Anne Marie Szilvay

The human immunodeficiency virus type 1 Rev protein shuttles between the cytoplasm and nuclear compartments.

A retroviral regulatory protein, Rev (regulator of virion protein expression), is made in cells infected by human immunodeficiency virus (HIV). Rev is essential for the completion of the retroviral life cycle and interacts with the host cell at some posttranscriptional step in order to express the incompletely spliced HIV mRNAs from which HIV structural proteins are translated. Neither the host cell components nor the mechanisms responsible for this important regulation have been defined. We now report that Rev is a nucleocytoplasmic shuttle protein which is continuously transported between the cytoplasm, the nucleoli, and nucleoplasmic speckles enriched in RNA splicing and processing factors. The results show that Rev has the potential to interfere specifically with the splicing of the HIV pre-mRNA in the nucleoplasm and, next, guide such mRNAs to the cytoplasm for translation.

Spatial association of HIV-1tat protein and the nucleolar transport protein B23 in stably transfected Jurkat T-cells

SummaryThe human immunodeficiency virus (HIV-1) encodes a transactivator protein, the product of the tat gene (tat), which is essential for virus replication. In this study, immunogold electron microscopy was used in a stably transfected Jurkat T-cell line that constitutively expresses HIV-1tat protein to determine the subcellular and intranuclear distribution oftat protein. Two nucleocytoplasmic shuttle proteins C23/nucleolin and B23 and a third nucleolar antigen that was detected by monoclonal antibody MAb 1277 were also examined. In addition, spatial association of C23 and B23 withtat protein at several subcellular locations was examined in dual-labeling experiments. The results showed thattat protein was found in both the cytoplasm and nucleus but was especially prominent within the dense fibrillar and granular components of the nucleolus. There was little labeling oftat protein in the fibrillar centers where MAb 1277 antigen was localized at a comparatively high level. The subcellular and intranucleolar distribution oftat protein was virtually identical to the pattern seen with C23 and B23. Although the intranuclear distributions of C23, B23 andtat protein were very similar, C23 andtat protein were seldom spatially associated. In contrast, B23 andtat protein were frequently spatially associated in the nucleolus and in several other subcellular locations including the cytoplasm, nucleoplasm, at the nuclear envelope and plasma membrane. While a physical association was not directly demonstrated in this study, the spatial association between B23 andtat protein strongly suggest that such an association may exist.

Probing the structure of HIV-1 Rev by protein footprinting of multiple monoclonal antibody-binding sites

Human immunodeficiency virus type 1 (HIV‐1) Rev is a small RNA‐binding protein which is essential for viral replication. To investigate the structure of Rev we have mapped the binding sites of a panel of monoclonal antibodies (mAb) by protein footprinting and identified a mAb protecting amino acids within both the N‐ and C‐terminal parts of Rev. Our mapping results support a previously proposed structure (Auer et al., Biochemistry, 33 (1994) 2988–2996) predicting that a helix–loop–helix motif in Rev brings the termini of the protein into proximity. Furthermore, we demonstrate that the binding sites mapped by protein footprinting are in agreement with conventional epitope mapping results and that this technique provides an advantageous strategy for mapping discontinuous sites.

B2 protein from betanodavirus is expressed in recently infected but not in chronically infected fish.

Betanodavirus infects both larvae and juvenile fish and can cause the disease viral encephalopathy and retinopathy (VER). During an acute outbreak of VER, infected individuals display several clinical signs of infection, i.e. abnormal swimming pattern and loss of appetite. Betanodaviruses can also cause chronic or persistent infection where the infected individuals show no clinical signs of infection. During infection the viral sub-genomic RNA3 and the RNA3-encoded B2 protein are expressed. Antibodies against the B2 protein from Atlantic halibut nodavirus were raised and used together with antibodies against the capsid protein to detect the presence of these 2 viral proteins in infected cells in culture and at different stages of infection in Atlantic halibut Hippoglossus hippoglossus and Atlantic cod Gadus morhua. The B2 protein was detected in recently infected, but not in chronically infected fish. Results suggest that the detection of B2 may be used to discriminate a recent and presumably active infection from a chronic and presumably persistent infection.

Co-expression of a trans-dominant negative mutant of the human immunodeficiency virus type 1 (HIV-1) Rev protein affects the Rev-dependent splicing pattern and expression of HIV-1 RNAs.

Trans-dominant negative mutants of the human immunodeficiency virus type 1 (HIV-1) regulatory protein Rev inhibit the function of wild-type Rev in a dose-dependent manner. This was previously shown to be caused by nuclear retention of the wild-type protein. In the present work, further analysis of the trans-dominant negative effect was performed using cotransfection experiments with different constructs encoding HIV-1 Rev and viral structural proteins together with a plasmid encoding a trans-dominant negative Rev mutant. Thus, one species of pre-mRNA was transcribed from the reporter plasmids. This pre-mRNA was then either spliced or exported by Rev as unspliced RNA for translation of the HIV structural proteins. An immunofluorescence assay and Western blot analysis were used for analysis of protein expression. In situ hybridization was applied for labelling of unspliced mRNA in transfected cells, and RNase protection analysis was used to determine the relative amount of unspliced versus spliced mRNAs. The experiments confirmed that the transdominant negative mutant inhibited nuclear export of unspliced mRNA. It was, in addition, demonstrated for the first time that the trans-dominant negative mutant also affected a Rev-dependent regulatory step connected with viral pre-mRNA splicing. As a consequence, proteins expressed from unspliced and singly spliced HIV mRNAs decreased while there was an increase in protein products encoded by spliced and alternatively spliced mRNAs.

HIV-1 Rev oligomerization is not obligatory in the presence of an extra basic domain

BackgroundThe HIV-1 Rev regulatory protein binds as an oligomeric complex to viral RNA mediating nuclear export of incompletely spliced and non-spliced viral mRNAs encoding the viral structural proteins. However, the biological significance of the obligatory complex formation of Rev upon the viral RNA is unclear.ResultsThe activity of various fusion proteins based on the negative oligomerization-defect Rev mutant M4 was tested using Rev dependent reporter constructs. An artificial M4 mutant dimer and an M4 mutant containing an extra basic domain from the HTLV-I Rex protein exhibited nearly full activity when compared to wild type Rev.ConclusionRev dimerization appears to be required to expose free basic domains whilst the Rev oligomeric complex remains bound to viral RNA via other basic domains.

Histographic recording of human immunodeficiency virus type 1 (HIV-1) regulatory protein Rev and nuclear factors

SummaryHeLa cells and HeLa cells expressing the HIV-1 regulatory protein Rev were immunostained for Rev and pre-mRNA processing factors and examined histographically by confocal laser scanning microscopy. Following short pulse-labelling with bromouridine tri-phosphate nascent RNA gave a granular nucleoplasmic staining increasing somewhat towards the periphery as did also the heterogeneous ribonucleoproteins (hnRNPs) Aff1 and particularly C1/C2, a distribution pattern which has not been described. The sm-antigen of the small ribonucleoprotein particle (snRNP) proteins U1, U2, U4/U6 and U5 stained the nucleoplasm diffusely in addition to speckles which co-localised with speckles of the non-snRNP splicing factor SC-35. Brominated RNA and the hnRNPs Aff1 and C1/C2 were to varying degrees excluded from the speckles. Rev concentrated in the nucleolus and often as a perinucleolar ring/zone. Rev also stained the nucleoplasm and cytoplasm without co-localising with the above-mentioned proteins or brominated RNA and was not enriched or excluded in SC-35 speckles. The nucleolar proteins B23 and C23, like Rev, gave primarily a perinucleolar ring and stained the nucleoplasm but did not otherwise co-localise with Rev or with nuclear proteins. Histographic recording of immunofluorescence images proved to be a valuable tool in the study of localisation of HIV-1 Rev and cellular components and of possible co-localisations. A parallel comparison of the subcellular patterns of pre-mRNA processing factors versus major nucleolar antigens is new and suggests that the factors are not strictly separated in the nucleoplasm.

Characterization of HIV-1 reverse transcriptase with antibodies indicates conformational differences between the RNAse H domains of p 66 and p 15

SummaryAntibody binding to the p66 and p 15 RNase H regions of HIV-1 reverse transcriptase was compared using a polyclonal rabbit immune serum raised against a synthetic peptide from the RNase H region of reverse transcriptase (aa 511–527) and six monoclonal antibodies binding to discontinuous epitopes in the RNase H region of p66. The antigens used in Western blot analysis included recombinantly expressed homodimeric p 66 digested with the HIV-1 protease for generation of the p 51 and p 15 polypeptides and two different length RNase H domains expressed as Trp E fusion proteins (aa410–560 and aa 441–560). The polyclonal rabbit antibody binding to a continuous epitope recognized both the Trp E-fusion proteins and also the polypeptides p 66 and p 15 generated by processing of homodimeric p66 with the viral protease. Two additional cleavage products with estimated molecular weights of 9 and 11 kDa were also detected. The anti-RNase H MAbs binding to discontinuous epitopes recognized only the RNase H domain of the p 66 polypeptide and the Trp E-RNase H fusion protein when this was expressed together with the C-terminal part of the polymerase domain. The results indicate conformational differences between the RNase H domain of the p66 subunit and the RNase H p 15 polypeptide.

Structural and functional similarities between HIV-1 reverse transcriptase and the Escherichia coli RNA polymerase β′ subunit

Four monoclonal antibodies (MAbs) recognizing HIV‐1 reverse transcriptase (RT) were shown here to cross‐react with the β′ subunit of Escherichia coli RNA polymerase (RNAP). The anti‐RT MAbs bind to a peptide comprising residues 294–305 of the RT amino acid sequence. Computer analyses revealed sequence similarity between this peptide and two regions of the RNAP β′ subunit. MAb‐binding studies using RT mutants suggested that the epitope is located to amino acids 652–663 of the β′ sequence. One of the MAbs which inhibited the polymerase activity of RT also mediated a dose dependent inhibition of the RNAP activity.

Functional rescue of an oligomerization-defective HIV-1 Rev mutant by fusion with an oligomeric tag.

SummaryThe HIV-1 wild-type Rev and the negative oligomerization-defective mutant RevM4 were fused to enhanced green fluorescent protein (EGFP) and the tetrameric red fluorescent protein (DsRed1) followed by examination of their intracellular localization and Rev activity. As previously shown, fusion of EGFP to Rev and RevM4 did not affect the biological activity. Fusion of DsRed1 to Rev inhibited activity, and interestingly, fusion of DsRed1 to RevM4 restored activity. Based on these results, a model is proposed suggesting how RevM4-DsRed1 is able to rescue Rev activity through oligomerization on the viral RNA.