Publication:
Replication of the 3' LUIII 3' subgenomic clone in human cells

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Authors
Vélez-Pérez, Lisandra
Embargoed Until
Advisor
Diffoot-Carlo, Nanette
College
College of Arts and Sciences - Sciences
Department
Department of Biology
Degree Level
M.S.
Publisher
Date
2004
Abstract
Parvoviruses are small, non-enveloped, icosahedral viruses that contain a linear, single-stranded DNA genome of approximately 5000 bp. Parvovirus LuIII is an autonomous parvovirus that shares up to 80% of sequence homology with the rodent parvovirus MVM and H1, but encapsidates equal amounts of plus and minus strand, identical to AAV and B19. To study the possibility that the termini determine the strand to be encapsidated, a plasmid containing two copies of the left end termini was initially constructed. However, this plasmid known as 3' LuIII 3' has an insertion of unknown origin between the two termini. To eliminate this stuffer sequence, three different strategies were followed. The first two strategies consisted in eliminating the stuffer sequence by digesting the plasmid independently with PmeI and MluI enzymes, and then re-circulating it, and the third strategy consisted in constructing the recombinant molecule de novo. Irrespective of the strategy used, the desired 3' LuIII 3' clone was not obtained. To determine if the original clone 3' LuIII 3', was able of replicating in eukaryotic cells, the plasmid was co-transfected into HeLa cells with the infectious clone pGLu883ΔXba. No evidence of 3' LuIII 3' replication was observed.

Los parvovirus son pequeños virus icosaédricos sin envoltura que contienen un DNA lineal de hebra sencilla de aproximadamente 5000 pb. El virus LuIII es un parvovirus autónomo que comparte hasta un 80% de homología con los parvovirus autónomos MVM y H-1, pero éste encapsida cantidades iguales de hebras positiva y negativa. Este patrón es idéntico a AAV y B-19. Para estudiar la posibilidad de que los terminales estén determinando la polaridad de la hebra que se encapsida, un plásmido que contiene dos copias del terminal 3' fue construido. Sin embargo, este plásmido llamado 3' LuIII 3', tiene una inserción de origen desconocido entre los dos terminales. Tres estrategias distintas se llevaron a cabo para eliminar esta secuencia. Las primeras dos estrategias consistieron en eliminar esta secuencia desconocida cortando el plásmido por separado con las enzimas PmeI y MluI. La tercera estrategia consistió en construir la molécula recombinante de novo. Independientemente de la estrategia utilizada, no se obtuvo un clon 3' LuIII 3' como fuera deseado. Para determinar si el clon original 3' LuIII 3' era capaz de replicarse en células eucarióticas, el plásmido fue co-transfectado con el clon infeccioso pGLu883ΔXba en células HeLa, no observándose evidencia de replicación para el plásmido 3' LuIII 3'.
Keywords
Parvoviruses,
Icosahedral viruses,
Parvovirus LuIII,
Autonomous parvovirus,
Rodent parvovirus MVM and H1,
Plasmid known as 3' LuIII 3'
Cite
Vélez-Pérez, L. (2004). Replication of the 3’ LUIII 3’ subgenomic clone in human cells [Thesis]. Retrieved from https://hdl.handle.net/20.500.11801/810