**1. Introduction**

Since the introduction of combination antiretroviral therapy (cART) against HIV-1 in the late 1990s, the number of overall new HIV infections and AIDS-related deaths has decreased [1]. Despite this, the development of resistance to antiretrovirals (ARVs) remains a barrier to the treatment of HIV infection, especially in low- and middle-income countries, and work is ongoing to produce drugs that target novel steps of the viral life cycle. One of the later classes of drugs to be approved for clinical use targets the integration of the virus into the host cell genome, specifically the DNA strand transfer reaction [2,3]. To date, there are five licensed integrase strand transfer inhibitors (INSTIs), raltegravir (RAL), elvitegravir (EVG), dolutegravir (DTG), bictegravir (BIC) and cabotegravir (CAB), that are available singly or as combination pills with RTIs [4].

Originally, RAL was used as part of a second-line treatment or salvage regimen for those failing previous lines of therapy [5]. However, InSTIs are now recommended for use as a third agen<sup>t</sup> in a nucleos(t)ide reverse transcriptase inhibitor (NRTI)-based first-line therapy [6,7]. Thus, it is highly likely that patients going onto InSTI-containing treatment will develop alongside or harbour viruses containing protease inhibitor (PI) and RTI resistance mutations. However, little is known about the full effects and coevolution of drug

**Citation:** Martin, S.A.; Cane, P.A.; Pillay, D.; Mbisa, J.L. Coevolved Multidrug-Resistant HIV-1 Protease and Reverse Transcriptase Influences Integrase Drug Susceptibility and Replication Fitness. *Pathogens* **2021**, *10*, 1070. https://doi.org/10.3390/ pathogens10091070

Academic Editors: Lawrence S. Young and Jochen Bodem

Received: 23 June 2021 Accepted: 12 August 2021 Published: 24 August 2021

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**<sup>\*</sup>** Correspondence: tamyo.mbisa@phe.gov.uk

resistance in the full-length HIV-1 *pol* gene, the target of the four main classes of ARVs used in cART: PIs, NRTIs, non-nucleoside reverse transcriptase inhibitors (NNRTIs) and InSTIs. This is because routine genotypic resistance testing in HIV-infected patients normally involves standard "bulk" population sequencing of viral genes of interest; usually protease (PR) and the N terminal of RT, or the catalytic core domain of integrase (IN) separately, or, alternatively, short read (75 to 300 bp) next-generation sequencing is used [8,9]. In addition, most phenotypic drug susceptibility assays have been developed to study the drug susceptibility of smaller patient-derived *gag-pol* fragments [10–13]. A limited number of studies have investigated the drug susceptibility of patient-derived full-length *pol* genes [14–16]. Consequently, not much is known about the effects of different combinations of PI, RTI and InSTI resistance mutations on overall drug susceptibility and viral fitness. These data may be important in informing how best to use drugs targeting all three genes in clinical practice. Here, we use a full-length *pol* single-genome sequencing assay to investigate the development, evolution and linkage of PI, RTI and InSTI resistance mutations in a patient undergoing InSTI-containing therapy. We also investigate the effect on InSTI susceptibility and viral fitness of patient-derived *IN* only or in combination with coevolved *PR* and *RT*, using a single-replication-cycle drug susceptibility assay.
