Insertions in the Reverse Transcriptase Increase both Drug Resistance and Viral Fitness in a Human Immunodeficiency Virus Type 1 Isolate Harboring the Multi-Nucleoside Reverse Transcriptase Inhibitor Resistance 69 Insertion Complex Mutation

Finger insertion mutations of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) (T69S mutations followed by various dipeptide insertions) have a multinucleoside resistance phenotype that can be explained by decreased sensitivity to deoxynucleoside triphosphate (dNTP) inhibition of the nucleotidedependent unblocking activity of RT. We show that RTs with SG or AG (but not SS) insertions have three-to fourfold-increased unblocking activity and that all three finger insertion mutations have threefold-decreased sensitivity to dNTP inhibition. The additional presence of M41L and T215Y mutations increased unblocking activity for all three insertions, greatly reduced the sensitivity to dNTP inhibition, and resulted in defects in in vitro DNA chain elongation. The DNA chain elongation defects were partially repaired by additional mutations at positions 210, 211, and 214. These results suggest that structural communication between the regions of RT defined by these mutations plays a role in the multinucleoside resistance phenotype.Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is responsible for replication of the HIV-1 genome and is therefore an important target for antiretroviral therapy. Many inhibitors of HIV-1 RT are nucleoside analogues that are converted to their active triphosphate forms by host cell kinases and are incorporated into the viral genome by HIV-1 RT. Because nucleoside analogues lack a 3Ј-OH group, their incorporation prevents further extension of the DNA chain. HIV-1 RT becomes resistant to nucleoside analogues primarily through two mechanisms (8, 12): (i) increased discrimination against the compounds (9, 13, 35) leading to decreased incorporation and less chain termination and (ii) increased ability to remove chain terminators from blocked DNA chains, allowing DNA synthesis to resume (1,3,19,20,22,26). The removal occurs through excision of the 3Ј-terminal nucleotide by transfer to one of several potential acceptor substrates, including pyrophosphate (PP i ) or ATP, generating the triphosphate form of the chain terminator or a dinucleoside polyphosphate, respectively. Most zidovudine (AZT) resistance mutations, including M41L, D67N, K70R, T215F/Y, and K219Q (29), confer increased removal, compared to wildtype (WT) RT (3,19,22,25,26). The effect of these mutations is greatest when a nucleoside triphosphate, such as ATP, is used as the acceptor substrate, whereas there is little if any effect of AZT resistance mutations on removal when a nucleoside diphosphate or PP i is used as the substrate (3,16,20,22,25).The efficiency of the removal reaction under physiological conditions will depend on the rate of the reaction and the ability of deoxynucleoside triphosphates (dNTPs) to inhibit it. HIV-1 RT bound to a chain-terminated primer-template will still bind the dNTP complementary to the next nucleotide on the template strand (34) to form a stable complex. Since chemical bond formation is prevented due to lack of a 3Ј-OH, this complex has been named a dead-end...

mentioning

confidence: 99%

Effects of Dipeptide Insertions between Codons 69 and 70 of Human Immunodeficiency Virus Type 1 Reverse Transcriptase on Primer Unblocking, Deoxynucleoside Triphosphate Inhibition, and DNA Chain Elongation

Meyer

Lennerstrand

Matsuura

et al. 2003

“…The inserted dipeptide is most often SS or a variation thereof and is present in approximately 2% of NRTI-treated individuals (9,(42)(43)(44). The insert provides a certain level of drug resistance, but it also has a negative impact on viral replication fitness in the absence of drugs (37).…”

mentioning

confidence: 99%

Increased Multinucleoside Drug Resistance and Decreased Replicative Capacity of a Human Immunodeficiency Virus Type 1 Variant with an 8-Amino-Acid Insert in the Reverse Transcriptase

Hoek¹,

Back²,

Jebbink³

et al. 2005

Resistance to antiretroviral drugs is generally conferred by specific amino acid substitutions, rather than insertions or deletions, in reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1). The exception to these findings is the amino acid insertions found in the ␤3-␤4 loop of the RT enzyme in response to treatment with nucleoside reverse transcriptase inhibitors. This insert consists most commonly of two amino acids, but we describe in detail the evolution of a variant with an 8-amino-acid (aa) insert in a patient treated with zidovudine (ZDV) and 2-3-dideoxycytidine (ddC). The 24-nucleotide insert is a partial duplication of local sequences but also contains a sequence segment of unknown origin. Extensive sequence analysis of longitudinal patient samples indicated that the HIV-1 population prior to the start of therapy contained not the wild-type amino acid 215T in RT but a mixture with 215D and 215C. Treatment with ZDV and subsequent ZDV-ddC combination therapy resulted in the evolution of an HIV-1 variant with a typical ZDV resistance genotype (41L, 44D, 67N, 69D, 210W, 215Y), which was slowly replaced by the insert-containing variant (41L, 44D, insert at position 69, 70R, 210W, 215Y). The latter variant demonstrated increased resistance to a wide range of drugs, indicating that the 8-aa insert augments nucleoside analogue resistance. The gain in drug resistance of the insert variant came at the expense of a reduction in replication capacity when assayed in the absence of drugs. We compared these data with the resistance and replication properties of 133 insertcontaining sequences of different individuals present in the ViroLogic database and found that the size and actual sequence of the insert at position 69 influence the level of resistance to nucleoside analogues.

“…These sets of primers and probes allowed subtype-specific PCR amplification and hybridization so that cross-hybridization between subtype B, A, and AE probes did not occur (30). A relative fitness value for each virus in the competition was estimated by using the production of each individual HIV-1 strain in the dual infection (24,25). A total relative fitness value was calculated as the average of the two relative fitness values corresponding to the competition between each subtype B HIV-1 isolate or recombinant virus and each of the nonsubtype B HIV-1 control strains.…”

mentioning

confidence: 99%

Role of the Human Immunodeficiency Virus Type 1 Envelope Gene in Viral Fitness

Rangel

Weber

Chakraborty

et al. 2003

Self Cite

A human host offers a variety of microenvironments to the infecting human immunodeficiency virus type 1 (HIV-1), resulting in various selective pressures, most of them directed against the envelope (env) gene. Therefore, it seems evident that the replicative capacity of the virus is largely related to viral entry. In this study we have used growth competition experiments and TaqMan real-time PCR detection to measure the fitness of subtype B HIV-1 primary isolates and autologous env-recombinant viruses in order to analyze the contribution of wild-type env sequences to overall HIV-1 fitness. A significant correlation was observed between fitness values obtained for wild-type HIV-1 isolates and those for the corresponding env-recombinant viruses (r ‫؍‬ 0.93; P ‫؍‬ 0.002). Our results suggest that the env gene, which is linked to a myriad of viral characteristics (e.g., entry into the host cell, transmission, coreceptor usage, and tropism), plays a major role in fitness of wild-type HIV-1. In addition, this new recombinant assay may be useful for measuring the contribution of HIV-1 env to fitness in viruses resistant to novel antiretroviral entry inhibitors.