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|Title:||Characterization of Pichinde Arenavirus Infection and Replication in Cell Cultures|
|Authors:||Polyak, John Stephen|
|Advisor:||Harnish, Delsworth G.|
|Keywords:||Medical Sciences;Medical Sciences|
|Abstract:||<p>In order to establish a model of arenavirus infection of monocytes, human peripheral blood monocytes (PBM), human promyelocytic HL60 cells and human THP-1 promoncytic cells were infected with Pichinde virus (PV). PV replication was analyzed using a variety of assays which detected viral antigen, RNA and progeny virus. While human PBM were susceptible to PV infection and replication, HL60 cells did not support PV replication, even when cells were induced to differentiate to monocytes with the phorbol ester, PMA. THP-1 cells supported high levels of PV replication only when cells were exposed to PMA. THP-1 cells not treated with PMA supported lower levels of PV replication. Infection of PMA treated THP-1 cells by PV was dependant on protein kinase C (PKC) activation and host cell transcription.</p> <p>The restriction of PV replication in untreated THP-1 cells was characterized further. Experiments with lysosomotropic compounds demonstrated that equal amounts of PV were bound and internalized by both THP-1 cells and PMA treated THP-1 cells. These studies also indicated that PV enters THP-1 cells by endocytosis into acidic vesicles. The expression of PV specific RNAs in PMA treated and untreated THP-1 cells were also examined. PV SRNA genomes, antiger omes, GPC mRNA, NP mRNA and L RNAs were expressed at higher levels in PMA treated THP-1 cells versus untreated THP-1 cells. Degradation of input viral S RNA could not account for the reduction of PV RNA replication in the untreated THP-1 cells. Increasing the multiplicity of infection of untreated THP-1 cells with PV was only able to partially overcome the restriction of virus multiplication. This suggested that the restriction of PV replication in the THP-cells occurred later than the initial binding and penetration stages but at, or just prior to, primary transcription of viral mRNAs. These studies supported a role host cell factors and a dependence on the activation or differentiation state of the THP-1 cell in order to support PV replication.</p> <p>In order to gain further insight into the mechanisms utilized by PV to initiate transcription and replication, the 5' termini of PV S RNA genomes, antigenomes, GPC mRNA and NP mRNA were characterized. All termini sequenced had at least one extra nontemplated base. In clones that contained a single extra nucleotide, this was invariably a G nucleotide. Clones containing single nontemplated G nucleotides were only the derived from PV infected total cellular RNA. The 5' termini of NP and GPC mRNAs had on average 4-8 nontemplated bases. In addition, on genomic sense RNAs the base a -1 did not appear to be conserved. These data have important implication with respect to the mechanisms of PV transcription and replication initiation and are discussed in the context of two possible models.</p>|
|Appears in Collections:||Open Access Dissertations and Theses|
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