Research within our lab group may be classified under one of five areas:

1. Roles of HIV-1 Reverse Transcriptase in viral genomic mutagenesis and cell-type specificity

We investigate mechanistic and structural characteristics of the HIV-1 RT active site related with enzymatic fidelity and dNTP substrate interactions.  We hypothesize that these enzymatic functions are related with unique genomic mutagenesis, evolution and cell type specificity of HIV-1.  Virological roles of the unique RT functions found in this biochemical study are also tested using various HIV-1 systems including vector systems and in vitro HIV-1 culture.

Selected publications:

·        Weiss, K. K., Bambara, R. A., and Kim, B. (2002) Mechanistic role of residue Gln151 in error prone DNA synthesis by human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). Pre-steady state kinetic study of the Q151N HIV-1 RT mutant with increased fidelity,J Biol Chem 277, 22662-9.

·        Weiss, K. K., Chen, R., Skasko, M., Reynolds, H. M., Lee, K., Bambara, R. A., Mansky, L. M., and Kim, B. (2004) A Role for dNTP Binding of Human Immunodeficiency Virus Type 1 Reverse Transcriptase in Viral Mutagenesis,Biochemistry 43, 4490-4500.

·        Weiss, K. K., Isaacs, S. J., Tran, N. H., Adman, E. T., and Kim, B. (2000) Molecular architecture of the mutagenic active site of human immunodeficiency virus type 1 reverse transcriptase: roles of the beta 8-alpha E loop in fidelity, processivity, and substrate interactions,Biochemistry 39, 10684-94.

2. Changes in fidelity and dNTP substrate interaction of HIV-1 RT during the course of viral infection

Our recent study suggested that the enzymatic fidelity and dNTP interaction of lentiviral RTs, which are hypothesized to be related with viral mutagenesis and cell tropism, can change in response to changes in host immune capability and viral cell type specificity during the course of viral infection. In this project, we characterize RT variants isolated from the HIV-1 infected patients at various infection stages.

Selected publications:

·        Diamond, T. L., Souroullas, G., Weiss, K. K., Lee, K. Y., Bambara, R. A., Dewhurst, S., and Kim, B. (2003) Mechanistic understanding of an altered fidelity simian immunodeficiency virus reverse transcriptase mutation, V148I, identified in a pig-tailed macaque,J Biol Chem 278, 29913-24.

·        Jamburuthugoda VK, Guo D, Wedekind JE, and Kim B (2005) Kinetic Evidence for Interaction of Human Immunodeficiency Virus Type 1 Reverse Transcriptase with the 3'-OH of the Incoming dTTP Substrate. Biochemistry 44(31):10635-43

·        Diamond, T. L., Kimata, J., and Kim, B. (2001) Identification of a simian immunodeficiency virus reverse transcriptase variant with enhanced replicational fidelity in the late stage of viral infection,J Biol Chem 276, 23624-31.

3. Mechanistic comparison between lentiviral and oncoretroviral RTs

Lentiviral RTs (HIV-1 and SIV RTs) and oncoretroviral RTs (MuLV and AMV RTs) have different fidelity and dNTP utilization efficiency.  We predict that these features may influence different mutation rate and cell tropism between these two groups of retroviruses.  We test the generality of these enzymatic differences by characterizing RTs from various viral sources.  We are also examining these differences using retroviruses derived from the same host species, such as in the feline system (i.e. FIV and FeLV) and bovine system (i.e. BIV and BLV).

Selected publications:

·        Operario DJ, Reynolds HM, and Kim B (2005). Enzymatic Comparison of DNA polymerase activities of Feline Immunodeficiency Virus and Feline Leukemia Virus. Virology. 335(1):106-21.

·        Skasko M, Weiss KK, Reynolds HM, Jamburuthugoda V, Lee K, and Kim B (2005) Mechanistic Differences in RNA dependent DNA polymerization and Fidelity between MuLV and HIV-1 Reverse Transcriptases. J. Biol. Chem. 280(13):12190-200.

4. Function analysis of HIV-1 accessory proteins
1. Vpr:  We are testing that HIV-1 Vpr, which induces G2 arrest in the infected T cells, also activate the DNA damage related cell cycle signals such as Chk1 and Cdc2.  We hypothesize that this event enhances cellular dNTP biosynthesis that stimulates viral replication process, particularly in nondividng cells (i.e. macrophages) containing little cellular dNTPs.
2. Tat: We hypothesize that the excretory HIV-1 Tat protein, which is responsible for apoptosis of brain cells and dementia in the HIV-1 infected patients, can activate DNA damage signals particularly in the nondividing bystander cells (i.e. microglia). This can induce higher susceptibility of these bystander cells to HIV-1.  This DNA damage response may also trigger the differentiation of the neighboring brain cells such as GRP cells.
3. Vif/CEM15: We are studying roles of a host antiviral protein, CEM15, in HIV-1 production and infection.

Selected publications:

·        Roshal, M., Kim, B., Zhu, Y., Nghiem, P., and Planelles, V. (2003) Activation of the ATR-mediated DNA damage response by the HIV-1 viral protein R,J Biol Chem 278, 25879-86.

·        Dehart JL, Andersen JL, Zimmerman ES, Ardon O, An DS, Blackett J, Baek Kim, Planelles V. (2005) The ataxia telangiectasia-mutated and Rad3-related protein is dispensable for retroviral integration. J. Virol. 2005 79(3):1389-96.

E. Impact of dNTP affinity change of HIV-1 RT on in vivo viralrecombination frequency

Reduction of the DNA polymerization rate by lowering dNTP substrate concentration promotes frequent stalling, subsequently elevating RNA template cleavage, template switching and viral recombination.  Our laboratory currently employs HIV-1 RT variants isolated from patient samples collected at various stages of HIV-1 infection in order to test our hypothesis that the dNTP interaction mechanism of HIV-1 RT directly contributes to viral genomic recombination, and that HIV-1 variants with varying viral recombination frequency can be produced during the course of viral infection due to the altered dNTP interaction of their RTs.

Selected publications:

·        Purohit V, Balakrishnan M, Kim B, Bambara RA (2005, in press) Evidence that HIV-1 reverse transcriptase employs the DNA 3' end-directed primary/secondary RNase H cleavage mechanism during synthesis and strand transfer. J Biol Chem 280(49): 40534-43.