Pablo Ferrer Campos

PhD Biochemistry, Clinic Hospital Universidad de Chile Molecular Medicine Laboratory, Section of Immunology, HIV and Allergies Department of Internal Medicine, Avenue Carlos Lorca Tobar 999, Independencia.

*Corresponding Author: Pablo Ferrer Campos, PhD Biochemistry, Clinic Hospital Universidad de Chile Molecular Medicine Laboratory, Section of Immunology, HIV and Allergies. Department of Internal Medicine, Avenue Carlos Lorca Tobar 999, Independencia.

Abstract
APOBEC3G is a cellular antiviral protein able to inhibit HIV replication and may protect from progression to AIDS. In this work, we showed the APOBEC3G activity in an asymptomatic HIV patient under therapy without complete viral suppression. The patient was a homosexual Chilean man with an initial CD4 count of 427 cells/mm³ and a viral load of 33,000 copies/mL. In March 2012 began therapy with ABC, 3TC, and EFV. Seven months later, the viral load was < 40 copies/mL. However, because of persistent low-level viremia reverse transcriptase (RT) and protease genes were investigated by PCR and sequencing using HIV proviral DNA. The sequences obtained were used for the resistance report and M184I and M230I mutations were found. M184I mutation causes high-level resistance to FTC and 3TC and low-level resistance to ABC. M230I was associated with low-level resistance to EFV, DOR, and ETR and intermediate resistance to NVP and RPV.

Surprisingly we detected three stop codons in positions 88, 153, and 212 that correspond to the tryptophan codon in the RT. In our patient, the three-stop codon detected at the tryptophan codon of RT suggests an APOBEC 3G hypermutation activity. These stop codons within RT of HIV are deleterious to replication and this could explain the persistent low viral load. In conclusion, using proviral DNA we detected APOBEC3G activity in an HIV patient which showed controllers. Finally, we also showed that proviral DNA is very useful in detecting resistances mutations in cases of low-level viremia and this allowed an earlier therapeutic intervention in the patient.

Keywords: HIV-1, APOBEC3G, cure, proviral DNA, resistance, stop codon, case report, endogenization HIV

Introduction
APOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like editing complex 3G) are enzymes important in both innate and adaptive immune responses against diverse kinds of viruses including HIV-1 [1]. APOBEC3G has the ability to change to A the G nucleotide present in the viral genome of HIV. Due to a G to A substitution in the TGG codon that coding for amino acid Tryptophan (W) APOBEC3G activity results in the introduction of a stop codon in all cases impairing the function of viral protein and HIV replication [2]. Thus, APOBEC3G reduces the viral load in infected patients conferring protection against progression to AIDS [3]. This strategy is an ancestral mechanism of retrovirus endogenization in humans and animals [4].

Material and Methods
The patient
In November 2011, a 29 years-old homosexual Chilean, was diagnosed HIV-positive with a CD4 cell count of 427 cells/mm³, HIV-RNA viral load (VL) of 33,000 copies/mL, and platelet count of 42,000 cells/mm³. This patient was not infected with other pathogens. At the time of diagnosis, this patient was under treatment with testosterone as a consequence of a previous testicular cancer treated by a surgical approach (2009). In March 2012, he began antiretroviral therapy (ART) with Abacavir (ABC), Lamivudine (3TC), and Efavirenz (EFV) because of the low CD4 cell count and the persistent thrombocytopenia. The clinical evolution was followed by five years with periodical medical checks at least once by year. The antiviral treatment reduces the viral load by 825-fold (2.9 log), achieving an undetectable status, however, this condition was not permanent along with the treatment, increasing progressively the VL, reaching a persistent low-level viremia (LLV) likely due to drug interaction between testosterone and EFV. Despite the LLV, the patient shows a low count of CD4 across all the treatments (Table 1). This work is part of Project AOIC#684/14. The patient was informed of the study’s scope and test limitations through pre-and post-assays counseling and voluntarily agreed to participate signing the informed consent approved by the Ethics Committee of the Hospital Clinico Universidad de Chile.

Table 1: Diagnosis, treatments, CD4 cell count and viral load evolution of the patient.

Abbreviations
HAART: Highly Active Antiretroviral Therapy Treatment.
ABC: Abacavir.
ATV/r: Atazanavir/ritonavir.
AZT: zidovudine.
EFV: Efavirenz.
3TC: Lamivudine.
TDF: Tenofovir.

Viral load assay
Determination of viral load was realized by Real-time type 1 (HIV-1) HIV assay (Abbott Diagnostics) that allows the quantification of HIV-1 RNA in plasma samples from infected patients, in the range of 40 to 10,000,000 copies/mL. It detects and quantifies the viral load of a large group of viruses, group M (subtypes A-H), group O, and group N. A viral load < 40 copies/mL is considered undetectable.

CD4 cell count
CD4 cell count and (%) was realized using blood/EDTA with The AQUIOSTM CL flow cytometer from Beckman Coulter.

HIV Resistance Test by PCR and Sequencing
To characterize if LLV is the consequence of HIV drugs resistance mutations (DRMs) that confer resistance against ART, we carried out an HIV resistance test. Total DNA human genomic (HIV proviral DNA is integrated into Human DNA) was extracted from 200 µL of fresh whole blood using QIAamp® DNA Mini Kit (Qiagen). Then full HIV reverse transcriptase (RT) and protease genes were amplified and sequenced. Amplifications were performed in duplicated (short and long fragments) by nested PCR using 5 µL of HIV proviral DNA [5]. Both PCR products were sequenced directly in Macrogen USA Co according to the method described previously and DRMs were identified using Stanford Database [6,7].

Results
The analysis of the HIV resistance test allowed identifying two mutations (M184I and M230I) in the coding region of the RT gene. Mutation M184I was associated with resistance to the nucleoside reverse transcriptase inhibitors (NRTIs): 3TC and FTC (high level) and ABC (low level), while mutation M230I to non-nucleoside reverse-transcriptase inhibitors (NNRTIs): EFV and ETR (low level), and NVP and RVP (intermediate level) (Table 2). Additionally, in the RT gene, we detected three mutations in the codon that codes for the amino acid tryptophan (W, in the nomenclature of one letter). A nonsense mutation is the substitution of a single base pair that leads to the appearance of astop codon where previously there was a codon specifying an amino acid. The presence of this premature stop codon results in the production of a shortened and likely nonfunctional protein. The three-stop codon was detected in the positions W88*, W153*, and W212*. This suggests the participation of APOBEC3G in the hypermutation observed in the RT gene. The muted HIV RT sequence was deposited in GenBank with accession number KX530453 (Figure 1). Then to reduce the VL, we changed the ART to Zidovudine (AZT)+Tenofovir (TDF)+ Atazanavir/ritonavir (ATV/r). Four months after we evaluated the VL, being < 40 copies/mL.

Table 2: Drugs resistance interpretation in Reverse Transcriptase HIV-1

RT comments (Standford University HIV Drug Resistance Database) (7)
NRTI
M184V/I cause high-level in vitro resistance to 3TC and FTC and low-level resistance to ddI and ABC. However, M184V/I are not contraindications to continued treatment with 3TC or FTC because they increase susceptibility to AZT, TDF and d4T and are associated with clinically significant reductions in HIV-1 replication.

NNRTI
M230I is an extremely rare mutation selected by RPV. Its effects on NNRTI susceptibility have not been well studied. It also often occurs because of APOBEC-mediated G-to-A hypermutation resulting in viruses that are likely to be noninfectious.

Dosage Considerations
This virus is predicted to have intermediate-level reduced susceptibility to RPV. The use of the combination of CAB/RPV should be contraindicated.

The three-stop codon were detected in the positions W88S*, W153L*, and W212S*. This suggests the participation of APOBEC3G in the hypermutation observed in the RT gene.