Open Access

Altered gene expression in asymptomatic SHIV-infected rhesus macaques (Macacca mulatta)

  • Erica E Carroll1,
  • Rasha Hammamieh1,
  • Nabarun Chakraborty1,
  • Aaron T Phillips1,
  • Stacy-Ann M Miller1 and
  • Marti Jett1Email author
Contributed equally
Virology Journal20063:74

https://doi.org/10.1186/1743-422X-3-74

Received: 06 July 2006

Accepted: 06 September 2006

Published: 06 September 2006

Abstract

Simian-Human immunodeficiency virus is a chimeric virus which, in rhesus macaques (Macacca mulatta) closely imitates immunodeficiency virus infection in human (HIV). A relatively new way to study pathogenesis of viral infection is to study alterations in host gene expression induced by the virus. SHIV infection with certain strains does not result in clinical signs. We hypothesized that alterations in gene expression relating to the immune system would be present in SHIV-infected animals despite the lack of clinical signs. Splenic tissue from four adult male Indian-origin Rhesus monkeys serologically positive for non-pathogenic SHIV 89.6 was processed by cDNA microarray analysis. Results were compared with the corresponding outcome using splenic tissues from four unexposed adult male Rhesus monkeys. Subsequent gene analysis confirmed statistically significant variations between control and infected samples. Interestingly, SHIV-infected monkeys exhibited altered expression in genes related to apoptosis, signal transduction, T and B lymphocyte activation and importantly, to immune regulation. Although infected animals appeared asymptomatic, our study demonstrated that SHIV-infected monkeys cannot reliably be used in studies of other infectious agents as their baseline gene expression differs from that of normal Rhesus monkeys. The gene expression differences in SHIV-infected animals relative to uninfected animals offer additional clues to the pathogenesis of altered immune function in response to secondary infection.

Background

Simian immunodeficiency virus (SIV) infection of rhesus macaques exhibits many similarities to human immunodeficiency viral (HIV) infection of humans. Most pathogenesis and vaccine studies for HIV-1 have been undertaken in either SIV-macaque or a chimeric simian-human immunodeficiency (SHIV)-macaque model [1]. SHIV strains have the viral envelope of HIV but the gag/pol genes of SIV. Pathogenesis is similar with respect to macrophage and T lymphocyte cell tropism, histopathologic changes, CD4-cell depletion and clinical signs of auto-immune deficiency syndrome (AIDS) in virulent strains. HIV and SIV additionally cause cognitive and motor impairments in infected patients and monkeys, respectively [2]. Host factors may play a role in degree of pathogenesis between varying SHIV constructs, as one study reported observing similar viral loads in rhesus monkeys infected with pathogenic and non-pathogenic SHIV constructs [1].

Gene expression studies have grown increasingly popular as a tool to mine large amounts of data from treated and control populations. Such data can be used to examine host factors involved in SHIV, and thereby HIV, pathogenesis. To our knowledge, microarray data from SHIV-infected Rhesus macaques have not yet been examined for genes affecting immune response and inflammation. Gene expression data have the potential to greatly expand the understanding of SHIV-host interaction beyond the limited number of cell types or cytokines generally examined.

In animals free of clinical signs of SHIV, altered baseline gene expression data may give clues to the pathogenesis of altered immune response to secondary infections. Studies involving HIV-infected humans demonstrated suppression of IL-2 in response to select antigens and increase in TNF-α even prior to the onset of CD4+ T-cell depletion [3, 4]. Gene expression data collected in this study from SHIV 89.6-infected monkeys demonstrate that these animals are not genetically 'normal' and cannot ethically be used for studies involving other infectious agents, if at all, without an explicit caveat listing their SHIV status. Comparison of gene expression patterns collected from SHIV-infected and uninfected animals to that of the matched animals exposed to select bacterial and viral agents would provide a more complete understanding of SHIV effect on immune response to particular infectious agents. Extrapolation to the HIV-patient response to secondary agents may then be attempted. Gene expression data may also provide clues to pathogenesis of cognitive and related ailments arising with HIV infection.

Results

Clinical history

A brief description of treated and control animals is given in Table 1. All monkeys were male; while two of them (one SHIV-positive, one SHIV-negative) were Herpes B-positive.
Table 1

An overview of the Rhesus macaques used in SHIV gene expression study

Animal ID

Gender

Age (yrs)

Geographic origin

Herpes B Status

SHIV 89.6 status

JGH

Male

7

Indian

positive

positive

PHB

Male

7

Indian

negative

positive

TTH

Male

7

Indian

negative

positive

FFG

Male

9

Indian

negative

positive

331

Male

adult

Indian

negative

negative

332

Male

adult

Indian

negative

negative

CJ2T

Male

4

Indian

negative

negative

EC49

Male

adult

Indian

negative

negative

DB87

Male

12.2

Indian

positive

negative

Table 2 summarizes abnormalities in clinical chemistries including complete blood counts of the SHIV-infected animals. Abnormalities were minimal. Attending veterinary clinicians considered these animals asymptomatic with respect to SHIV infection.
Table 2

Clinical pathology of SHIV-positive rhesus macaques

Animal ID

Abnormal findings in complete blood count and serum chemistry analysis.

FFG

Sodium 144 mg/dl (reference range 147–158)

Chloride 108 mg/dl (range 110–120)

Lymphocytes 65.4% (reference range 14–64%)

PHB

Sodium 146 mg/dl (range 147–158)

Carbon dioxide 29 mmol/L (range 19–29)

Total protein 6.4 g/dl (range 6.7–8.0)

ALT 113 U/L (range 20–91)

LDH 538 U/L (range 638–3012)

TTH

Sodium 145 mg/dl (range 147–158)

Chloride 110 mg/dl (range 110–120)

AST 29 U/L (range 29–64)

JGH (Herpes B+)

Sodium 147 mg/dl (range 147–158)

Chloride 109 mg/dl (range 110–120)

Carbon dioxide 30 mmol/L (range 19–29)

Triglycerides 18 mg/dl (range 35–137)

Total protein 6.6 g/dl (range 6.7–8.0)

AST 26 U/L (range 29–64)

Table 3

The sequences of the primers used in the present project

Name

Gene Bank ID

Description

Sequence

Product Size

ANLN

R16712

Anilin

5'-TCC AAG TCC TGT GTC TCC TC-3'

 
   

5'-TCT TGA GTT CAG CCC TCT CC-3'

109 bp

Bit1

AI339248

CGI-147 protein

5'-TGG CTG TTG GAG TTG CTT G-3'

 
   

5'-TGT GTG TCT TGC TCG TCT TG-3'

93 bp

CLCA2

AI675394

chloride channel. calcium activated, fam

5'-CAA CCA AGA AGC ACC AA CC-3'

 
   

5'-CAT CCA GCA CTA AAC AGA CCA C-3'

179 bp

 

AA922998

postmeiotic segregation increased 2-like

5'-GTT TCA GGC AAT GGA TGT GG-3'

 
   

5'-CAT GGC AGG TAG AAA TGG TG-3'

178 bp

COL15A

AA455157

collagen, type XV, alpha 1

5'-CCA CCT ACC GAG CAT TCT TAT C-3'

 
   

5'-CAA TAC GTC TCG ACC ATC AAA G-3'

197 bp

IL2RA

AA903183

interleukin 2 receptor, alpha

5'-CTG AGA GCA TCT GCA AAA TGA C-3'

 
   

5'-GGC CAC TGC TAC TTG GTA CTC T-3'

242 bp

PDCD4

N71003

programmed cell death 4

5'-CCG GTG ATG AAG AAA ATG CT-3'

 
   

5'-TGG TTG GCA CAG TTA ATC CA-3'

207 bp

ADORA2

N57553

adenosine A2a receptor

5'-TCA ACA GCA ACC TGC AGA AC-3'

 
   

5'-ATG GCA ATG TAG CGG TCA AT-3'

220 bp

RBM9

AA451903

RNA binding motif protein 9

5'-AAC TCC TGA CTC AAT GGT TC-3'

 
   

5'-CAT TTT GTG TGC TGG GTG AG-3'

194 bp

MAP2K7

H85962

mitogen-activated protein kinase kinase

5'-ACC AGG CAG AAA TCA ACG AC-3'

 
   

5'-GAT GAA CGT CCC AAA GCA CT-3'

224 bp

COL7A1

AA598507

collagen, tykpe VII, alpha 1 (epidermolysin)

5'-AGC CCA GAT GTT TCC ACT CA-3'

 
   

5'-ACA AGA GGC AAT CCT TGG AGA-3'

239 bp

Micro-array analysis of SHIV-infected versus uninfected

Using the 38 most varying genes between SHIV-infected and SHIV-uninfected animals, we performed Principle Component Analysis, a non-hierarchal clustering tool, to revalidate the t-test result. Figure 2 demonstrates that the SHIV positive and negative groups were clustered together, keeping a significant distance between them along the first principal component (X-axis), which shared the highest fraction of group variation. The pattern of clustering also suggested that the gene expression variability was independent of the animals' Herpes B status.
Figure 1

Hieratically clustered Tree-view of genes differentially expressed between the SHIV positive and negative animals.

Figure 2

Principal component analysis was performed over the SHIV infected and non-infected population. Though the animals were clinically reported asymptomatic, the SHIV treated and control samples cluster far from each other along PCA1 axis. The result also suggests that the Herpes B status does not affect the outcome. Here PCA1 has 61.7% population, while PCA2 and PCA3 shares 12.6% and 8.56% of the population respectively.

Gene ontology study, using GeneCite [6], associated the members of the differentially expressed genes to a range of important biological and pathological functions including immune defense, cell death or apoptosis, cell growth, signal transduction and others. Table 4 represents the functional classification of some of the genes of interest.
Table 4

The list of some of the genes of interest.

Gene ID

Symbol

Gene Name

Fold Change

Cellular defense immunity:

   

AA424786

GOLGA2

golgi autoantigen, subfamily a2

2.802126

AA664195

HLA-DRB3 (HLA-DRB1)

major histocompatibility complex, class II, DR beta 1

0.202677

AI815229

LILRB3

leukocyte immunoglobulin-like receptor, subfamily B, member 3

0.074432

H96643

FOSL1

FOS-like antigen-1

0.284931

Cell growth/proliferation:

   

AA035384

SDHD

succinate dehydrogenase complex

0.287502

AA521228

HIBCH

3-hydroxyisobutyryl-Coenzyme A hydrolase

4.260302

AA699573

TCF2

hepatic transcription factor 2

4.223543

AI220577

TNP2

transition protein 2

0.262051

H06676

ALDH5A1

aldehyde dehydrogenase 5 family

2.381241

AI798238

P2RY11

peter pan homolog

0.174406

Cell death/Apoptosis:

   

AA458838

NOXA

phorbol-12-myristate-13-acetate-induced protein 1

3.872641

AI339248

Bit1

CGI-147 protein

0.337378

AI972925

API5

apoptosis inhibitor 5

0.17877

Molecular binding/Adhesion:

   

AA167269

NAP1L1

nucleosome assembly protein 1-like 1

0.272199

AA424824

DSTN

destrin

2.876146

AA669637

PNRC1

proline rich 2

0.142976

AA676840

UTRN

utrophin

2.340295

AI769340

HRC

histidine-rich calcium-binding protein

0.220777

R16712

ANLN

anillin

0.280955

T60070

RAB40B

GTP-binding protein, member RAS oncogene family

2.649082

AA426374

TUBA2

alpha tubulin 2

0.112352

AA055163

CASQ2

calsequestrin 2

0.531223

AA521350

Sep15

15 kDa selenoprotein

0.33198

AA633747

COL6A2

collagen, type VI, alpha 2

2.061697

AA634218

PRAF2

JM4 protein

0.35942

AA922998

PMS2L5

postmeiotic segregation increased 2-like 5

0.289763

AI364103

CINP

cyclin-dependent kinase 2-interacting protein

3.399017

AI653424

NUFIP1

nuclear fragile X mental retardation protein interacting protein 1

0.15423

W32272

IQGAP2

IQ motif containing GTPase activating protein

3.137166

Signal Transduction:

   

AA427491

TRAC

T-cell receptor active alpha-chain

0.145492

AI401275

CALCR

calcitonin receptor

0.329203

AA421819

CDH6

K-cadherin

0.241252

Transport:

   

AI675394

CLCA2

calcium activated chloride channel

3.802165

W94331

CTNS

nephropathic cystinosis

0.212335

N46828

ITPKC

inositol 1,4,5-trisphosphate 3-kinase C

5.969257

Biogenesis:

   

AA056013

MAGP2

Microfibril-associated glycoprotein-2

2.312604

AA629189

KRT4

keratin 4

0.227523

H27864

 

secretogranin II

0.089345

The first, second and third columns list the GeneBank ID, Symbol and Gene Name respectively. The Fourth column stands for the corresponding fold change of SHIV positive animal with respect to that of the control animal, averaged over the entire population, i.e. (Average fold change for all SHIV positive animals)/(Avg FC for all control animals)

Confirmation of gene expression changes by Real-Time PCR analysis

Ten genes were selected for real-time polymerase chain reaction (PCR). They are RNA binding motif protein 9 (AA451903), collagen, type XV, alpha 1 (AA455157), collagen, type VII, alpha 1(AA598507), interleukin 2 receptor, alpha (AA903183), Chloride channel, calcium activated, family member 2 (AI675394), mitogen-activated protein kinase kinase (H85962), adenosine A2a receptor (N57553), programmed cell death 4 (N71003), postmeiotic segregation increased 2-like (AA922998), Bcl-2 inhibitor of transcription (AI339248) and Anillin (R16712). Figure 3 illustrates that the real-time PCR expression profiles for the selected genes are well correlated with the corresponding microarray results.
Figure 3

A comparative analysis of four selected genes using array analysis and Real-time PCR. RNA binding motif protein 9 (AA451903), collagen, type XV, alpha 1 (AA455157), collagen, type VII, alpha 1(AA598507), interleukin 2 receptor, alpha (AA903183), Chloride channel, calcium activated, family member 2 (AI675394), mitogen-activated protein kinase kinase (H85962), adenosine A2a receptor (N57553) and programmed cell death 4 (N71003) were up regulated in SHIV infected animals while postmeiotic segregation increased 2-like (AA922998), Bcl-2 inhibitor of transcription (AI339248) and Anillin (R16712) were down regulated.

Discussion

Simian immunodeficiency virus (SIV), previously referred to as simian T-cell lymphotropic virus type III (STLV-III), induces an AIDS-like disease in its natural host, rhesus macaques. HIV and SIV, members of the lentivirus subfamily of retroviruses, not only resemble each other by their antigenicity, but also bear remarkable similarity in their biological properties, such as cytopathic effect and tropism for CD4-bearing cells. These criteria render the chimeric SHIV the best animal model currently available for HIV study.

In this study, we examined gene expression in SHIV- infected male rhesus macaques of Indian origin using a genomic perspective and compared the results to uninfected age, gender and Herpes B-status-matched controls. Although infected animals were without clinical signs related to SHIV infection, a significant number of genes exhibited significantly altered expression concurrent with SIV infection.

Ontological research revealed that several genes, namely FOS-like antigen 1 (FOSL1, ID: H96643), golgi autoantigen (GOLGA2, ID: AA424786), major histocompatibility complex (MHC), class II, DR beta 1 (HLA-DRB1, ID: AA664195) and leukocyte immunoglobulin-like receptor (LILRB3, ID: AI815229) are associated with human immune defense. LILRB3 is a leukocyte inhibitory receptor which, upon binding to MHC Class I molecules, transmits inhibitory signals to the nucleus. HLA-DRB1, down regulated by SIV infection, is a cell-surface-associated immunoregulatory protein. Interestingly, this human leukocyte antigen (HLA)-associated gene has been correlated with non-responsiveness to recombinant hepatitis B virus (HBV) vaccine but does not alter susceptibility to viral persistence [6]. Another MHC protein binding unit, T cell receptor alpha locus (TRAC, ID: AA427491) is ontologically related to signal transduction.

Gene ontology investigation classified a significant subset of the genome of interest as a regulator of cell growth and apoptosis. SIV infection results in down-regulation of apoptosis inhibitor 5 (API5, ID: AI972925) and up-regulation of pro-apoptotic protein phorbol-12-myristate-13-acetate-induced protein 1 (NOXA, ID: AA458838) [8]. These alterations in gene expression might instigate opportunistic infections by inducing apoptosis among T-helper lymphocytes. Likewise, SIV infection alters several metabolism and cell growth regulating factors. For example, SIV-infected genome contains upregulated aldehyde dehydrogenase 5 family member A1 (ALDH5A1, ID: H06676); and concurrent down regulated succinate dehydrogenase complex, subunit D (SDHD, ID: AA035384) and nephropathic cystinosis (CTNS, ID: W94331). Reports suggest that overexpressed ALDH5A1 changes the concentration of gamma-aminobutyric acid (GABA) and glutamate, commencing henceforth excitotoxic damage, a well-established clinical marker of HIV activity [9]. Underexpressed SDHD and CTNS are associated with immunodeficiency through curbed monocyte and CD4+ T cell -induced immunoregulation [10], respectively.

Several entries of the present genome are functionally related to cellular and molecular transportation and binding. Interestingly, five actin-binding genes appeared in the list; namely: anillin (ANLN, ID: R16712), destrin (DSTN, ID: AA424824), utrophin (UTRN, ID: AA676840), cyclin-dependent kinase 2-interacting protein (CINP, ID: AI364103) and IQ motif containing GTPase activating protein 2 (IQGAP2, ID: W32272). Actin, the ubiquitously present cellular protein, has been reported to guide the direct cell-to-cell HIV-1 propagation by making of a stable adhesive junction at the target-effector cell interface [11]. Table 4 displays the down regulation of another molecular binding protein, 15 kDa selenoprotein (SEP15, ID: AA521350). Reduced level of selenoprotein in cells is a known marker of in vitro infection of SHIV [12]. Our data also supports the fact that immunodeficiency is correlated with altered calcium ion binding (UTRN, ID: AA676840; CDH6, ID: AA421819, CASQ2, ID: AA055163) and also is influenced by calcium- activated chloride channels (CLCA2, ID: AI675394) of host cells. Those are well established pathoregulating markers of activ HIV-1 negative factor (Nef) [1315].

In summary, in this small sample of SHIV-infected Rhesus macaques, expression was consistently altered in specific groups of genes which regulate a broad range of biochemical functions. A few important members of the genome of interest are discussed here. The present study, along with correlating some genes with SHIV and HIV model, identifies several novel genes as potential therapeutic markers for immune deficiency studies. Furthermore, results of this study suggest that SHIV infection of rhesus macaques may influence immune response to a second agent, even if baseline levels of clinical measurements appear normal. This study substantiates and validates the concern that an infected (i.e., antibody-producing) but asymptomatic animal should not be used in any other study involving infectious agents unless the pattern of gene expression to that agent is compared to normal animals' pattern, one agent at a time.

Note: microarray data have been submitted to the Gene Expression Omnibus (GEO) and can be searched using the Platform ID: GPL3395.

Materials and methods

Animals and virus

Four adult (7–8 years old) male Rhesus macaques (one Herpes B-positive and three Herpes B-negative) that were previously exposed to SHIV 89.6 strain (Animal identifications: FFG, JGH, PHB and TTH) were euthanized due to being declared 'excess' and no longer usable due to their serologically positive SHIV status. Splenic tissue was collected from each animal upon euthanasia and immersed in RNA Later® for 30–60 minutes before freezing at -80C.

SHIV 89.6, like all SHIV strains, has the env gene from the HIV-1 strain. All four animals had been challenged with 1.0 ml intravenous SHIV 89.6, a non-pathogenic strain, and became seropositive. Previous studies by the same researchers showed that seropositive animals were PCR positive as well (WRAIR Protocol TO03-98). All animals remained free of clinical signs. Complete blood counts and serum chemistry profiles were performed on the SHIV-positive animals and were within or very close to normal limits. The negative control animals were Indian-origin adult male Herpes B-negative Rhesus macaques. Splenic tissues were kindly provided by Scripps Institute, the National Institute of Health, and the Oregon National Primate Research Center. Tissue from a SHIV-negative animal (DB-87, provided by the Tulane National Regional Primate Research Center) was Herpes B-positive to control for the Herpes B-positive status of one SHIV-infected animal Table 1 represents an overview of the Rhesus macaques used in this study. Table 2 shows the clinical results of the SHIV-positive rhesus macaques

RNA isolation

Splenic tissue samples stored in RNALater® (Ambion, TX) at -80C were thawed in 1.5 mL tubes on ice. Tissue was submerged in Trizol ™ (Invitrogen, CA) solution and RNA isolation was carried out paccording to the Trizol ™ Reagent manufacturer's recommended instructions. RNA was ethanol-precipitated, air-dried and re-suspended in 20 ul/sample of nuclease-free water. RNA quantity was measured via spectrophotometry followed by analysis with a Bioanalyzer 2100 (Agilent Technologies, CA)

Custom made cDNA microarray SlidePreparation and hybridization

The gene library for the present project was commercially obtained from Research Genetics (Invitrogen, CA), containing 7489 genes, including 7019 known genes, 249 unknown genes and 110 expressed sequence tagged genes (ESTs). Superamine coated Telechem slides (Telechem Inc., OR) were used for printing the cDNA clones using 12 × 4 pin format, on a Virtek chip writer professional microarrayer in KemTek, Inc, MD. The printed slides underwent UV cross-linking, followed by post-processed by succinic anhydride treatment. The Micromax™ Tyramide Signal Amplification (TSA)™ Labeling and Detection Kit (PerkinElmer, Inc., MA) was used as directed by the manufacturer to determine relative gene expression of the collected samples. Custom-made reference RNA was prepared by combining aliquots of RNA from 33 normal Rhesus tissues and was used on every slide as the array controller, to check overall sensitivity of array printing, and to monitor reverse transcription, labeling and hybridization efficiency. Sample hybridization was carried out at 55°C for sixteen hours. A laser detection system was used (GenePix 4000b, Axon Instruments, CA) to scan the finished slides. Intensity of the scanned images was digitalized through Genepix 4.0 software (Axon Inc., CA).

Microarray analysis

Data cleansing and statistical analysis was carried out using Genespring® 7.0 (Agilent Tech., CA). Local background was subtracted from individual spot intensity. Genes that failed this 'background check' in any of the eight given experiments were eliminated from further analysis. Each chip was next subjected to intra-chip normalization (LOWESS). The genes that varied most between control and treated sample sets were selected via t-test analysis. The p-value cutoff was set at 0.05. Four hundreds and thirty two genes were differentially expressed between SHIV -infected and control uninfected animals with p < 0.05.

The pattern of gene expression variability of the experimental set having reduced dimension was evaluated using principal component analysis (PCA) classifying SHIV positive and negative samples as the two variable classes [16].

Real Time PCR

The t-test result was corroborated through real time polymerized chain reaction (Real-time PCR). A web-based primer designing tool was used to design the primers for the selected genes [17]. The specificity of each primer sequence was further confirmed by running a blast search. Reverse transcription and Real-time PCR reactions were carried out using reverse transcription kit (Invitrogen, CA) and Real-time PCR kit (Roche, IN), respectively. Each reaction with five technical duplicates was run in I-Cycler machine (Bio-Rad, CA). Each sample was also amplified against the house-keeping probe of the experiment: glyceraldehyde 3 phosphate dehydrogenase (GAPDH). The resultant cycle threshold data from each real-time-PCR 'run' was converted to fold-change using an established algorithm [5].

Quantitative and qualitative verification of the PCR product was accomplished by performing 1% agarose gel electrophoresis using SYBR Green I (Kemtek, Rockville, MD). Gel images were captured using PharosFX Molecular Imager system (Bio-Rad, CA) scanner and analyzed using Quantity One software (Bio-Rad, CA).

Notes

Declarations

Acknowledgements

EEC wants to extend thanks to LTC Gary D. Coleman and LTC Keith E. Steele for giving her the time to devote to this project in the face of other equally pressing mission requirements.

Authors’ Affiliations

(1)
Division of Pathology, Walter Reed Army Institute of Research

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Copyright

© Carroll et al; licensee BioMed Central Ltd. 2006

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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