Expression kinetics and subcellular localization of HIV-1 regulatory proteins Nef, Tat and Rev in acutely and chronically infected lymphoid cell lines

Abstract

Information concerning the expression kinetics and subcellular localization of HIV regulatory proteins is of importance in understanding the viral pathogenesis and may be relevant for drug and vaccine development, as well. We have used combined immunocytochemistry and in situ hybridization to study firstly, the order of expression of regulatory HIV-1 proteins Nef, Rev and Tat in relation to non-spliced and spliced mRNA expression and secondly, the subcellular localization of these proteins in acutely and chronically infected human T-cell lines. We used monoclonal antibodies against HIV-1 Nef, Tat, Rev and gp160, and RNA probes reacting either with all mRNAs (nef) or only with the full-length mRNA (gag-pol). In acutely infected MT-4 and H9 cells, four distinct phases of infection could be defined. In the first phase lasting from 0 to 6 h post-infection, only incoming virus could be demonstrated by gp160 immunocytochemistry. During the second, regulatory phase (6-9 h), abundant cytoplasmic expression of Nef, Rev and Tat proteins and a positive in situ RNA hybridization with the nef probe was seen, while the in situ hybridization with full-length mRNA probe and immunohistochemistry for gp160 were still negative. The productive phase (12-48 h) was characterized by abundant expression of full-length mRNA and gp160, and by the nuclear localization of Nef and Tat proteins. In contrast, an antibody that recognized the RRE binding region of the Rev protein localized Rev in the cytoplasm both during the regulatory and productive phase. During the fourth, cytopathic phase, the expression of mRNA or viral proteins decreased and the regulatory proteins studied were again mainly localized in the cytoplasm. Based on the results, we speculate that HIV Nef may function as a nuclear factor, and that Tat is possibly bound by cellular proteins before its transport to the nucleus.

Nef-Mediated Resistance of Human Immunodeficiency Virus Type 1 to Antiviral Cytotoxic T Lymphocytes

“…Antagonizing the function of Nef pharmacologically could be a means of increasing the efficiency of protective CTL responses. Because the Nef protein is expressed early in the viral life cycle (45), it is unclear whether Nef-specific CTL could be more effective than other CTL, by recognizing infected cells earlier and before the onset of Nef-mediated effects. Another strategy could be to produce or enhance MHC-I C-restricted CTL responses.…”

Human immunodeficiency virus type 1 Rev- and Tat-specific cytotoxic T lymphocyte frequencies inversely correlate with rapid progression to AIDS.

“…Secondly, in the replicative cycle, Rev and Tat are expressed earlier than the structural proteins. This may allow specific CTL to kill infected cells well before release of progeny virus (Ranki et al, 1994 ;Riviere et al, 1994). Although both considerations would also hold true for Nef, specific CTL responses to this protein were observed in most progressors.…”

“…Recent reports on virus escape from HIV-1-specific CTL responses clearly indicate that CTL exert pressure on virus replication in vivo, but that the overall CTL efficacy may be dependent on the (lack of) conservation in the epitope sequences (Borrow et al, 1997 ;Goulder et al, 1997). In addition, it has been suggested that CTL responses against early expressed epitopes may be more efficacious than those against late expressed epitopes (Ranki et al, 1994 ;Riviere et al, 1994). Qualitative and quantitative analyses of specific CTL responses, before the immune status has deteriorated, may identify the requirements for a CTL response to be protective.…”

Improved Vaccine Protection from Simian AIDS by the Addition of Nonstructural Simian Immunodeficiency Virus Genes

“…HIV-1 encodes an additional protein, Vpu, but lacks the open reading frame for Vpx. After viral entry, reverse transcription, and integration of the viral genome, the first genes expressed by HIV/ SIV are completely spliced mRNAs that encode for the nonstructural Tat, Rev, and Nef proteins (12)(13)(14). Only when Rev reaches a sufficient level are the incompletely spliced Env mRNA and the genomic RNA encoding for Env and Gag-Pol, respectively, transported to the cytoplasm for translation.…”