 5, 224–233), affecting our understanding of the potential relationship of viral replicative fitness with HIV-1 disease. It has been demonstrated previously that both pol and env genes play a major role in HIV-1 replicative fitness of clinical isolates. Therefore, measuring clinically relevant replicative fitness using recombinant viruses where a single mutation and/or viral gene have been introduced does not seem like a reasonable approach in this era of multi-target antiretroviral therapy. A novel method was developed to measure HIV-1 replicative fitness based on recombinant viruses expressing the enhanced green fluorescent (EGFP) or the Discosoma sp. red fluorescent (DsRed2) proteins in a HIV-1NL4-3 backbone. Contrary to previous designs to analyze HIV-1 fitness, these replication competent viruses were created in an intact viral genetic background (without deleting or affecting the expression of any viral gene). This new system was used to evaluate the contribution of drug-resistance mutations in the pol and env genes to overall viral replicative fitness (in the presence and absence of drug pressure) using direct growth competition experiments. Mutations in pol showed a stronger effect on HIV-1 replicative fitness than mutations in the env gene associated with resistance to enfuvirtide, corroborating the plasticity of the later gene to accept mutations and the sensibility of the protease and reverse transcriptase enzymes to drug-associated primary mutations. In conclusion, a new protocol was used to measure HIV-1 replicative fitness in either the presence or absence of antiretroviral drugs, which may be used as a high-throughput assay to help us understand the clinical significance of viral fitness.

Introduction

All currently available classes of antiretroviral drugs, i.e. protease (PI), reverse transcriptase (RTI), and fusion inhibitors (FI) can select for mutations within the target gene that confer high-level drug resistance (Gallant et al., 2003, Menendez-Arias and Este, 2004). Although these mutations result in an increased ability of the variant to replicate in the presence of drug, they often reduce the in vitro replicative fitness of the virus compared with wild-type strains in the absence of drug pressure (Quinones-Mateu and Arts, 2001). Many studies have examined the potential relationship of HIV-1 replicative fitness with plasma viral load, drugs resistance and disease progression [reviewed in Buhler et al. (2001), Nijhuis et al. (2001), Quinones-Mateu and Arts (2001), Quinones-Mateu and Arts (2002)]. Most of them, however, have used different methods to measure viral replicative fitness in vitro (e.g. catalytic activity of HIV-1 enzymes, viral growth kinetics, single-cycle infection assays, and growth competition experiments), which thwart comparisons of fitness values obtained in different studies (Clavel et al., 2000, Nijhuis et al., 2001, Quinones-Mateu and Arts, 2001, Quinones-Mateu and Arts, 2002).

A single-cycle infection assay developed as a variant of the Phenosense™ phenotypic assay (ViroLogic) is currently one of the methods most frequently used to estimate HIV-1 replication capacity (Barbour et al., 2004, Bates et al., 2003, Deeks et al., 2001, Deeks et al., 2003, Petropoulos et al., 2000, Wrin et al., 2001). This technique, although usually comparable to in vitro dual infections (Prado et al., 2002, Prado et al., 2005, Weber et al., 2003b), is not able to detect small differences in viral replication, which are clearly better discriminated using growth competition experiments (Abraha et al., 2005, Arien et al., 2005, Domingo et al., 1999, Holland et al., 1991, Quinones-Mateu et al., 2000, Rangel et al., 2003, Weber et al., 2003b). However, the main attribute that renders growth competitions as the gold standard to estimate viral fitness in vitro (i.e. the ability to compete two viruses in a single environment), makes it more laborious and difficult to analyze than single-cycle or growth kinetics experiments. For example, to distinguish two different HIV-1 strains in a dual infection the competing viruses need to be identified by specific characteristics. Original studies on fitness of RNA viruses were based on growth competitions followed by sequencing of specific genomic regions (Clarke et al., 1994, Duarte et al., 1992, Novella et al., 1995). Initially used on HIV-1 fitness studies (Croteau et al., 1997, Harrigan et al., 1998, Imamichi et al., 2000, Martinez-Picado et al., 1999a, Martinez-Picado et al., 2000, Sharma and Crumpacker, 1997), this tedious methodology was replaced by more rapid and efficient techniques to estimate the frequency of the two viruses in the competition, e.g. heteroduplex tracking assay (Nelson et al., 2000, Quinones-Mateu et al., 2000, Resch et al., 2001, Yuste et al., 1999) or real-time PCR (Collins et al., 2004, De Ronde et al., 2001, Lu and Kuritzkes, 2001, Weber et al., 2003b).

More recently, recombinant viruses carrying specific markers (i.e. reporter genes) have proved to be useful for assessing the effect of drug resistance mutations on viral replicative fitness in a similar genetic background, eliminating the confounding effects of mutations or polymorphisms located in other genomic regions. More important, these reporter genes facilitate the detection and quantitation of the two viruses in growth competition experiments. A variety of markers have been used in recombinant viruses, some of them aimed at measuring HIV-1 replicative fitness, for example: chemiluminescent markers such as firefly and Renilla luciferase (Deeks et al., 2001, Mo et al., 2004, Petropoulos et al., 2000), fluorescent proteins (Kutsch et al., 2002, Neumann et al., 2005, Zhang et al., 2004), or combinations of reporter genes such as Salmonella typhimurium histidinol dehydrogenase and human heat-stable placental alkaline phosphatase (Lu and Kuritzkes, 2001) or murine CD24 receptor and Thy 1.2 reporter (Ali et al., 2003). However, all these approaches have in common the deletion of a viral gene to facilitate the cloning and expression of the reporter gene. Although perhaps not relevant for studying the effect of specific mutations in a single genetic backbone, it is possible that altering the expression of certain viral genes may affect intra-genomic interactions (i.e. epistasis) important for overall HIV-1 replicative fitness.

In this study we have developed and validated a novel system to quantify HIV-1 replicative fitness in vitro based on recombinant viruses expressing the EGFP or DsRed2 fluorescent genes without affecting the expression of any HIV-1 gene. This method allowed the analysis of replicative fitness of viruses pseudotyped simultaneously with pol and env sequences using growth competition experiments in the presence and absence of antiretroviral drugs. Direct pairwise competitions based on this new assay showed that drug-resistance associated mutations in the pol gene seem to have a greater effect on viral replicative fitness than mutations in the env gene known to confer resistance to the fusion inhibitor enfuvirtide.

Section snippets

Plasmids, cells, and viruses

Plasmids p83-2 and p83-10, containing the 5′-half (i.e. 5′ LTR, complete gag, pol, vif coding regions and part of the vpr gene) and the 3′-half (i.e. partial vpr and complete tat, rev, vpu, env, nef genes, as well as the 3′ LTR), respectively, of the HIV-1NL4-3 genome were obtained through the AIDS Research and Reference Reagent Program (ARRRP), Division of AIDS, NIAID, NIH from Dr. Ronald Desrosiers and Mr. Dean Regier. MT4, MT2, PM1, Jurkat, TZM-bl, and U87.CD4.CXCR4 cells were obtained from

HIV-1 EGFP- or DsRed2-tagged viruses are intact and able to multiple rounds of replication

Different replicative competent viruses carrying a marker, either fluorescent or chemiluminescent, have been created by deleting a non-essential gene in the HIV-1 genome (Ali et al., 2003, Deeks et al., 2001, Lu and Kuritzkes, 2001, Mo et al., 2004, Neumann et al., 2005, Petropoulos et al., 2000, Zhang et al., 2004). Thus, one of the main objectives of this study was to develop a novel assay to quantify HIV-1 replicative fitness based on intact recombinant viruses expressing two different

Discussion

In vitro assays using HIV-1 isolates or recombinant viruses provide useful models for viral replication in a fixed environment and for the behavior of drug-resistant variants. As established with other RNA viruses, the “gold standard” assay to measure viral replicative fitness is based on growth competition experiments (Quinones-Mateu and Arts, 2001, Quinones-Mateu and Arts, 2002). However, growth competition assays are usually complex and expensive, which limit clinical HIV-1 fitness assays to

Acknowledgements

Work at M.E.Q-M laboratory was supported by research grants NIH-HL-67610, NIH-DE-015510, NIH-AI-36219 (Center for AIDS Research at Case Western Reserve University), and unrestricted research grants from Agouron Pharmaceuticals, Inc—A Pfizer Company. M.C. was supported by a Short-Term Fellowship from Fogarty International Center/NIH grant through the AIDS International Training and Research Program. J.M-P was supported by contract 99/3132 from the Spanish Ministry of Health, and research grants

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    This research was presented in part at the XIII International HIV Drug Resistance Workshop: basic principles and clinical implications, Tenerife, Canary Islands, Spain, June 8–12, 2004.

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