Effects of Korean Red Ginseng and HAART on vif Gene in 10 Long-Term Slow Progressors over 20 Years: High Frequency of Deletions and G-to-A Hypermutation

To investigate if Korean red ginseng (KRG) affects vif gene, we determined vif gene over 20 years in 10 long-term slowly progressing patients (LTSP) who were treated with KRG alone and then KRG plus HAART. We also compared these data with those of 21 control patients who did not receive KRG. Control patient group harbored only one premature stop codon (PSC) (0.9%), whereas the 10 LTSP revealed 78 defective genes (18.1%) (P < 0.001). The frequency of small in-frame deletions was found to be significantly higher in patients who received KRG alone (10.5%) than 0% in the pre-KRG or control patients (P < 0.01). Regarding HAART, vif genes containing PSCs were more frequently detected in patients receiving KRG plus HAART than patients receiving KRG alone or control patients (P < 0.01). In conclusion, our current data suggest that the high frequency of deletions and PSC in the vif gene is associated with KRG intake and HAART, respectively.


Introduction
Panax ginseng has a long history of medicinal use in Asia. At present, ginseng is the best-selling herbal medicine in the world [1]. About 200 constituents of Korean ginseng have been isolated and characterized. Its major components include ginseng saponins and polysaccharides. The major pharmacological effects of ginseng include adaptogenic effects [2]; that is, ginseng nonspecifically increases the resistance to physical, chemical, and biological stress by immunomodulation of the hypothalamic-pituitary-adrenal axis [3]. Recent studies have also demonstrated ginseng's potential use in adjuvant and immunotherapies [4][5][6][7][8].
Persistent immune activation and inflammation despite sustained antiretroviral therapy (ART)-mediated viral suppression have emerged as a major challenge in the modern era of HIV treatment [9]. In particular, the saponin fraction of ginseng downregulates proinflammatory mediators in LPS-stimulated cells and protects mice against endotoxic shock [10][11][12]. The absence of microbial translocation and immune activation in well-adapted, natural hosts with simian immunodeficiency virus [13] is a very important mechanism in our understanding of the slow progression of HIV-1infected patients who have been treated with Korean red ginseng (KRG) [14].
We previously reported that KRG induces gross deletions in the nef gene [14] and frequent genetic defects in the 5 LTR/gag gene [15]. Interestingly, the detection of genetic defects was inhibited during the administration of highly active antiretroviral therapy (HAART) [16]. However, there are only a few studies that have reported the gross deletion of the vif gene because it is the second most highly conserved gene after pol [17][18][19]. Hence, to determine if KRG affects the vif gene, as shown for the nef and gag genes [14][15][16], we amplified vif gene in peripheral blood mononuclear cells (PBMCs) obtained over 20 years from 10 long-term slowly progressing (LTSP) patients. It appears from our analyses that KRG intake might induce gross and small in-frame deletions.

Effects of HAART on PSC.
The 10 LTSP patients we included in our present study were untreated before December 1991, were treated with only KRG between 2004 and 2009, and have been treated with KRG plus HAART since 2009 ( Figure 1). We analyzed 432 vif genes over 20 years in these 10 LTSP patients. Among these, 15 vif genes (3.5%) demonstrated PSC. In total, 275 and 157 vif genes were obtained during KRG and KRG plus HAART, respectively. Each group demonstrated 3 (1.1%) and 12 (7.6%) vif genes with PSCs, respectively. When receiving only KRG, three patients (90-50, 93-04, and 93-60) demonstrated PSC at 14 years, 1 year and 9 months, and 7 years after starting to receive KRG, respectively. The frequency was significantly higher when receiving KRG plus HAART than KRG alone ( < 0.01; Table 2). This suggests that HAART itself might induce Gto-A hypermutation, thereby resulting in PSC and, possibly, lethal hypermutations. This is consistent with the findings of other studies [22]. However, we found no difference in the frequency of PSCs between our KRG (3 of 275 genes) and control groups (1 of 106 genes). Interestingly, of the 15 vif genes that demonstrated PSCs in our present analysis, eleven did not satisfy the criteria of Hypermut 2.0 in comparison with the earliest sequences obtained from each patient ( < 0.05; Figure 3).

Effects of KRG and HAART on Genetic Defects.
We detected 11 gross deletions in 432 amplicons obtained from our 10 LTSP patients (Table 2; Figure 2). No deleted genes, including gross deletions, were detected in the control patients. In total, five patients demonstrated gross deletions after >19 months of KRG intake. Four patients demonstrated gross deletions during KRG intake prior to HAART, although the frequency was very low (1.5%). Specifically, a sequence containing a gross deletion and duplication/recombination (KC247159) was identified in patient 87-05. A 362-base pair  Table 2). Each deletion was one (JQ327723) of 3 amplicons and one (KC247314) of 2 amplicons obtained between August 2008 and October 2010 and all 4 amplicons, respectively ( Figure 1). This frequency is the highest among the related studies to date, although we found no significant differences in terms of the frequencies of gross deletions between patients who received KRG (1.5%) or KRG plus HAART (4.5%; Table 2) in our present study. If both PSCs and gross deletions are only defined as nonfunctional vif genes, the proportion of nonfunctional genes is 2.5% (7 of 275 genes) during KRG and 12.1% (19 of 157 genes) during KRG plus HAART ( < 0.01).  Figure 3) demonstrated PSCs. The frequencies of the PSCs in 5 tryptophan residues (21,38,70,89, and 174) were 3, 7, 13, 2, and 7, respectively. In the control group, one patient demonstrated stop codons at residues 21, 70, and 174 (JQ066980).

Small in-Frame Deletions Are Associated with KRG Intake.
We did not find any specific changes in the nucleotide or amino acid sequences due to KRG intake. However, interestingly, two of our patients demonstrated small deletions during KRG intake; one patient  Figure 2). The frequencies of these deletions were similar between patients who received KRG (10.5%; 29 of 275 genes) and KRG plus HAART (15.9%; 25 of 157 genes). However, the frequency of these deletions was zero (0 of 52 genes, as determined using RT-PCR) during pre-KRG and 10.5% (29 of 275) during KRG only without HAART ( < 0.01). These deletions manifested after 6.3 and 12.0 years of KRG intake in patients 90-18 and 92-13, respectively. Interestingly, patient 90-18 also demonstrated a 6 bp deletion in the nef gene after 11 years of KRG intake with HAART, although we did not categorize this as a gross deletion [14]. Deletions were conserved during KRG plus HAART therapy ( Figure 2) and comprised a higher proportion than wild-type alleles. In particular, three amplicons containing PSC also demonstrated small deletions in patient 90-18. The presence of both wild-type and mutant alleles in the same samples differed from the findings for the samples obtained from patients who were not treated with KRG, in which all amplicons contained deletions in five patients (data not shown). In addition, there were no such deletions in the control group (0 of 106 genes).  [26]. Overall, the proportions of defective genes identified in our present study were significantly lower than the proportions of defective nef (94 of 479 genes, including      Figure 1: Changes in the CD4+ T-cell count, plasma viral load, and genetic defects in terms of Korean red ginseng (KRG) intake and highly active antiretroviral therapy (HAART). The durations of KRG intake and HAART are indicated by the bars. Solid and dotted lines denote good (>90%) and poor (<90%) compliance according to self-administered responses, respectively. The upward arrow (↑), downward arrow (↓), plus sign (+), and asterisk ( * ) denote the sequences of the vif gene, gross deletions, 3-and-6-base pair (bp) in-frame insertions, and stop codons, respectively. gΔ and sΔ denote gross deletion and in-frame small deletion, respectively. HAART: highly active antiretroviral therapy; ND: not determined. a Two out of three genes contained both stop codon and sΔ. b < 0.01. < 0.01 for the sum of 3 kinds of defective genes (36/275 versus 42/157). Fifty-two vif genes at baseline obtained from serum using RT-PCR were all wild types. * Control patients ( = 21) revealed premature stop codon in one out of 106 vif genes. stop codons; < 0.05) and 5 LTR/gag genes (71 of 189 genes; < 0.001) identified in the same patients [14,15].

Discussion
To                                In total, five of our patients demonstrated in-frame deletions of the vif gene of the 75 KSB-infected patients who were treated with KRG. Also in our present study cohort, five patients, including 90-18 and 92-13, demonstrated deleted vif genes (small in-frame and gross deletions) during KRG intake, whereas all of our 10 LTSP patients demonstrated deletions in the nef gene ( < 0.05) [14]. In addition, compared with the reported deletions of the nef and 5 LTR/gag genes, the locations of the deleted vif gene were positioned within a narrow range at the terminus [15,16,23]. Regarding gross deletions, to date, only two patients have demonstrated gross deletions in the vif gene [17,18] and only one patient has demonstrated the insertion of two amino acids, and these patients were part of a nonprogressing mother-child pair [28]. However, in our present study, of the 10 LTSP patients who were assessed, four patients demonstrated gross deletion in the vif gene during KRG intake prior to receiving HAART (1.5%). This frequency is significantly lower than the previously reported incidence of 10 of 10 patients demonstrating gross deletions in the nef (18.7%) and 5 LTR/gag genes (37.6%; < 0.01) [27,28]. The frequency of deletion in the nef gene was reported to significantly decrease during KRG plus HAART ( < 0.01) [16]. In contrast to previous studies on the nef and gag genes [16,29], the detection of in-frame deletions in the vif gene was not suppressed in the patients receiving HAART in our present cohort. The reasons for this include the following: (1) the proportion and frequency (approximately two-thirds) of deleted vif genes after the first occurrence were much higher than in the nef and 5 LTR/gag genes (<20 and 30%, resp.); (2) small deletions in the vif gene occurred as a single band, whereas gross deletions in the nef and 5 LTR/gag genes were mainly detected as two bands (wild type and short) per PCR reaction; and (3) the number of amplicons indicating PSC due to G-to-A hypermutation was significantly higher in patients receiving KRG plus HAART than patients receiving only KRG. This phenomenon is consistent with our previous data in 5 LTR/gag gene [29]. However, the frequency of G-to-A hypermutation was significantly lower in vif gene than that in 5 LTR/gag gene [29]. Thus, for the vif gene, the detection of small deletions might be less affected by limit-diluting effects, as has been shown for the nef gene [29]. Taken together, these additional defects in the vif genes in the same patients might be related to the replicative impairment in vif defective HIV-1, which ultimately results in defects in the synthesis of viral DNA [30].
ApoBec3G-induced hypermutation in LTNPs has been reported previously [33]. Indeed, it has been reported also that LTNP patients harbor PSC-containing vif genes more frequently than progressors [25], although the reported frequencies of defective genes vary among studies [34]. In our current analyses, vif gene-containing PSC due to G-to-A hypermutation was found to be significantly higher during KRG plus HAART than only KRG intake prior to HAART. This finding is consistent with our previous data obtained from a cohort of the Korean hemophiliacs (2.1% incidence