ANALYSIS OF THE AVIAN SARCOMA-LEUKOSIS VIRUS OR, AN RNA ELEMENT ESSENTIAL FOR VIRAL REPLICATION: COMPLEMENTATION AND SPONTANEOUS MUTATIONS RESTORE REPLICATION IN A VIRAL VECTOR LACKING THE DR
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Author/Creator ORCID
Date
2000-06
Department
Hood College Biology
Program
Biomedical and Environmental Science
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Abstract
The genomes of avian sarcoma-leukosis viruses (ASLVs) have at least one copy of
an essential element called the direct repeat (DR). The DR, which is located in the 3'
untranslated region of the genome, is required for the production of infectious virus
particles and may have a role in controlling the processing, transport, and packaging of
viral RNA. ASLVs are normally restricted to avian hosts. Previous studies suggest that
the ability of host cell factors to recognize the DR may have a role in the failure of
ASLVs to replicate in mammalian cells. Elements equivalent to the DR have not been
identified in other retroviruses. In contrast to ASLVs, amphotropic murine leukemia
virus (MLV ampho) is capable of replicating in cells from a variety of species,
including mouse, human, and chicken. It is likely that RNA sequences or structures in
the genome of MLV ampho are recognized by factors in all of these cell types. I have
found that a 96-bp sequence from the U3 region of the MLV ampho LTR can substitute
for the DR in an ASLV vector, RCASBP(A), in DF-1. (chicken) cells. Preliminary
experiments indicate that this U3 sequence is required for replication of MLV ampho in
mammalian cells but not in DF-1 cells. After selection, the RCASBP vector with the
MLV U3 element can replicate in human cells expressing the ASLV subgroup (A)
receptor [293R(A) cells]. The chimeric virus was passaged between DF-1 and 293R(A)
cells to improve the efficiency of replication in 293R(A) cells. Passage of the virus
selected for point mutations in the ASLV gag gene, but the virions produced by
293R(A) cells were poorly infectious. Analysis of viral RNA demonstrated that these
particles contain both unspliced and aberrantly spliced RNAs. Insertion (integration) of
DNA copies of the aberrantly spliced RNAs into the genomes of infected cells reduces
the amount of infectious virus produced by 293R(A) cells. These results suggest that
modifications in the sequence of ASLV RNA can affect the recognition and handling of
the RNA by cellular factors.