HIV-1 Protease, Reverse Transcriptase, and Integrase Variation

Abstract

HIV-1 protease (PR), reverse transcriptase (RT), and integrase (IN) variability presents a challenge to laboratories performing genotypic resistance testing. This challenge will grow with increased sequencing of samples enriched for proviral DNA such as dried blood spots and increased use of next-generation sequencing (NGS) to detect low-abundance HIV-1 variants. We analyzed PR and RT sequences from >100,000 individuals and IN sequences from >10,000 individuals to characterize variation at each amino acid position, identify mutations indicating APOBEC-mediated G-to-A editing, and identify mutations resulting from selective drug pressure. Forty-seven percent of PR, 37% of RT, and 34% of IN positions had one or more amino acid variants with a prevalence of ≥1%. Seventy percent of PR, 60% of RT, and 60% of IN positions had one or more variants with a prevalence of ≥0.1%. Overall 201 PR, 636 RT, and 346 IN variants had a prevalence of ≥0.1%. The median intersubtype prevalence ratios were 2.9-, 2.1-, and 1.9-fold for these PR, RT, and IN variants, respectively. Only 5.0% of PR, 3.7% of RT, and 2.0% of IN variants had a median intersubtype prevalence ratio of ≥10-fold. Variants at lower prevalences were more likely to differ biochemically and to be part of an electrophoretic mixture compared to high-prevalence variants. There were 209 mutations indicative of APOBEC-mediated G-to-A editing and 326 mutations nonpolymorphic treatment selected. Identification of viruses with a high number of APOBEC-associated mutations will facilitate the quality control of dried blood spot sequencing. Identifying sequences with a high proportion of rare mutations will facilitate the quality control of NGS.IMPORTANCE Most antiretroviral drugs target three HIV-1 proteins: PR, RT, and IN. These proteins are highly variable: many different amino acids can be present at the same position in viruses from different individuals. Some of the amino acid variants cause drug resistance and occur mainly in individuals receiving antiretroviral drugs. Some variants result from a human cellular defense mechanism called APOBEC-mediated hypermutation. Many variants result from naturally occurring mutation. Some variants may represent technical artifacts. We studied PR and RT sequences from >100,000 individuals and IN sequences from >10,000 individuals to quantify variation at each amino acid position in these three HIV-1 proteins. We performed analyses to determine which amino acid variants resulted from antiretroviral drug selection pressure, APOBEC-mediated editing, and naturally occurring variation. Our results provide information essential to clinical, research, and public health laboratories performing genotypic resistance testing by sequencing HIV-1 PR, RT, and IN.

Antiviral activity of HIV‐1 integrase strand‐transfer inhibitors against mutants with integrase resistance‐associated mutations and their frequency in treatment‐naïve individuals

“…On average, 11.6 polymorphisms were present in the HIV‐1 IN sequence of each subject (range: 2‐30; compared with the HIV‐1 NL4‐3 reference sequence). Using a cutoff of 1%, natural polymorphisms were detected at 99 of the 288 amino acid positions of IN (Figure ), in close agreement with a report by Rhee et al that found 98 polymorphic residues using the same 1% cutoff. Of the remaining 189 residues of IN, 146 positions were polymorphic below 1% and 43 positions were fully conserved.…”

HIV-1 drug resistance and resistance testing

“…Indeed, the information present in an electropherogram provides intrinsic quality control data including the level of background “noise” as indicated by low signal intensities and numerous poorly defined peaks. In addition, a high number of electrophoretic mixtures, atypical mutations, and APOBEC mutations observed in a nucleotide sequence can signal quality control problems (Rhee et al, 2016; Shafer et al, 2000). Furthermore, phylogenetic analysis and distance measurements of sequences can be used to detect possible laboratory contamination or specimen mislabeling at the point of collection.…”

“…Indeed, more than 80 non-polymorphic PI-selected mutations have been reported (Rhee et al, 2016; Shahriar et al, 2009). The majority of these reduce in vitro susceptibility to one or more PIs (Rhee et al, 2010).…”

Viral evolution in the cell‐associated HIV‐1 DNA during early ART can lead to drug resistance and virological failure in children

“…We defined treatment‐relevant mutations as described in the IAS‐USA 2017 mutations list as signature mutations (mutations in bold) in the RT gene selected by the NRTIs (zidovudine, stavudine, tenofovir, and lamivudine) and NNRTIs (nevirapine and efavirenz), initiated in the cohort, that alone or in combination with other mutations result in substantial reduction in drug susceptibility . Signature mutations associated with host antiviral defense protein APOBEC3G and 3F mediated G‐to‐A editing of the RT gene was identified as D67N, E138K, M184I, G190S/E, and M230I . Hypermutation was defined as having > 3 characteristic mutations in the RT gene associated with APOBEC3G/F activity …”

“…40 Signature mutations associated with host antiviral defense protein APOBEC3G and 3F mediated G-to-A editing of the RT gene was identified as D67N, E138K, M184I, G190S/E, and M230I. 41 Hypermutation was defined as having > 3 characteristic mutations in the RT gene associated with APO-BEC3G/F activity. 22…”