HIV-1 drug resistance

The first HIV-1 antiretroviral drug resistance was described in 1989 by Larder et al.

when it was noted that patients become less likely to respond to AZT therapy after prolonged treatment.

Drug resistance is the reduction of drug susceptibility of a virus compared to the wild-type virus by the presence of genetic mutations. Drug resistance mutations occur in the protease and reverse transcriptase genes and can be attributed to the poor proofreading ability of the RT enzyme during the replication cycle that causes genetic variation in HIV-1. The average error rate of the enzyme is 1 error per 2000-5000 nucleotides polymerized (Menéndez-Arias, 2008) and it has been estimated that single

point mutations occurs 10 000 to 100 000 times per day in an untreated HIV-1 positive person. The number of HIV-1 variants is further increased by the extremely high rate of replication of HIV-1, up to 10¹⁰ viral particles produced per day (Coffin, 1995). This contributes to the high degree of diversity of HIV-1 during the course of an infection.

Single point mutations, deletions and insertions during the HIV-1 replication cycle lead to the development of drug resistance mutations. The size and variation of the HIV-1 population within an individual, the suppression of viral replication during antiretroviral therapy, the path by which a mutation is acquired, the effect that the mutation will have on viral fitness and the influence that the mutation will have on drug susceptibility, can all play a role in the rate and amount of drug resistant mutations that develop (Shafer, 2002).

There is a standard numbering system for drug resistance mutations based on their amino acid positions within the protease and reverse transcriptase gene. The subtype B consensus sequence is used as the reference sequence. This consensus sequence was formed from the alignment of sequences available in the HIV Sequence Database (Kuiken et al, 1999) at the Los Alamos National Laboratory. Mutations are described by using the first letter of the consensus subtype B amino acid, followed by the position of the mutation in the sequence, followed by the letter of the amino acid that indicate the mutation e.g. M184V. When a mixture of more than one amino acid is reported at one position, the different amino acid letters are written after the position separated by a forward slash e.g. M184M/V indicating a mixture of the wild-type amino acid and the mutant amino acid (Shafer, 2004).

Some resistance mutations, can on their own, cause resistance to one or more drugs, e.g. M184V, and are referred to as primary or major resistance mutations. Other mutations only cause resistance if present in combination with major resistance mutations e.g. E138A, and are refered to as secondary or minor mutations (Shafer, 2004).

The accumulation of mutations such as M41L, D67N, K70R, L210W, T215F or T215Y and K219Q or K219E can cause high level of resistance against the NRTIs.

These mutations are known as the thymidine analogue mutations (TAMs) as they are found most often when the patients are on a regimen containing AZT and d4T

(Shafer, 2004). These mutations are frequent in low-income countries, like South Africa that use thymidine analogues in the first and second line regimens (Shafer and Schapiro, 2008). There are two patterns seen for the accumulation of TAMs. The type 1 pattern includes mutations M41L, L210W and T215Y and the type 2 pattern includes mutations D67N, K70R, T215F and K219Q/E. The type 1 TAMs cause higher levels of resistance than type 2 TAMs (Shafer and Schapiro, 2008).

HIV-1 diversity also plays a role in the development of resistance mutations. It was found that naturally occurring polymorphisms in non-B HIV-1 subtype can contribute to drug resistance (Cornelissen et al, 1997). Polymorphisms in non-B subtype HIV-1 sequences also create shorter pathways for the selection of drug resistance mutations.

The pathway for the selection of the V106M mutation, that causes resistance to NNRTIs, is shorter in HIV-1 subtype C than in subtype B viruses. The polymorphism at codon 106 of GTG, found in 94% of HIV-1 subtype C sequences, can be changed to ATG with a single mutation. This polymorphism is rarely seen in the HIV-1 subtype B viruses where the codon is GTA and 2 mutations are needed for the V106M resistance mutation to develop (Brenner et al, 2003). The implications of the effect of subtype associated polymorphism on the algorithms used for drug resistance and the treatment options for patients with non-B subtypes HIV-1 still are not fully understood.

1.11.1 Protease inhibitor (PI) resistance

The PR targets the cleavage sites on the Gag and Gag-Pol polyproteins to produce the structural proteins and enzymes of the virus. More than 30 mutations are associated with PI resistance (Johnson et al, 2008). Resistance to PI’s develops due to structural changes that reduce the binding of the protease to the inhibitor.

1.11.2 Nucleoside analogue Reverse Transciptase (NRTI) resistance

The mechanism of NRTIs is to block the elongation of the proviral DNA during reverse transcription and terminate the DNA chain formation. A drug enters the host cell where it is phosphorylated and then competes with natural dNTPs for incorporation into the newly synthesised DNA chains. This incorporation terminates the elongation of the chain and inhibits the viral replication. Resistance to NRTI’s develops via two biochemical processes. One method is to decrease the incorporation of NRTI’s by the action of mutations that allow RT to discriminate between NRTI’s

and natural dNTPs during phosphorylation and thus prevent their incorporation into the newly formed DNA chain. Such discriminatory mutations are the M184V, Q151M, K65R and L74V. The second process is phosphorolytic removal of the incorporated NRTI’s from the newly formed chain to allow continued polymerisation and strand elongation. Mutations that facilitate phosphorylitic removal of incorporarated NRTIs are the TAMs, the T69 insertion and accessory mutations (Shafer, 2004, Shafer and Schapiro, 2008).

1.11.3 Non nucleoside analogue Reverse Transciptase (NNRTI) resistance The NNRTIs bind to the HIV-1 reverse transcriptase in the hydrophobic pocket located between the 6- 10- 9 and 2- 13- 14 sheets of the p66 subunit. Binding of the NNRTIs to this site displaces the catalytic aspartate residues relative to the polymerase-binding site and inhibits HIV-1 replication. Resistance arises with the development of mutations in or next to the NNRTI binding site. Only one or two mutations in this area are necessary for the development of high level resistance, therefore HIV has a low genetic barrier to the development of NNRTI resistance.

These mutations often lead to a high level of cross resistance between the different NNRTI’s. Resistance occurs due to mutations causing a reduction in the binding potential of the NNRTI’s (Y181C and Y188C/I/H), stabilizing of the RT conformation (K103N) or reduction of protein inhibitor contact (L100I, V106A and V108I) and charge repulsion (K101E, E138K) (Ren and Stammers, 2008).

Mutations for NNRTI resistance can be categorized into 4 groups: 1) major NNRTI resistance mutations that cause resistance to one or more NNRTI’s and that develop early, 2) minor NNRTI resistance mutations that occur in combination with major NNRTI resistance mutations, 3) minor non-polymorphic mutations that occur alone or in combination with other mutations that cause low-level NNRTI resistance and 4) polymorphic mutations that influence the effect of other NNRTI resistance mutations (Shafer and Schapiro, 2008).

Mutations in the envelope gene and the integrase gene that confers resistance to the entry inhibitors and the integrase inhibitors have been identified but will not be discussed as the inhibitors are not feely available in South Africa and have no bearing on the project.