A first case of protease codon 35 amino acid insertion in a HIV-1 subtype B sequence detected in the Bauru Region , State of São Paulo , Brazil : case report

Amino acid insertions in the protease have rarely been described in HIVinfected patients. One of these insertions has recently been described in codon 35, although its impact on resistance remains unknown. This study presents a case of an HIV variant with an insertion in codon 35 of the protease, described for the first time in Bauru, State of Sao Paulo, Brazil, circulating in a 38-year-old caucasian male with asymptomatic HIV infection since 1997. The variant isolated showed a codon 35 insertion of two amino acids in the protease: a threonine and an aspartic acid, resulting in the amino acid sequence E35E_TD.

The principal objective of antiretroviral therapy in HIV infection is to suppress the plasma viral load to undetectable levels using combinations of drug classes 1 , which slow the progression of the infection 2 .
The antiretroviral drug selection pressure associated with a high mutation rate 3 has led to the emergence of resistant HIV variants.Resistant strains can result from amino acid changes or an insertion/ deletion in the viral sequence, leading to an alteration in enzyme kinetics or antiretroviral drug accessibility 4 .
Amino acid insertions in the protease (PR) have rarely been described in HIV-infected patients, occurring in 0.1 to 4.6% of patients naïve to or on treatment with protease inhibitors 5 .These insertions have been reported mainly in codons 17, 18, 22 to 25, 31 to 31, 35 to 38, 70, 71, 95 and 96 6 .
Insertions in codon 35 of PR have recently been reported [7][8][9] , but their impact on enzymatic activity, viral biology and resistance to protease inhibitors (PI) remains unknown 8 .These variants could show an advantage in proliferation in the presence of drug selection pressure 5,8,9 .
The purpose of this study was to report a case of an HIV variant with an insertion in codon 35 of the protease, described for the first time in Brazil, which was identified circulating in an HIV-infected patient on treatment in Bauru, State of São Paulo.
The patient is a 38-year-old caucasian male with asymptomatic HIV infection since 1997 and no history of the conditions associated with AIDS.He had been previously treated with different drug combinations (Table 1), including didanosine, estavudine, zidovudine, lamivudine and efavirenz, while nowadays using lamivudive, tenofovir, atazanavir and ritonavir.The use of protease inhibitors was initiated in 1998 and continues up to the present, although their use was interrupted for four years (between 2001 and 2005).Since diagnosis, the patient never presented with a CD4 cell count lower than 200cells/mm 3 and the plasma viral load showed wide variation.After one therapeutic scheme that was not tolerated and two that led to failure, a drug resistance test (TRUGENE® HIV-1 The viral strain was subtype B (93% similarity in protease gene), according to the Viral Genotyping Tool available at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/projects/genotyping/formpage.cgi).The sequence obtained showed a codon 35 insertion of two amino acids in the protease gene, a threonine, encoded by ACA and ACG, characterizing a dual viral population, and a single GAT sequence encoding aspartic acid, resulting in the sequence E35E_TD (Figure 1).
The mutation profile included the mutations I135T, D177E, Q197K, R211K and V245K in the reverse transcriptase gene and I13V, ins35TD, M46I, I62V, L63H, I64V and I72V in the protease gene, according to the Genotypic Resistance Interpretation Algorithm (HIVdb program) available at the Stanford University site (http://hivdb.stanford.edu/pages/algs/sierra_sequence.html).Codon 35 of the protease has recently been described as a site at which insertions can occur.A two-amino acid insertion in protease codon 35, producing the sequence ETNLNL, circulating in a PI-naive patient and in his partner, was described in 2001.This strain has shown a normal replication capacity and susceptibility to protease inhibitors 7 .In our laboratory, of the 472 infected patients tested over a three-year period, only one showed an insertion in protease codon 35, suggesting that insertions in this region are rare.The sequence obtained in our laboratory (with ins35TD) was submitted to the Blastn algorithm (Blast Basic Local Alignment Search Tool) available at NCBI Blast (http://blast.ncbi.nlm.nih.gov/Blast.cgi).A 95% identity was determined, though no perfect alignment was obtained due to the presence of the insertion.

T G A
Various plasma viral load levels and CD4 T counts have been reported for viral variants with insertions in codon 35 of the protease gene, suggesting no relation with disease progression 10 .In this study, the isolated viral variant was detected using plasma viral RNA as the source, suggesting that this strain may be stable and manageable with HAART.
The presence of the insertions ins35G and ins35TN alone were not associated with decreased drug susceptibility.In the presence of the ins35TN and resistance mutations, the replication level was lower than when the resistance mutations were present with ins35G 8 .On the other hand, studies have shown that ins33L and ins35E can favor resistance to protease inhibitors 9 .The virus circulating in our patient did not exhibit a high resistance level to any protease inhibitor, but showed intermediate resistance to nelfinavir and potentially low-level resistance to atazanavir/r, fosamprenavir/r, indinavir/r and lopinavir/r.The mutation M46I decreases susceptibility to IDV/r, NFV, FPV/r, LPV/r, and ATV/r, when present with other mutations, suggesting that the low and intermediate resistance verified could be related to the presence of the M46I mutation.However, one study suggests that the ins35TD may have been selected during protease inhibitor therapy 5 .
Viral and host conditions may be required for the emergence of insertions 5 .Our patient could not be evaluated before the initiation of therapy, and, thus, further studies of ins35TD are required to determine the factors that contribute to the selection of this variant.

FIGURE 1 -
FIGURE 1 -Part of the electropherogram of the HIV-1 protease genomic sequence, showing the insertion of two amino acids between codon 35 and 36.The first amino acid is encoded by ACR, the R represents dual population: A, in green and G, in black (see arrow) and; the other amino acid encoded by GAT.

TABLE 1 -Drugs combination used by the patient, showing the onset and end of each combination and the reasons for change.
Genotyping Test on an OpenGene® DNA Sequencing System, Siemens Healthcare Diagnostics, Deerfield, IL , USA) was performed in the Molecular Biology Laboratory of the Blood Donor Center at the Botucatu School of Medicine, São Paulo State University (Universidade Estadual Paulista, UNESP), a laboratory of the Brazilian National Network for HIV-1 Genotyping (RENAGENO).At the time of writing this paper, the patient's CD4 cell count and the plasma viral load were 415 cells/mm 3 and 3.34 log, respectively.