T-cell activation influences initial DNA synthesis of simian immunodeficiency virus in resting T lymphocytes from macaques

Abstract

The relationship between T-cell activation and early events in the replication cycle of simian immunodeficiency virus (SIV) was analyzed in resting T lymphocytes from macaques. We used the polymerase chain reaction to detect an early product of reverse transcription (R/U5) and almost complete viral DNA (long terminal repeat/gag). We found that SIV can enter resting T lymphocytes and initiate replication but that the reverse transcription process is not efficient and proceeds slowly in resting cells. Cross-linking the CD3/T-cell receptor complex with monoclonal antibodies, unlike cross-linking either the CD28 or CD2 accessory receptor and like phorbol myristate acetate, induced a rapid increase in viral R/U5 DNA detected within 3 to 6 h postinfection. Anti-CD3 or phorbol myristate acetate induced replication of full-length viral DNA within 6 to 9 h postinfection, but full-length SIV DNA was not detectable at earlier time points. We then compared various inhibitors of T-cell activation for their effects on viral initiation and complete replication. Cyclosporin A, an inhibitor of a distal step in T-cell activation, blocked anti-CD3-induced T-cell proliferation and completion of SIV DNA replication but had no effect on induced increases in SIV R/U5 DNA. By contrast, initial SIV DNA synthesis was partially blocked by inhibitors of very early steps in T-cell activation, including herbimycin A, an inhibitor of protein tyrosine kinases, and by two different inhibitors of protein kinase C, H7 and staurosporine. Since resting T cells do not efficiently complete SIV DNA synthesis and cyclosporin A can block the formation of complete viral DNA induced in activated T cells, a cellular factor(s) present in activated T cells appears to be required for the formation of full-length SIV DNA.

Immunodeficiency virus cDNA synthesis in resting T lymphocytes is regulated by T cell activation signals and dendritic cells

“…The molecular events required for human immunodeficiency virus (HIV) or simian immunodeficiency viruses (SIV) to replicate and to be established as proviruses in resting CD4' T cells are not well defined [ 171. However, completion of viral DNA synthesis in the cytoplasm, transport to the nucleus, chromosomal integration, and viral production do not occur unless the T cells are activated [2,4,21,25]. Both linear viral DNA as well as forms of circular DNA can be detected in the nucleus of infected cells [9].…”

Formation of simian immunodeficiency virus long terminal repeat circles in resting T cells requires both T cell receptor- and IL-2-dependent activation.

“…Compared with unstimulated controls, resting macaque T cells stimulated with PMA, anti-CD3, or anti-CD3 plus anti-CD28 mAbs at the time of SIV infection had increased * Dense macaque T cells were cultured for 24 h with FN18 anti-CD3 mAb alone or with 9.3 anti-CD28 mAb in the presence or absence of 1.0/zg/ml CsA. The ratio of env/LTR DNA was calculated as described (10). infected with SIWMne and either were not stimulated (Medium) or were stimulated with CD3 mAb alone as in Fig.…”

“…Thus, each phase of the viral life cycle in T cells is affected by lymphocyte activation signals. Agents such as CsA, rapamycin, or CTLA4.Ig fusion proteins that inhibit distinct steps in T cell activation may be worth considering in combination to block establishment of immunodeficiency virus infection and viral spread (10,29).…”

“…However, the amount of R / U 5 DNA detected after CD3 stimulation alone vs ligation of both CD3 and CD28 receptors was similar. Full-length viral DNA was detectable 6-9 h after infection in stimulated cells but not in unstimulated cells (10).…”

“…A sample dilution of the first round was used in the second round with primers inside the LTR (RO1/U51). One oligonucleotide of each complementary pair was 5' end-labeled with [32p]ATP, and the 32P-labeled PCR products were analyzed by electrophoresis and visualized by autoradiography as described (10). The copy numbers of SIV included in the standard curve generally ranged from 30 to 3,000 copies in the reaction, and the individual standard curves used are indicated in the figures.…”