Molecular biotechnology and retrovirology

Dag E. Helland

UoB

Science
    Faculty

MBI

Research
- Fjose
- Fladmark
- Goksøyr
- Helland
- Jensen
- Lillehaug
- Male
- Raae
- Seo
- Ziegler
- Walther
- Aasland

General background for the research activities.
The main research interest is primarily on the molecular biology of HIV (Human Immunodeficiency Virus). The goal is to increase the knowledge of how the virus replicates in order to support drug development. This virus is studied by a number of different strategies including comparing HIV replication with other lentiviruses. The work is conducted by strong interactions with research groups in Norway and abroad.

It is now 24 years since AIDS (Acquired Immune Deficiency Syndrome) was first described in a publication from the Centers of Disease Control and Prevention in the US. The epidemic caused by HIV (Human Immunodeficiency Virus) is far greater today than what was predicted a decade ago. Almost 25 million people have died from AIDS and more than 30 millions are currently living with HIV. Each day 18000 new infections occur according to WHO which is monitoring the epidemic on worldwide bases.

Existing therapies have shown some promising results in delaying the most severe effects caused by the virus, but these therapies are only available to a small fraction of those living with HIV. The current therapies have their limitations since they are highly toxic and expensive. Also the drugs in use today are unable to purge the body of the virus and often rapid rebound of plasma viraemia is observed.

Development of an effective vaccine against HIV infections remains a pressing challenge. However, how this can be achieved is still today an open question since the last report spring 2003 from a large vaccine trial in humans showed no effect in preventing infections.

Since so little of the pathogenesis of the virus is understood and infection is not neutralized by the strong immune response following the first infection by the virus in addition to the fact that not only neutralizing antibodies but also enhancing antibodies are being produced following infection some scientists are skeptical toward a successful development of an effective preventive or even a therapeutic vaccine.

In addition to research on vaccines, development of new antiviral drugs reducing development of full blown AIDS following HIV infection has to be a top priority for the scientific community and for the supporting and funding agencies.
Development of new drugs requires strong interactions between academic researchers and pharmaceutical industry. The academic research will be laying the theoretical framework for further drug developments.

The aim of this research group in Bergen is to contribute to the enhancement of such a theoretical framework by using the knowledge and research interest in studying functional aspects of the HIV-1 gene products in particular and also by comparing these with gene products from other lentiviruses. As discussed below this will be achieved by a number of different approaches and by conducting the research in collaboration with strong international groups and institutions.

Although the AIDS epidemic is mainly a serious problem in Africa and Asia it represents also a general and serious public health issue for the industrialized countries since HIV infections affect immunization programs.
The multivalent pneumococcal vaccine is ineffective in Africans infected with HIV and live attenuated vaccines such as vaccinia, measles and oral polioviruses may become dangerous pathogens when administered to immune suppressed persons. Furthermore, HIV can convert acute infections into a persistent infection allowing new variants of the infectious agent (e.g. influenza) to develop. HIV positive individuals can also act as evolutionary incubators for microbes like canine Toxoplasma or other microbes that normally depend on an animal reservoir allowing development of new human pathogens. Especially is this a risk when these patients are being treated insufficiently for their opportunistic infections by different antibiotics!

HIV is also an ideal organism for studying functional genomics and proteomics. With all different mutants or variants of HIV or the other lentiviruses identified with nearly the same replication fitness science have a ready-made maximal mutagenesis system at its hands.

HIV and lentiviral replication
Compared to other biological systems HIV and the other lentiviruses have a high frequency of genetic variation.
Since we started working on HIV in 1987 several aspects of this virus have been studied by our group. Currently the main activity is on mechanistic aspects of reverse transcription and integration studied both by in vitro and in vivo systems. The scientific goal is to understand the interactions between the primer (Lys3-tRNA), template, reverse transcriptase (RT) and integrase both trough the early phase of replication as well as in the maturation process of virus particles. Why this polymerase is a heterodimer is also one of the aspects we are studying. Assay systems to screen for inhibitors have been established and several inhibitors identified as part of practical aspects of the studies.

The structure of reverse transcriptase from HIV-1 has been determined by X-ray crystallography. The enzyme is a heterodimer consisting of one p66 subunit and one p51 subunit. The p51 subunit is generated from the p66 subunit by a specific cleavage by the viral protease (PR). The p66 has in addition to the DNA polymerase domain an RNaseH (RH) domain. In spite of having same sequence from the N-terminus the subunits are folded differently. Both subunits have the thumb, finger and palm domains found in all DNA polymerases. The contact between the two subunits is large and mainly hydrophobic.

RT from HIV has low fidelity and it is general thought that this is causing the high mutation rate observed in HIV replication.
The replication of viral RNA to double stranded proviral DNA occurs in a complex consisting of both viral and cellular components called the preintegration complex (PIC). Integration is also thought to occur while the proviral DNA is in such a complex.
The ultimate goal of our research interest is to understand in detail the composition, structure and functions of this complex from infection to integration

We are applying the yeast two-hybrid system to study interaction between the two monomers of the heterodimeric lentiviral reverse transcriptase. Different approaches and strategies are being followed. Currently we are studying formation of dimers between SIV and HIV. This will be extended to study dimer formation between monomers from different lentiviruses. The genetic studies will be complemented with bioinformatic analysis of the sequences from the different lentiviral RTs.
Site directed mutation analysis combined with in vitro coupled transcription and translation is used to study processing and folding of the pol-gene product. If resources get available the group will extend its studies to fidelity of lentiviral reverse transcriptases.

A common characteristic for all the different lentiviruses is that they encode a protein called Tat.
Tat is encoded by the HIV genome and is associated with HIV-LTR driven transcription. We have demonstrated that Tat is secreted from cells and can also be taken up by cells, a process we have defined as transcellular transactivation. Work is in progress to understand the mechanisms behind the transport of this protein across membranes and the effect this protein has on cellular gene activation. We think that Tat plays an important role in the progress of the disease caused by HIV.
The two-exon virus encoded HIV-1 Tat is a multifunctional protein essential for viral replication. After synthesis it is imported to the nucleus where it interacts with TAR, a secondary RNA structure located in the viral long terminal repeat (LTR). The interaction with TAR and cellular factors enhance the transcription of full length viral mRNA by several hundred folds. We were the first to show that Tat can also be released from HIV-1 infected cells and taken up by neighboring cells where it is imported to the nucleus and can perform TAR dependent transactivation. We called this phenomenon transcellular transactivation.

The most studied form of Tat has been the 86-amino acid form of Tat where residues 1-72 are encoded by the first exon. Tat contains several important domains. The area between the amino acids 49-72 is responsible for binding to TAR RNA and includes the basic nuclear localization signal (NLS). Tat is primarily localized to the nucleoplasm and nucleolus of infected or transfected cells. The 86-amino acid form of Tat exists in a few laboratory-passaged virus strains, but represents a truncated form. The Tat form found in most viral sequences encodes a 101-amino acid protein, where residues 73-101 are encoded by the variable second exon. A few isolates contain a 115-amino acid variant.

The aim of our studies is to compare the different forms of Tat and additional mutants with respect to nuclear compartmentalization, oligomerization, nuclear import/export, nucleocytoplasmic shuttling and expression of cellular and viral genes. To conduct these studies a number off different approaches are taken.
The study of cellular localization is accomplished by fusion of the different forms and mutants of Tat to the green fluorescent protein (GFP) or red fluorescent protein (RFP). The two-exon coding GFP-coupled variants; 86, 101 and 115 showed a predominantly nuclear localization, with concentrated nucleolar staining and a more diffuse and speckled nucleoplasmic staining. This speckled pattern was compared to the speckled pattern seen for the splicing factor SC35, and colocalization was observed between Tat speckles and SC35 speckles. The one-exon (72-amino acids) coupled to GFP localized in the same pattern as the two-exon variants. All Tat constructs were tested for functionality by using a stable cell line containing the provirus with a mutation in the tat gene. No virus and syncytia is observed without the addition of Tat. In a heterokaryon assay the GFP-Tat86, GFP-Tat101, GFP-Tat115 and GFP-Tat-72 all showed nucleocytoplasmic shuttling capabilities.

The two-exon double spliced form of Tat is expressed early in the viral infection, while the single spliced form is expressed later. We have discovered that HIV-1 Tat 101 contains two nuclear localization signals (NLS) located in exon 1 and exon 2, receptively. The human ribosomal protein L5 has also been shown to contain two NLSs. The reason for two NLSs are not known and a Tat101 mutant has been constructed to further investigate this phenomena.

A Tat deletion mutant missing the first 20 aa, thus the proline rich area, was unable to induce syncytia formation in the functionality assay.
Maedi Visna Virus (MVV) is a retrovirus of the lentivirus family, which infect sheep. The two main clinical disorders in animals infected with MVV is a chronic interstitial pneumonia or slow progressive encephalitis, which may evolve to total paralysis and death.
The MVV Tat protein is encoded by one exon in contrast to the HIV Tat protein that is encoded by two. The MVV Tat protein, like the HIV Tat protein, is in involved in stimulation of gene expression directed by the viral long terminal repeat (LTR). The MVV Tat protein mediates the accumulation of viral mRNA via the AP-1 (activator protein-1) and AP-4 binding sites in the U3 region of the LTRs or via cellular factors such as c-Fos and c-Jun. The mechanism of transcriptional transactivation by MVV Tat, however, is not well understood and MVV Tat belongs to a group of Tat proteins characterized by a weak transactivation potential. Unlike HIV Tat, which strongly transactivates their LTR by binding to a TAR (transactivation response element) sequence, MVV does not have a TAR-like structure in its LTR region and MVV Tat has not been found to bind either RNA or DNA directly. A leucine-rich domain present in MVV Tat is likely to be responsible for targeting the Tat protein at AP-1 sites in the viral LTR. MVV Tat is not able to transactivate the HIV-1 LTR.

The localization of the MVV Tat protein has not been much studied so the aim of this study is to present the intracellular localization of MVV Tat by construction of a Green Fluorescent Protein (GFP) fusion protein, identify its nuclear localization signal, investigate the role of its leucine rich domain in nuclear export and its ability for nucleocytoplasmic shuttling. The GFP-MVV-Tat is concentrated in the nucleus with an evenly distribution in the nucleolus and the nucleoplasm. In addition it is seen diffusely in the cytoplasm, in cytoplasmic dots and in connections between cells. Two mutants have been made and fused to the Red Fluorescent Protein (RFP). Arginines have been changed to alanines in the first mutant in order to interrupt MVV Tat´s unknown NLS, in the second mutant the leucine rich area have been mutated. The localization of these mutants is ongoing studies. For test of functionality an MVVLTR-RFP-CAT construct was made.

Gene therapy and DNA mediated vaccination
Intrabodies made from the different antibodies raised against HIV protein in our laboratory are being used to construct single chain antibodies. The effects of these different intrabodies on HIV replication are studied in tissue culture systems. Inhibition of replication has been obtained with some intrabodies.
Our studies of retrovirus replication are also of general interest for development of gene therapy since retroviruses might be important vectors for delivering genes in such therapy.

Immunizing mice with plasmids containing a eukaryotic promoter and the sequences encoding the protein of interest is an established method developed over the last years. We are interested in this both as a method to generate antibodies of interest for other projects, to study the potential of this method as a vaccine strategy and to study safety aspects of DNA vaccination. Currently we are focusing on the horizontal and vertical transformation of plasmid injected into animals and the genetic stability of the plasmids.

AIDS vaccines
Currently we are participating in a large European consortium with groups from Italy and Sweden in developing new strategies for an AIDS vaccine. The project is funded by through EU as part of the STREP program and called VIAV (Very innovative AIDS vaccine). The funding is for 2005 and 2006.

Molecular systematics.
Together with members of the Institute of fisheries and marine biology. Center of Virology and Department of Botany we are establishing an activity in molecular systematic. The organisms studied so far include HIV, maedi virus, influenza virus, algae, legions and higher plants
The research has been supported by The Norwegian Research Council (NFR), The Norwegian Cancer Society and "L. Meltzer's Legat" and grants from the University of Bergen.

Recent desertations from the group:

PhD-thesis:
Vibeke Andresen, December 2004; “The subcellular localization,function and trafficking of lentiviral Tat proteins”

Master thesis:

Siri Bakke, June 2005; “A genetic approach to study heterodimerization of HIV-1 Reverse Transcriptase by site-directed mutagenesis using the yeast-two-hybrid system: A special emphasis on the role of the RNase H domain”

Silje S. Markussen, June 2005; “Establishing a Yeast Three-Hybrid System to study Ternary Complex formation between Nucleocapsid and the Heterodimer Reverse Transcriptase of HIV-1”

Christel Gill Haanshuus, August 2005; “A Study of Heterodimerization of HIV-1 Reverse Transcriptase Using Deletion Mutations in the RNase H Domain”

Selected references:
Helland, D.E., Welles, J.L., Caputo, A. and Haseltine, W.A. (1991) Transcellular Transactiviation by the HIV-1 tat Protein. J. Virol., 65: 4547 – 4549

Szilvay, A.M., Nornes, S., Haugan, I.R., Olsen, L., Prasad, V.R., Endresen, C., Goff, S.P., and Helland, D.E. (1992) Epitope mapping of HIV-1 reverse transcriptase with monoclonal antibodies which inhibit the polymerase and the RNaseH activities. J. Acq. Imm. Def. Syndr. 5: 647-657

Shilatifard, A., Merkle, R.M., Helland, D.E., Welles, J.L., Haseltine, W.A., Cummings, R.D. (1993) The Complex-Type N-Linked Oligosaccharides of gp120 from HIV-1 Contain Sulfated N-Acetylglucosamine Residues. J. Virology 67: 943-952

Mhashilkar, A.M., Bagley, J., Chen, S.Y., Szilvay, A.M., Helland, D.E., Marasco, W.A. (1995) Inhibition of HIV-1 Tat-Mediated LTR Transactivation and HIV-1 Infection by Anti-Tat by Anti-Tat Single Chain Intrabodies. EMBO J. 14, 1542-1551

Valvatne, H., Szilvay, A.M., and Helland, D.E. (1995) A monoclonal antibody defines a noval HIV- Type 1 Tat domain involved in trans-cellular trans-activation. AIDS Research and Human Retroviruses, 12: 611 - 619

Nilsen, B.M., Haugan, I.R., Berg, K., Olsen, L., Brown, P.O., and Helland, D.E. (1996) Monoclonal antibodies raised against HIV-1 integrase: epitope mapping and differential effects on integrase activities in vitro. J. Virol. 70, 1580-1587

Szilvay AM, Stern B, Blichenberg A and Helland DE (2000) Structureal and functional similarities between HIV-1 reverse transcriptase and the Escherichia coli RNA polymerase _´ subunit. FEBS letters, 24219, 1 – 5