Studies of <i>deltaretrovirus</i> RNA packaging, infectivity and drug susceptibility
Deltaretrovirus is a genus of the Retroviridae that includes bovine leukemia virus (BLV) and human T-cell leukemia virus types 1 and 2 (HTLV-1 and HTLV-2). This dissertation extends the knowledge for three aspects of deltaretrovirus replication: (1) the identification of a deltaretrovirus RNA packaging signal that is necessary and sufficient for packaging of heterologous RNAs; (2) construction of indicator cell lines to detect deltaretrovirus infectivity; and (3) the sensitivity of BLV to nucleoside reverse transcriptase inhibitors (NRTIs).
A minimal BLV RNA packaging sequence (E) required for packaging of heterologous RNAs into BLV particles was analyzed. The hypothesis that a region previously mapped to contain the BLV primary and secondary packaging signal (BLV E) was necessary and sufficient for RNA packaging of heterologous RNA into BLV particles was tested. The BLV E was inserted into a non-viral vector, pLacZ, in order to determine if packaging of the non-viral vector RNA would occur. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of viral RNA from virus particles revealed that non-viral RNA containing the BLV E was packaged into BLV particles, indicating that the BLV E is necessary and sufficient to allow for packaging of a non-viral vector RNA.
In the second study, mammalian cell lines were created to facilitate analysis of deltaretrovirus replication. The hypothesis tested in this study was that the long terminal repeats (LTRs) of BLV, HTLV-1 or HTLV-2 would drive expression of the green fluorescent protein gene ( gfp) in cells when transactivated by the BLV, HTLV-1 or HTLV-2 Tax proteins, respectively. The BLGFP, H1GFP and H2GFP cell lines detect virus infection by the expression of GFP due to transactivation of the LTR via the respective Tax proteins.
In the third study, the sensitivity of BLV to NRTIs was analyzed. The hypothesis tested was that BLV is susceptible to some nucleoside reverse transcriptase inhibitors that are used to inhibit human immunodeficiency virus type 1 (HIV-1) replication. BLV was found to be sensitive to low concentrations of dideoxyadenosine (ddA), dideoxyinosine (ddl) and 3′-azido-3′ -deoxythymidine (AZT). Interestingly, we observed that BLV was sensitive to (−)2′,3′dideoxy-3 ′-thiacytidine (3TC) even though it was predicted to be resistant.
In summary, this dissertation has determined that a minimal BLV E is necessary and sufficient for RNA packaging of heterologous RNAs into BLV particles. Deltaretrovirus Tax proteins were shown to transactivate each others LTRs and result in GFP expression, which allowed for the development of indicator cell lines for virus infectivity. Finally, this work has demonstrated that BLV is susceptible to NRTIs. (Abstract shortened by UMI.)