Journal of Clinical Trials, Pathology and Case Studies | Volume 1 | Issue 1 | 2016-08-30

Co-variation between HERV-K RNA Expression and CD4 Cell Counts in Untreated HIV-1 Infected Individuals

Jianli Dong

Abstract

Human endogenous retroviruses (HERVs) have been implicated in the etiology of cancer, chronic inflammation, and other diseases, and emerging data supports the regulation and functional interaction between K type HERV (HERV-K) and HIV-1. In this study, we performed a reverse transcription-polymerase chain reaction (RT-PCR) assay using DNase I digestion to remove contaminating HERV-K genomic DNA and examined HERV-K RNA expression in plasma samples from 54 untreated, newly diagnosed, HIV-1 infected individuals. We found that, HERV-K expression did not correlate with HIV-1 viral load. However, the mean CD4 cell count was significantly lower in HERV-K RNA positive specimens. Further studies are necessary to examine the regulatory relationship between HERV-K and CD4 cell counts in HIV-1 infected individuals.

Keywords: HERV-K expression, HIV-1 viral load, CD4 cell count, DNase I digestion

Full text

The human genome has approximately 400,000 genetic loci, half of which are composed of elements that consist of DNA transposons and retroelements [1,2] . Human endogenous retroviral (HERV) sequences are categorized as types of retro elements that comprise approximately 8.3% of the human genetic sequence [3,4] . According to a previous hypothesis, HERVs were once exogenous retroviruses that were incorporated into the human germ-line resulting in vertical transmission and becoming endogenous [5] . HERVs share sequence homology with animal retroviruses such as the mouse mammary tumour virus and the murine leukemia virus; however, they are found exclusively in catarrhines (old world monkeys, apes, and humans) [6] . Moreover, there are specific HERV loci that are exclusive to humans, such as the AF001550 LTR cluster, the AC003023 LTR cluster, and others [7,8] . The genetic composition of HERV elements is similar to that of HIV-1, HIV-2, HTLV 1 and HTLV 2. A complete HERV strain consists of genes that code for a group-specific antigen (GAG) a retroviral capsid protein, protease (PRO), a reverse transcriptase domain (POL) and an envelope protein (ENV), flanked by regulatory sequences called long terminal repeats (LTRs). The majority of HERV elements found within the genome are degenerated and defective due to a number of mutations within their sequences; that said, some are believed to have biological activities and retain relatively complete genetic sequences. According to studies regarding HERV expression, when present, it can be related to an active role in the pathogenesis of cancer, chronic inflammation, and other diseases [9] . In addition, these elements have been associated with playing a functional role in the regulation of innate immune pathways, as well as in the regulation of host gene encoding immunity factors [10] . Retroelements can be divided in two different categories: one with LTR and one without LTR; with HERVs falling in the category that contains LTRs in their genetic structure [9] . The discovery of HERVs results from various experimental approaches. One approach focused on using probes derived from animal retroviruses with conserved POL-regions to screen human genomic libraries under low-stringency conditions [11,12] . Other approaches used oligonucleotides with homology to viral primer binding sites [13] , and the use of cross-reacting antibodies to detect highly conserved Gag retroviral proteins [14] . Still, some HERV families are detected by chance [15-17] . Asides from the presence of LTRs, HERVs are grouped into three different classes: Class I, most closely related to gamma retroviruses, including HERV-W and HERV-H; Class II, most closely related to beta retroviruses, including various strains of HERV-K (one of the most complete and active HERVs in the human genome); and Class III, most closely related to spuma retroviruses, including HERV-K and HERV-S [2-7] . Asides from such classifications, in regards to protein biosynthesis, HERVs are classified within that category based on their tRNA primer binding site if they were to be replicating viruses, HERV-K would use lysine, and HERV-W would use tryptophan [18] . The HERV-K strand is then classified in 10 different families, ranging from HERV-K human MMTV-like (HML)-1 to HML-10 [19,20] . Among HERV-K families, HERV-K HML-2 has been the focus of recent investigations due to its open reading frames for all major retroviral proteins: Gag, Pro, Pol, and Env. HERV-K HML-2 also carries a sequence specific nuclear RNA export factor, Rec, that is functionally analogous to the HIV-1 Rev protein and the HTLV Rex protein [21] . HERV expression is mostly negative, however, factors such as UV radiation, cytokines or hormones have been reported to activate HERV expression [22-25] . It has been well established that HERV-K mRNA and proteins are expressed in many tissues. As mentioned prior, enhanced expression is present in a wide range of pathological conditions including: viral infections, multiple sclerosis, HIV-1, and melanomas [9,26,27] . The focus of this study was to examine the possible correlation of HERV-K RNA expression in relation to HIV-1 clinical phenotypes, in this case viral load and CD4 cell count. Materials and Methods Patient specimen HERV-K expression was measured in plasma from 54 untreated, newly diagnosed, HIV-1 infected individuals submitted for HIV-1 clinical testing in the Molecular Diagnostics Laboratory at The University of Texas Medical Branch (UTMB) between May and August 2013. The study was approved by the UTMB Institutional Review Board. HIV-1 viral load Plasma HIV-1 RNA assay with a lower limit of quantification of 75 copies/mL was performed by the VERSANT HIV-1 RNA 3.0 Assay (bDNA) using the standard procedure according to manufacturer instructions (http://www.fda.gov/downloads/BiologicsBloodVaccines/ BloodBloodProducts/ApprovedProducts/PremarketApprovalsPMAs/UCM091276.pdf , Siemens Healthcare Diagnostics, Washington, DC). CD4 cell count CD4 cell count was quantified by flow cytometric method using BD FACSCanto II Flow Cytometer following standard manufacturer instructions (http://www.uni-regensburg.de/Fakultaeten/Medizin/Pathologie/pdf/CantoManual.pdf , BD Biosciences, San Jose, CA). RNA extraction and DNase I digestion RNA was extracted from patient samples with the use of HIV-1 ViroSeq RNA preparation method (Abbott Molecular, Des Plaines, IL) [13,28] . 500 L plasma were extracted for every patient sample, with final RNA diluents of 100 l for the samples. Samples were then stored at -80 C. A DNase I Digestion Kit (New England Biolabs, Ipswich MA) was used to eliminate residual DNA from RNA diluents. 4 l of DNase buffer, 1 l of DNase I and 15 l of ddH 2 O were combined with 20 l of extracted RNA samples prior to the digestion process. A sample totaling 40 l was placed in a GeneAmp 9700 Thermocycler for 1 hr at 37 C. Sample was then removed and left for 1 hr at room temperature (15 - 25 C). At 50 minutes, 0.4 l EDTA was added to prevent RNA degradation. Tubes were then placed in 75 C heat block for heat inactivation. Tubes were then allowed to cool to room temperature prior to storage at -80 C. HERV-K PCR and HERV-K reverse transcriptase-PCR Proceeding DNase I digestion, two separate reaction protocols were followed in order to assure no residual DNA remained in the sample and to confirm positive HERV expression: HERV-K PCR and reverse transcriptase-PCR (RT-PCR). PCR: 5 l PCR buffer, 1 l forward primer, 1 l reverse primer, 2 l dNTPs, 0.25 l Taq polymerase, and 13.75 l ddH 2 O were combined with 2 l of digested sample. The following standard PCR reaction protocol was used on a GeneAmp 9700 Thermocycler: 95 C (5 min); 95 C, 56 C, 72 C (35 cycles at 1 min each); 72 C (10 min), 40 C (sample preservation temperature). Reverse transcriptase-PCR: 5 l of RT-PCR buffer, 1 l forward primer, 1 l reverse primer, 2 l dNTPs, 1 l Qiagen One Step RT-PCR enzyme mix, 13 l ddH 2 O were combined with 2 l of digested sample. The following standard RT-PCR protocol was used on a GeneAmp 9700 Thermocycler: 50 C (30 min); 95 C (5 min); 95 C, 56 C, 72 C (35 cycles at 1 minute each); 72 C (10 min), 4 C (sample preservation temperature). Gel Electrophoresis: PCR and RT-PCR products were analyzed by fractionation in 1% agarose gel and visualized by GelRed DNA stain (Phenix Research Products, Candler, NC). Images were then captured using Fotodyne FOTO/Analyst Workstation (Fotodyne Incorporated, Hartland, WI). HERV-K expression was compared to 100 bp DNA Ladder (Promega, Madison, WI) with designated parameters: -, +/-, +, ++, +++, ++++. Statistical analysis: The relationships among HERV-K, HIV-1 viral load, and CD4 cell counts were examined by pooled t-test and Pearson correlation coefficients using SAS 9.4 (SAS Institute Inc., Cary, NC). Results and Discussion After sample analysis, we detected HERV-K RNA positivity expression following RT-PCR in 35% (19 of 54) of plasma specimens from newly diagnosed, HIV-1 infected individuals (Table 1). Of note, patients in this study were previously untreated HIV-1 infected individuals. This is in contrast to previous reports where patients absence of antiretroviral treatment history was not considered [29] . Table 1: HERV-K RNA detection in 54 untreated HIV-1 infected individuals. Sample HERV-K Interpretation HIV-1 Viral Load (copies/ml) CD4 Count (cells/ l) PCR RT-PCR HERV-K RNA 47 - (++++) Positive 209881 4 43 - (++) Positive 57133 36 18 - (+/-) Positive 11037 232 84 - (++) Positive 205104 247 55 - (+) Positive 31301 261 41 (+) (++) Positive 2522 271 52 - (+++) Positive 26172 294 64 - (+) Positive 34242 304 49 - (+) Positive 1788 331 57 - (+) Positive 1930 346 17 - (+++) Positive 1106 351 63 - (+) Positive 8356 351 46 - (+++) Positive 14505 440 50 (++) (++++) Positive 13237 454 19 - (+) Positive 9773 499 83 - (++) Positive 4282 508 20 - (+) Positive 10691 510 44 - (++) Positive 11375 621 51 (+) (++++) Positive 2126 650 72 - Negative 41397 8 54 - Negative 1696 42 24 (+/-) - Negative 8597 138 76 - Negative 20352 142 32 (+/-) - Negative 2413 200 28 (+) - Negative 40885 224 71 - Negative 14671 344 5 (+) - Negative 108 345 14 (+++) (++) Negative 26870 345 2 - - Negative 44075 349 85 - Negative 18154 374 78 - Negative 93 379 74 - Negative 33935 386 15 (+) - Negative 33071 405 81 - Negative 2330 426 75 - Negative 339 429 33 (++++) (+) Negative 188623 434 22 (++++) (+) Negative 1451 487 21 - - Negative < 75 494 82 - Negative 1558 577 69 - Negative 3537 578 61 - Negative < 75 583 29 (++) - Negative 1390 596 23 (++) - Negative 17680 618 70 - Negative 86238 624 73 - Negative 14188 643 26 (+/-) - Negative 1291 646 31 (+/-) - Negative 958 660 77 - Negative 22468 671 79 - Negative 1628 693 59 - Negative 1495 710 27 (+/-) - Negative < 75 747 40 (+) (+/-) Negative 1226 776 25 (+/-) - Negative 111 919 30 (+) - Negative 163 1039 RNA extraction was performed with the use of HIV-1 ViroSeq RNA preparation method (Abbott Molecular, Des Plaines, IL). DNase I digestion was performed utilizing the previously described protocol to remove contaminating cellular DNA. As stated in other reports, positive HERV-K expression is possible due to the presence of circulating cell-free RNA that is active within patient samples [27] . Several factors can contribute to variation in the expression of HERV-K RNA; such as, different HERV-K assay sensitivity, difference in HIV-1 infection progression, exogenous factors as mentioned earlier, variation in patient medical history and background, and sub-population polymorphisms of HERV-K sequences. The positivity rate can also be attributed to the DNase I protocol used to further reduce contaminating HERV-K genomic DNA in RNA extractions. In HERV-K RNA positive subjects, mean CD4 cell count was 358 cells/ l, ranging from 4 to 650 cells/ l, and mean viral load was 34,736 copies/ml, ranging from 1,106 to 209,881 copies/ml (Table 1). We found no significant difference of mean HIV-1 viral load in HERV-K RNA positive and negative specimens (p < 0.2995, pooled t-test), consistent with our previous report [29] . Interestingly, mean CD4 cell count was significantly lower in HERV-K RNA positive than negative specimens (p < 0.0335, pooled t-test). Further studies are necessary to examine the co variation between HERV-K expression and CD4 cell count. If confirmed, it should warrant investigation of the biological significance and regulatory relationship between HERV-K and CD4 cell counts in HIV-1 infected individuals. The pathogenic involvement of HERV-K remains unknown and warrants further study. HERV-K activation has been associated with malignancies, autoimmune disorders, and neuropathological conditions [9] . Methodology optimization is a key to assess HERV-K positive expression to properly understand its role in disease pathogenesis. 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