Abstract
BackgroundPrevious studies have suggested that the process of HIV-1 tRNA primer selection and encapsidation of genomic RNA might be coupled with viral translation. In order to further investigate this relationship, proviruses were constructed in which the primer-binding site (PBS) was altered to be complementary to elongator tRNAMet (tRNAMet(e)) (HXB2-Met(e)) or initiator tRNAMet (tRNAMet(i)) (HXB2-Met(i)). These tRNAMet not only differ with respect to the 3' terminal 18-nucleotides, but also with respect to interaction with host cell proteins during protein synthesis.ResultsConsistent with previous studies, HXB2-Met(e) were infectious and maintained this PBS following short-term in vitro culture in SupT1 cells. In contrast, transfection of HBX2-Met(i) produced reduced amounts of virus (as determined by p24) and did not establish a productive infection in SupT1 cells. The low infectivity of the virus with the PBS complementary to tRNAMet(i) was not due to differences in endogenous levels of cellular tRNAMet(i) compared to tRNAMet(e); tRNAMet(i) was also capable of being selected as the primer for reverse transcription as determined by the endogenous reverse transcription reaction. The PBS of HXB2-Met(i) contains an ATG, which could act as an upstream AUG and syphon scanning ribosomes thereby reducing initiation of translation at the authentic AUG of Gag. To investigate this possibility, a provirus with an A to G change was constructed (HXB2-Met(i)AG). Transfection of HXB2-Met(i)AG resulted in increased production of virus, similar to that for the wild type virus. In contrast to HXB2-Met(i), HXB2-Met(i)AG was able to establish a productive infection in SupT1 cells. Analysis of the PBS following replication revealed the virus favored the genome with the repaired PBS (A to G) even though tRNAMet(i) was continuously selected as the primer for reverse transcription.ConclusionThe results of these studies suggest that HIV-1 has access to both tRNAMet for selection as the replication primer and supports a co-ordination between primer selection, translation and encapsidation during virus replication.
Highlights
Previous studies have suggested that the process of human immunodeficiency virus type 1 (HIV-1) tRNA primer selection and encapsidation of genomic RNA might be coupled with viral translation
Construction of HIV-1 proviruses with primer-binding site (PBS) complementary to tRNAMet(e) or tRNAMet(i) In previous studies, we have described the isolation and characterization of a HIV-1 mutant in which the PBS was complementary to tRNAMet(e) (HXB2-Met(e)) [20,22,31]
Infectivity of HIV-1 with PBS complementary to tRNAMet(e) or tRNAMet(i) To characterize the effects of the PBS mutations on HIV-1 replication, we first analyzed the production of infectious virus following transfection of wild type and mutant proviral genomes. 293T cells were transfected with equal amounts of proviral DNA and the supernatants analyzed for the production of infectious virus using the JC53-BL assay [33]
Summary
Previous studies have suggested that the process of HIV-1 tRNA primer selection and encapsidation of genomic RNA might be coupled with viral translation. In order to further investigate this relationship, proviruses were constructed in which the primer-binding site (PBS) was altered to be complementary to elongator tRNAMet (tRNAMet(e)) (HXB2-Met(e)) or initiator tRNAMet (tRNAMet(i)) (HXB2-Met(i)). These tRNAMet differ with respect to the 3' terminal 18-nucleotides, and with respect to interaction with host cell proteins during protein synthesis. The initiation of reverse transcription occurs at a site near the 5' end of the viral RNA genome termed the primer-binding site (PBS) [3,4,5]. Murine leukemia virus selects tRNAPro, avian leukosis virus selects tRNATrp while lentiviruses, including human immunodeficiency virus type 1 (HIV-1), select tRNALys, as the primer for reverse transcription [8,9,10,11]
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