HTLV-1 p12I and p30II proteins in viral persistence and pathogenesis

CASE REPORTS

HTLV-1 p12^I and p30^II proteins in viral persistence and pathogenesis

Genoveffa Franchini; Risaku Fukumoto; Mirek Dundr; Valerio William Valeri

HTLV-1 is the only retrovirus known to be the etiologic agent of a human cancer, adult T cell leukemia/lymphoma (ATLL). HTLV-1 as well as some DNA viruses cause lifelong infections. In the case of DNA viruses, the inability of the host's immune response to clear virus-infected cells has been associated with viral latency and/or the ability of viral-encoded proteins to interfere with the host's immune response to the virus, particularly to evade CTLs, which play a major role in cellular immunity against cell-associated viruses.

We hypothesized that this may be the case also for HTLV-1 and focused on both the p12^I and p30^II proteins encoded by alternatively spliced mRNAs from ORFs I and II, respectively, within the 3' end of the viral genome. The HTLV-1 p12^I protein localizes to the ER and Golgi, exhibits weak oncogenic activity, shares aa similarities with the bovine papillomavirus type 1 E5 oncoprotein, and binds to the IL-2R $ and (_c chains. The spliced mRNA encoding p12^I has been detected in in vitro and ex vivo HTLV-1-infected T cells and macrophages. Sera from rabbits experimentally infected with HTLV-1, as well as sera from humans infected with HTLV-1, have been shown to recognize the ORF I product , and a CTL response to ORF I products can be detected in HTLV-1-infected individuals. Ectopic expression in Hela-Tat cells or overexpression of p12^I in PBMCs is associated with enhancement of STAT5 activation and decreased IL-2 requirement for proliferation. This effect however is strictly dependent on activation of T cells by both ligation of TCR and PHA stimulation and is not observed in HTLV-1-immortalized T cells since culture conditions (IL-2 addition) likely compensate for ORF I expression. The work of others has demonstrated that, in the presence of PMA, p12^I induces calcium release and NFAT activation and transcriptional modulation of cellular genes. A seminal finding was that the ablation of p12^I in an infectious clone impairs viral infectivity of primary lymphocytes in vitro and, importantly, in rabbits in vivo. A recent report suggested that p12^I may not be essential for viral transmission. However, this conclusion was drawn from the finding of in vivo p12^I mutations that preserve 84% of the p12^I protein. The HTLV-1 p30^II is a nuclear/nucleolar protein ⁵ and contains a highly conserved bipartite nuclear localization signal (NLS) between aa 71 and 98, which can be functionally substituted for the NLS of Rex. In addition, p30^II contains serine- and threonine-rich regions that share distant homology to the activation domain of transcriptional activators, such as Oct-1/2, Pit-1, and POU-1, that modulates cell gene expression and is important for in vivo infectivity. Thus, both p12^I and p30^II play important roles in viral infectivity in vivo. There is the need for further investigation on which functions of p12^I and p30^II contribute to impaired infectivity in vivo as these proteins may play a key role in viral persistence and mitotic propagation of the virus (reviewed in X).

p12^I binds to the newly synthesized MHC class I heavy chain (MHC class I-Hc) before its association with $₂-microglobulin and targets it for degradation by the proteasome. As a result of the Hc-p12^I interaction, there is a disruption of intracellular trafficking of the MHC class I complex and a significant decrease in MHC class I at the surface of human T cells. We hypothesized that decreased density of MHC class I-viral peptide complexes on the cell surface may impair recognition of HTLV-1-infected cells by CTLs. To test this hypothesis, a relevant system composed of B cells from an MHC class I A2-positive individual and CD8⁺ CTL clones able to recognize the Tax peptide presented by the MHC class I A2.01 was used. Both p12^I and Tax can be expressed in the B cell line as demonstrated by the dose-dependent increase of Tax-induced transactivation of an HTLV-1 LTR-Luc construct. As expected, the expression of p12^I was associated with decreased MHC class I A2 expression on the surface of the human B cells. Cytolytic activity on targeted B cells was observed when more than 0.5 mg of Tax were transfected and p12^I inhibited CTL activity in a dose-dependent manner, demonstrating that p12^I expression can result in CTL killing of human target cells expressing Tax.

Others have shown that p12^I increases calcium release and NFAT activation upon stimulation of T cells with PMA. This effect bypasses antigen receptor ligation, as p12^I increases PMA-induced NFAT activation also in LAT-deficient T cells. Following ligation of TCR by antigen or Abs, TCR signaling occurs through a cascade of events that involves, at first, phosphorylation of protein tyrosine kinase, Lck, and Fyn that, in turn, phosphorylate the tyrosines in the cytosolic domain of TCRz and CD3, which become the docking sites for ZAP70. Activated ZAP70 phosphorylates LAT that binds Grb2, PLC-g1, and the p85 subunit of phosphatidylinositol 3-kinase and probably indirectly Vav, Cb1, and SLP76. The recruitment of these critical molecules to the membrane ultimately causes calcium release, dephosphorylation and nuclear translocation of NFAT, enhancement of transcription, cytokine production, and T cell proliferation. Interestingly, while PMA stimulation of T cells increases NFAT activity, which is enhanced by p12^I-induced calcium release, we observed that, following ligation of the TCR with aCD3e alone, expression of p12^I in Jurkat T cells resulted in a decrease in NFAT activity. In the same experiment, PMA stimulation of cells resulted in increased NFAT activity, as previously described. Both the negative effect of p12^I on TCR signaling and the positive effect following PMA stimulation were dose-dependent. Costimulation with the aCD28 Ab did not restore downregulation of TCR signaling. To investigate the mechanism of p12^I-induced downregulation of NFAT activity, we assessed phosphorylation of cellular proteins following ligation of the TCR. A decreased phosphorylation of several cellular proteins in the range of 100 to 210 kDa was found, and, among them, PLC-g1, Vav, and Cbl; ZAP70, however, was not affected, consistent with a possible effect of p12^I on LAT. Indeed, we found that p12^I binds preferentially to nonphosphorylated LAT. As LAT resides in the rafts ³⁰, we assessed whether p12^I is also localized to the lipid rafts. Flotation assay demonstrated that p12^I is enriched in fractions containing Lck, a raft-associated protein in Jurkat T cells. Similar results were obtained when p12^I was expressed in 293T cells whereby p12^I colocalized with Lyn, a protein found in the raft. To confirm by an independent approach p12^I raft localization, p12^I was expressed in Jurkat T cells, and these were stained with FITC-conjugated cholera toxin (Co-Tx) (FITC-Co-Tx) that binds to the raft ³¹. The Ab to p12^I stained a subset of cells that were also costained by FITC-Co-Tx, and image merging demonstrated colocalization of p12^I and Co-Tx. Thus, p12^I localizes not only to the ER-Golgi but also in lipid microdomains. Antigen-presenting cells (APCs), such as B cells, transduce signals to T cells through the TCR. The TCR located in the rafts translocates to the central core of the T cell supramolecular activation cluster within the cell-cell junction termed the immunological synapse (IS). Because p12^I is found in the rafts and affects TCR signaling, we hypothesized that p12^I may be recruited in the IS. To test this, Jurkat T cells and Raji cells were used as an in vitro model of T cell and APC interaction that involved the TCR and MHC class II. Indeed, polarization of the TCR was seen within 15 min of cocultivation of Jurkat T cells with superantigen staphylococcal enterotoxin E (SEE)-prepulsed Raji B cells. In the same experimental conditions, within 15 min, the p12^I protein expressed in Jurkat T cells coactivated with SEE-prepulsed Raji B cells, polarized together with CD3e to the IS. IS enriched for p12^I was also observed at 30 min from cocultivation. As expected, IS formation was not observed in the absence of SEE.

HTLV-1-Encoded p30^II is a Post-Transcriptional Negative Regulator of Viral Replication

The stoichiometry and the catalytic activity of Tax, the viral transactivator, determine T cell progression through the G1 phase of the cell cycle (reviewed in X). The Rex protein promotes viral production by regulating the transport of genomic and envelope viral mRNAs to the cytoplasm and influences the expression of other cellular genes (reviewed in X). Because Tax is highly immunogenic, it was hypothesized that HTLV-1 in addition to interfering with CTL recognition through p12^I may have evolved a dedicated genetic function to reduce the expression of viral proteins (including Tax) and become transiently dormant. This would help the virus-infected cells evade host immune surveillance, a strategy commonly used by DNA viruses. To this end, the effect on viral replication of the HTLV-1 p30^II protein, encoded by the doubly spliced mRNA from ORF II, was investigated. p30^II was chosen because the nuclear and nucleolar localization of this protein suggested that it might have regulatory function(s). We demonstrated indeed that HTLV-1 has evolved a genetic function to restrict its own expression by a novel post-transcriptional mechanism. The HTLV-1-encoded p30^II is a nuclear-resident protein that binds to and retains in the nucleus the doubly spliced mRNA encoding the Tax and Rex proteins. Because Tax and Rex are positive regulators of viral gene expression, their inhibition by p30^II reduces virion production, and this is true also in human T cell lines HUT102, C91PL, and MT2 chronically infected with HTLV-1. In a collaborative study with P.L. Green and M.D. Lairmore, we also showed that the HTLV-1 p30^II represses by a post-transcriptional mechanism HTLV-2 expression and that HTLV-2 also encodes a protein (p28^II) able to suppress both HTLV-2 and HTLV-1 replication.

The HTLV-1 p30^II has a negative effect on viral replication. Therefore, it could be thought of as a "latency protein." Mechanistic studies on p30^II function and regulation and whether inhibition of p30^II function may reveal hidden infected cells to host immune surveillance are subjects that need further investigation. Our demonstration that p30^II binds to the splice junction for the Tax/Rex mRNA and not to the p21^Rex RNA demonstrates specificity. However, the precise nature of this interaction has not been unveiled.

Publication Dates

• Publication in this collection
  06 Jan 2006
• Date of issue
  Oct 2005