Journal of Undergraduate Research
Volume 4, Issue 2 - October 2002

An Investigation of CD8+ T Cell Ability to Suppress Feline Immunodeficiency Virus Replication through Inhibition of the Viral LTR Region

Jennifer Minton

ABSTRACT

Biological and morphological similarities between feline immunodeficiency virus (FIV) and HIV have led FIV to be frequently used as an animal model for studying the antiviral responses mediated by CD8+ T cells during HIV infection. Two types of CD8+ mediated antiviral responses have been described in FIV. One involves cytolysis of the infected cells and the other results in inhibition of viral replication in the absence of cell killing. Recent studies suggest that CD8+ soluble factor(s) target stages of the HIV life cycle corresponding with transcription and early proviral gene expression. This project determined the molecular mechanism(s) involved in CD8+ mediated suppression of FIV infection by investigating the role that CD8+ soluble factors play in limiting viral transcription of the Long Terminal Repeat (LTR). CD8+ cells were magnetically sorted from the blood of FIV infected asymptomatic cats. The cells were cultivated with Crandell feline kidney (CrFK) cells transfected with a Chloramphenicol Acetyltransferase (CAT) expression cassette under the control of the FIV LTR. Enzyme levels were measured to quantify transcription. These data were compared to the level of inhibition of productive infection mediated by CD8+ cells in culture. As expected, low levels of CAT were detected in the assay and these correlated with reduced virus replication, showing that CD8+ cells are effective in limiting transcription of the FIV LTR. By understanding the precise mechanisms involved in CD8+ suppression, therapies and drugs could be developed to inhibit viral replication through a similar mechanism.

INTRODUCTION

Feline immunodeficiency virus (FIV) is a retrovirus belonging to the genus Lentivirus that affects mainly domestic cats. FIV was first isolated in 1986 and has become an important area of medical research.7 As a result of biological and morphological similarities between FIV and human immunodeficiency virus (HIV), FIV infection of domestic cats is considered an important small animal model for developing various therapeutic methods against HIV infection.6

The main cellular targets of FIV infection are the CD4+ T-lymphocytes.6 The life cycle of FIV begins with viral attachment to specific cellular receptors known as CXCR4 antigens on the cell surface.14 Once inside the cell, the enzyme reverse transcriptase (RT) converts FIV RNA into DNA before the virus reaches the cell nucleus. In the nucleus, viral DNA is integrated into the cellular DNA, creating an FIV provirus. At this point, the proviral DNA is either transcribed to form genomic or subgenomic RNA and translation occurs, or the virus establishes a latent or persistent form of infection lasting months or years.5

FIV infection progresses through three general stages defined by clinical signs, levels of viremia and CD4+ lymphocyte to CD8+ T lymphocyte ratios (CD4:CD8). A detectable viremia and a sudden decline in CD4+ lymphocytes in the peripheral blood characterize the first stage. The second stage is a clinically asymptomatic phase of variable duration in which low CD4+ cell counts are sustained. The final stage of FIV infection is identified by the classical form of feline acquired immunodeficiency syndrome (AIDS).7

Figure 1 illustrates the genomic structure of FIV. Flanking the viral genes on the 3' and 5' ends of the genome are two Long Terminal Repeats (LTRs). Normally formed during reverse transcription, LTRs serve a variety of functions in viral replication. The LTR contributes to the packaging of viral RNA during virus assembly as well as aiding in insertion of viral DNA into the chromosomal DNA of the host cells. Most importantly, however, the LTR functions to regulate transcription of viral RNA. The FIV transcriptional promoter and regulatory elements are located within the 5' LTR, making it the prime target for CD8+ suppression of virus replication.2

Figure 1. Genomic Structure of FIV

Figure 1. Genomic Structure of FIV

The purpose of this project is to determine the role that CD8+ soluble factors play in inhibiting FIV transcription. Specifically, we hope to determine whether these factors reduce the transcriptional activity of the viral LTR or work at some other region to limit RNA synthesis.

MATERIALS AND METHODS

Animals and Blood Collection

Peripheral blood samples were obtained by phlebotomy from two separate, asymptomatic FIV infected cats designated as 6J2 and 7K11. Both were infected with the FIV-NCSU1 stock of the virus.1 Between forty and fifty milliliters of blood were gathered at each of seven collection periods. The blood was collected in tubes treated with EDTA to prevent agglutination. Peripheral blood mononuclear cells (PBMC) were isolated by discontinuous gradient centrifugation, as described elsewhere.15

Magnetic Separation

Aliquots of PBMC cell suspensions were fractionated into T-cell subsets by use of MiniMACS and MidiMACS immunomagnetic bead-sorting techniques (Miltenyi Biotech) following manufacturer instructions. In short, the lymphocytes were suspended in sorting buffer (Phosphate Buffered Saline [PBS] supplemented with 2mM EDTA, 0.5% bovine serum albumin, and 0.01% sodium azide [pH 7.4]). PBMCs were incubated with monoclonal antibody (MAb) to feline CD8 (3.357-FITC). After washing, the labeled cells were incubated with anti-FITC magnetic beads and were applied to a magnetized column for positive selection of CD8+ T lymphocytes. PBMCs, NonCD8+ PBMCs, and CD8+ lymphocytes were incubated overnight in RPMI 1640 media supplemented with 100U/ml human recombinant interleukin-2.

Flow Cytometry

CD8+ T cell purity and CD4+ to CD8+ ratios were analyzed by dual fluorescence flow cytometry before and after magnetic separation. Unfractionated PBMCs, NonCD8+ PBMCs and CD8+ T lymphocytes were incubated with a combination of MAb CAT30A-bio (developed with streptavidin-phycoerythrin) and MAb 3.357-FITC. Flow cytometry was performed as described elsewhere.8 In each instance CD8+ T cell purity was above eighty-four percent.

Cell Co-culture and Reverse Transcriptase (RT) Assay

The suppressive ability of CD8 cells was assayed by co-cultivation with CD4+ cells. CD8+ cells and NonCD8+ cells, as well as the CD4+ virus infected and non-infected cells were suspended in complete RPMI 1640 media supplemented with 100U/ml human recombinant interleukin-2. A ratio of 4 to 1 CD8+ or NonCD8+ cells to CD4+ cells was established and aliquots of 100mL (10^5 cells) were placed in triplicate in 96-well microtiter plates. The plates were incubated at 37ºC in 5% CO2 for 7 days and then centrifuged at 1000 RPM for 5 minutes for harvesting the supernatants. Supernatants were assayed for Mg++-dependent RT activity as described.9 RT activity corresponds to the level of productive infection in the animals.

LTR-CAT assay

Crandell Feline Kidney cells were transfected overnight with an LTR-CAT gene construct. The cells were then plated in duplicate in 96-well plates and CD8+ T-cells or NonCD8+ cells were added to yield a 1:1 dilution. All cells were suspended in complete media and allowed to incubate for 48 hours. CAT levels were measured by performing a CAT assay as described elsewhere.3

RESULTS

CD8+ T cells and Cell Associated Factors (CAF) suppress productive infection with FIV. In each of the co-culture trials, CD8+ T cells showed an increase in suppressive activity over NonCD8+ PBMCs. This suppressive activity was determined by observation of a lower viral load present in cultures containing CD8+ T-cells.

In some trials, CD8+ cells marked 10-fold greater suppressive ability. In other trials, CD8+ inhibition was only slight, but overall suppressive ability was observed in cultures that contained CD8+ T cells as compared to cultures that only contained infected CD4+ cells (Figure 2).

Figure 2. Average Viral Load Present in Cell Cultures for All Animals (6J2 and 7K11).

Figure 2. Average Viral Load Present in Cell Cultures for All Animals (6J2 and 7K11).

Figure 3. Levels of Chloramphenicol Acetyltransferase (CAT) Produced in the Presence and Absence of CD8 + T-cells in animal 6J2.

Figure 3. Levels of Chloramphenicol Acetyltransferase (CAT) Produced in the Presence and Absence of CD8 + T-cells in animal 6J2.

Results of the LTR-CAT assay confirmed that virus suppressions was mediated in part by a reduction of LTR-mediated transcription. Figure 3 illustrates that levels of Chloramphenicol Acetyltransferase were lower in suspensions containing CD8+ cells than in suspensions where they were absent, demonstrating that transcription occurred at a slower rate in these cultures.

DISCUSSION

Two forms of CD8+ cell mediated antiviral functions are induced during FIV infection. One involves the cytolysis of infected cells. The other results in the suppression of viral replication in infected cells.4 Research has suggested that during HIV infection, a secreted CD8+ cell antiviral factor (CAF) mediates viral suppression.10 This noncytolytic inhibition is important in HIV because a correlation has been found between CD8+ cell response and good clinical prognosis.13

CD8+ noncytotoxic activity involves a non-HLA restricted mechanism which is most active during the asymptotic phase of viral infection.11 In HIV infected patients, CAF-containing fluid suppressed LTR-driven transcription of a luciferase reporter gene in a study very similar to ours.12 As stated before, HIV and FIV are similar enough biologically that it would not be difficult to expect a similar decline in transcription with FIV infection. Since the virus is being inhibited, but not killed by CD8+ cells, the number of FIV-infected cells is not decreased. This fact many be the reason that viral loads are still high in some CD8+ cultures where higher suppressive activity was expected.

These data show that although CD8+ cells are responsible for FIV suppression, NonCD8+ cells also inhibit viral production to some degree (figure 2). Moreover, CD8+ T cell suppression of productive FIV infection is mediated at the level of the viral LTR through reduced transcriptional activation. With this new knowledge, drugs and immune-based therapies could be designed to inhibit viral replication through a similar mechanism.

ACKNOWLEDGEMENTS

We thank Dr. Ayalew Mergia, Ms. Tina Ciccarone, Mr. George Papadi and Ms. Jennifer Mathews for technical support and Mr. Neal Benson for performing flow cytometry for this project.

REFERENCES

  1. English RV, Johnson CM, Gebhard DH, Tompkins MB. In vivo lymphocyte tropism of feline immunodeficiency virus. J. Virol 1993;67:5175-86. Back
  2. Fields, N.B., et al. Virology. New York: Raven Press, Ltd., 1990. Back
  3. Chen, C.H., Weinhold, K.J., Barlett, J.A., Bolognesi, D.P., Greenberg, M.L. AIDS Res. Hum. Retroviruses 1993;9:1079-1086. Back
  4. Gebhard DH, Dow JL, Childers TA, Alvelo JI, Tompkins MB, Tompkins WA. Progressive expansion of an L-selectin-negative CD8 cell with anti-feline immunodeficiency virus (FIV) suppressor function in the circulation of FIV-infected cats. J Infect Dis 1999;180:1503-13. Back
  5. Greene WC. The molecular biology of human immunodeficiency virus type 1 infection. The New England Journal of Medicine 1991;324:308-17. Back
  6. Hohdatsu T, Miyagawa N, Ohkubo M, Kida K, Koyama H. Studies on feline CD8+ cell non-cytolytic anti-feline immunodeficiency virus (FIV) activity. Arch Virol 2000;145:2525-38. Back
  7. Johnson CM, Bortnick SJ, Crawford PC, Papadi GP. Unique susceptibility of the fetal thymus to feline immunodeficiency virus infection: An animal model for HIV infection in utero. AJRI 2001;45:273-88. Back
  8. Johnson CM, Papadi GP, Tompkins WA, et al. Biphasic thymus response by kittens inoculated with feline immunodeficiency virus during fetal development. Vet Pathol 1998;35:191-201. Back
  9. Johnson C, Papadi G, Benson N,. Apoptosis and CD4 lymphocyte depletion following feline immunodeficiency virus infection. Vet Pathol 1996:33:195-203. Back
  10. Knipe, D.M., et al. Fields Virology. New York: Lippincott Williams & Wilkins, 2000. Back
  11. Levy JA, Hsueh F, Blackbourn DJ, Wara D. Weintrub PS. CD8 cell noncytotoxic antiviral activity in human immunodeficiency virus-infected and -uninfected children. J. Infect. Dis 1998;177:470-2. Back
  12. Mackewicz CE, Blackbourn DJ, Levy JA. CD8+ T cells suppress human immunodeficiency virus replication by inhibiting viral transcription. Proc Natl Acad Sci USA 1995;92:2308-12. Back
  13. Stranford SA, Skurnick J, Louria D, Osmond D, Chang S, Snonsky J, Ferrari G, Weinhold K, Lindquist C, Levy JA. Lack of infection in HIV-exposed individuals is associated with a strong CD8+ cell noncytotoxic anti-HIV response. Proc Natl Acad Sci USA 1999;96:1030-35. Back
  14. Tomaras GD, Lacey SF, McDanal CB, Ferrari G, Weinhold KJ, Greenberg ML. CD8+ T cell-mediated suppressive activity inhibits HIV-1 after virus entry with kinetics indication effects on virus gene expression. Proc Natl Acad Sci USA 2000;97:3503-8. Back
  15. Tompkins MB. Ogilvie GK, Gast AM, Franklin R, Weigel R, Tompkins WA. Interleukin-2 suppression in cats naturally infected with feline leukemia virus. J Biol Response Mod 1989;26:86-96. Back

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