216 research outputs found
The G490E mutation in reverse transcriptase does not impact tRNA primer selection by HIV-1 with altered PBS and A-loop
AbstractThe initiation of HIV-1 reverse transcription utilizes a cellular tRNALys,3 as a primer. The 3′ terminal 18-nucleotides of the cellular tRNALys,3 are complementary to a region on the viral genome, designated as the primer binding site (PBS). Previous studies have shown that forcing HIV-1 to utilize alternative tRNA primers through alteration of the PBS results in viruses that revert to utilize tRNALys,3 following in vitro replication. In some instances though, HIV-1 has been shown to select alternative tRNAs for initiation of reverse transcription if additional mutations upstream in the U5 region (A-loop) were made to be complementary to these alternative tRNAs. Recently, an HIV-1 has been described in which the U5 region distinct from the A-loop, designated as the primer activation site (PAS), was mutated in conjunction with the PBS to force the virus to use tRNALys1,2 as a primer. An additional mutation in reverse transcriptase (RT), G490E, was found to facilitate the forced use of tRNALys1,2 as the primer. In the current study, we have investigated the impact of the G490E mutation in RT on the selection and use of alternative primers by HIV-1. Viruses were first constructed in which the PBS and A-loop region were made complementary to tRNATrp. Previous studies from our laboratory have shown that these viruses are unstable and mutate to select tRNAMet or tRNALys1,2. Analysis of the replication of viruses with the U5 and PBS complementary to tRNATrp with or without the G490E mutation revealed no significant differences with respect to infectivity and viral growth in SupT1 or peripheral blood mononuclear cells (PBMC). In addition, the presence of the G490E mutation did not influence the capacity of this virus to revert to utilize tRNAMet as the primer for initiation of reverse transcription. In a previous study, we have described an HIV-1 that has been forced to utilize tRNALys1,2 through mutations in the A-loop and PBS. The G490E RT mutation in this virus did not impact on the virus infectivity or growth in SupT1 or PBMC. We did not find a significant fitness advantage to viruses in which the A-loop and PBS were made complementary to tRNALys1,2 that also contained the G490E mutation in RT. The results of these studies then establish that HIV-1 can be forced to use alternative primers through mutations in the U5 (PAS or A-loop) for certain tRNAs. Furthermore, for mutations in the A-loop and PBS, the RT does not play an important role in dictating the selection of the alternative primers to be used for initiation of reverse transcription
Mutations within the Primer Binding Site of the Human Immunodeficiency Virus Type 1 Define Sequence Requirements Essential for Reverse Transcription
AbstractThe primer binding site (PBS) is involved in two stages during the reverse transcription of the retroviral RNA genome. In the early stage, the PBS provides complementary sequences through which tRNALys,3binds the viral RNA genome to initiate minus-strand DNA synthesis; in the later stages, complementarity between the plus- and minus-strand copies of the PBS is required to facilitate the second template transfer needed to complete reverse transcription. We previously constructed a mutant HIV-1 proviral genome, designated as pHXB2PBS(pheC + 5) (now referred to as pheC + 5), which was used to identify regions of the PBS involved in the initiation and second template transfer steps of reverse transcription. To further define the sequence requirements of the PBS for the initiation of reverse transcription, we have made single nucleotide substitutions within the first six nucleotides of the pheC + 5 PBS. Our results demonstrate that mutations within the first five nucleotides of the PBS which disrupt base paring with tRNALys,3-PBS results in an noninfectious virus; a G-U base pair at position six of the tRNALys,3-PBS complex was tolerated. In contrast to the requirements for initiation, we found that complementary binding between only three base pairs of the plus- and minus-strand PBSs was required for the extension of plus-strand DNA during the second template transfer. Furthermore, regions of the minus-strand DNA of up to 24 nucleotides could be looped-out to facilitate the complementarity required for the completion of plus-strand DNA synthesis. Taken together, the results of our studies demonstrate that different features of the PBS with respect to RNA:RNA and DNA:DNA interactions are required for initiation of reverse transcription and the completion of plus-strand DNA synthesis, respectively
Analysis of the replication of HIV-1 forced to use tRNA(Met(i) )supports a link between primer selection, translation and encapsidation
BACKGROUND: 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 tRNA(Met )(tRNA(Met(e))) (HXB2-Met(e)) or initiator tRNA(Met )(tRNA(Met(i))) (HXB2-Met(i)). These tRNA(Met )not only differ with respect to the 3' terminal 18-nucleotides, but also with respect to interaction with host cell proteins during protein synthesis. RESULTS: Consistent 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 tRNA(Met(i) )was not due to differences in endogenous levels of cellular tRNA(Met(i) )compared to tRNA(Met(e)); tRNA(Met(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 tRNA(Met(i) )was continuously selected as the primer for reverse transcription. CONCLUSION: The results of these studies suggest that HIV-1 has access to both tRNA(Met )for selection as the replication primer and supports a co-ordination between primer selection, translation and encapsidation during virus replication
Mutations in the TΨC Loop of E. coli tRNA(Lys,3 )Have Varied Effects on In Trans Complementation of HIV-1 Replication
BACKGROUND: Human immunodeficiency virus (HIV-1) exclusively selects and utilizes tRNA(Lys,3 )as the primer for initiation of reverse transcription. Several elements within the TΨC stem loop of tRNA(Lys,3 )are postulated to be important for selection and use in reverse transcription. The post-transcriptional modification at nucleotide 58 could play a role during plus-strand synthesis to stop reverse transcriptase from re-copying the tRNA primer. Nucleotides 53 and 54 within the TΨC stem loop of the tRNA have been shown to be important to form the complex between tRNA and the HIV-1 viral genome during initiation of reverse transcription. RESULTS: To further delineate the features of the TΨC stem loop of tRNA(Lys,3 )in reverse transcription, we have developed a complementation system in which E. coli tRNA(Lys,3 )is provided in trans to an HIV-1 genome in which the PBS is complementary to this tRNA. Successful selection and use of E. coli tRNA(Lys,3 )results in the production of infectious virus. We have used this single round infectious system to ascertain the effects that different mutants in the TΨC stem loop of tRNA(Lys,3 )have on complementation. Mutants were designed within the TΨC loop (nucleotide 58) and within the stem and loop of the TΨC loop (nucleotides 53 and 54). Analysis of the expression of E. coli tRNA(Lys,3 )mutants revealed differences in the capacity for aminoacylation, which is an indication of intracellular stability of the tRNA. Alteration of nucleotide 58 from A to U (A58U), T54G and TG5453CC all resulted in tRNA(Lys,3 )that was aminoacylated when expressed in cells, while a T54C mutation resulted in a tRNA(Lys,3 )that was not aminoacylated. Both the A58U and T54G mutated tRNA(Lys,3 )complemented HIV-1 replication similar to wild type E. coli tRNA(Lys,3). In contrast, the TG5453CC tRNA(Lys,3 )mutant did not complement replication. CONCLUSION: The results demonstrate that post-transcriptional modification of nucleotide 58 in tRNA(Lys,3 )is not essential for HIV-1 reverse transcription. In contrast, nucleotides 53 and 54 of tRNA(Lys,3 )are important for aminoacylation and selection and use of the tRNA(Lys,3 )in reverse transcription
Importance of A-loop complementarity with tRNA(His )anticodon for continued selection of tRNA(His )as the HIV reverse transcription primer
BACKGROUND: Human immunodeficiency virus (HIV-1) preferentially selects tRNA(Lys,3 )as the primer for reverse transcription. HIV-1 can be forced to select alternative tRNAs through mutation in the primer-binding site (PBS) and a region upstream of the PBS designated as the A-loop. Alteration of the PBS and A-loop to be complementary to the 3' terminal nucleotides and anticodon of tRNA(His )results in HIV-1 that can stably utilize this tRNA for replication. RESULTS: In the current study, we have investigated the effect that mutations within the A-loop have on the stability of HIV-1 with a PBS complementary to tRNA(His). For these studies, we have altered the A-loop to be complementary to tRNA(Met), tRNA(Gln), tRNA(Ile), tRNA(Thr )and tRNA(Ser). All substitutions of the A-loops with the PBS complementary to tRNA(His )resulted in a reduction of infectious virus obtained following transfection of proviral genomes in the 293T cells. Virus replication in SupT1 cells was also impaired as a result of the alteration of the A-loop. Viruses with the A-loop complementary to tRNA(Lys,3 )and tRNA(Ser )reverted to utilize tRNA(Lys,3 )following in vitro replication. In contrast, viruses with the A-loop complementary to the other tRNAs remained stable and continued to use tRNA(His). RNA modeling of the stem-loop structure revealed that nucleotides were displayed on the loop region that could potentially interact with the anticodon of tRNA(His). To further explore the effects of the A-loop mutations on virus replication, the A-loops complementary to tRNA(Ser )or tRNA(His )were cloned into the wild type genome with the PBS complementary to tRNA(Lys,3). Transfection of proviral genomes which contained the wild type PBS and A-loops complementary to tRNA(Ser )or tRNA(His )into 293 T cells did not impact on the production of viruses as measured by p24 antigen ELISA. However, viruses with the A-loop complementary to tRNA(His )had greatly reduced infectivity and replicated poorly in SupT1 compared to the wild type or viruses with the A-loop complementary to tRNA(Ser). CONCLUSION: These studies demonstrate that complementarity of A-loop region with the anticodon of tRNA(His )has a pronounced effect on the capacity of HIV-1 to utilize tRNA(His )as the primer for reverse transcription. Complementarity between A-loop and anticodon of the tRNA then is important for the selection of the tRNA primer used for reverse transcription
HIV-1 designed to use different tRNA(Gln )isoacceptors prefers to select tRNA(Thr )for replication
BACKGROUND: Previous studies have shown that infection with human immunodeficiency virus type 1 (HIV-1) causes acceleration of the synthesis of glutamine tRNA (tRNA(Gln)) in infected cells. To investigate whether this might influence HIV-1 to utilize tRNA(Gln )as a primer for initiation of reverse transcription, we have constructed HIV-1 proviral genomes in which the PBS and the A-loop region upstream of the PBS have been made complementary to either the anticodon region of tRNA(Gln,1 )or tRNA(Gln,3 )and 3' terminal 18 nucleotides of each isoacceptor of tRNA(Gln). RESULTS: Viruses in which the PBS was altered to be complementary to tRNA(Gln,1 )or tRNA(Gln,3 )with or without the A-loop all exhibited a lower infectivity than the wild type virus. Viruses with only the PBS complementary to tRNA(Gln,1 )or tRNA(Gln,3 )reverted to wild type following culture in SupT1 cells. Surprisingly, viruses in which the PBS and A-loop were complementary to tRNA(Gln,1 )did not grow in SupT1 cells, while viruses in which the PBS and A-loop were made complementary to tRNA(Gln,3 )grew slowly in SupT1 cells. Analysis of the PBS of this virus revealed that it had reverted to select tRNA(Thr )as the primer, which shares complementarity in 15 of 18 nucleotides with the PBS complementary to tRNA(Gln,3). CONCLUSION: The results of these studies support the concept that the HIV-1 has preferred tRNAs that can be selected as primers for replication
HIV gene expression from intact proviruses positioned in bacterial artificial chromosomes at integration sites previously identified in latently infected T cells
AbstractHIV integration predominantly occurs in introns of transcriptionally active genes. To study the impact of the integration site on HIV gene expression, a complete HIV-1 provirus (with GFP as a fusion with Nef) was inserted into bacterial artificial chromosomes (BACs) at three sites previously identified in latent T cells of patients: topoisomerase II (Top2A), DNA methyltransferase 1 (DNMT1), or basic leucine transcription factor 2 (BACH2). Transfection of BAC-HIV into 293T cells resulted in a fourfold difference in production of infectious HIV-1. Cell lines were established that contained BAC–Top2A, BAC–DNMT1, or BAC–BACH2, but only BAC–DNMT1 spontaneously produced virus, albeit at a low level. Stimulation with TNF-α resulted in virus production from four of five BAC–Top2A and all BAC–DNMT1 cell lines, but not from the BAC–BACH2 lines. The results of these studies highlight differences between integration sites identified in latent T cells to support virus production and reactivation from latency
Nucleotide Sequences within the U5 Region of the Viral RNA Genome Are the Major Determinants for an Human Immunodeficiency Virus Type 1 to Maintain a Primer Binding Site Complementary to tRNAHis
AbstractThe initiation of reverse transcription of the human immunodeficiency virus type 1 (HIV-1) genome requires cellular tRNALys,3as a primer and occurs at a site in the viral RNA genome, designated as the primer binding site (PBS), which is complementary to the 3′-terminal 18 nucleotides of tRNALys,3. We previously described an HIV-1 virus [designated as HXB2(His-AC)], which contained a sequence within the U5 region complementary to the anticodon region of tRNAHisin addition to a PBS complementary to the 3′-terminal 18 nucleotides of the tRNAHis. That virus maintained a PBS complementary to tRNAHisafter extendedin vitroculture (Wakefieldet al., J. Virol.70, 966–975, 1996). In the present study, we report that subcloning a 200-base-pair DNA fragment encompassing the U5 and PBS regions from an integrated provirus of HXB2(His-AC) back into the wild-type genome (pHXB2) resulted in an infectious virus, designated as HXB2(His-AC-gac), which again stably maintained a PBS complementary to tRNAHis. DNA sequence analysis of the 200-base-pair region revealed only three nucleotide changes from HXB2(His-AC): a T-to-G change at nucleotide 174, a G-to-A change at nucleotide 181, and a T-to-C change at nucleotide 200. The new mutant virus replicated in CD4+Sup T1 cells similarly to the wild-type virus. Comparison of the nucleotide sequence of nucleocapsid gene of the wild-type and HXB2 (His-AC-gac) virus revealed no differences. Although we found numerous mutations in the reverse transcriptase gene in proviral clones derived from HXB2 (His-AC-gac), no common mutations were found among the 13 clones examined. Comparison of the virion-associated tRNAs of HXB2(His-AC-gac) with those of the wild type revealed that both viruses incorporated a similar subset of cellular tRNAs, with tRNALys,3being the predominant tRNA found within virions. There was no selective enrichment for tRNAHiswithin virions of HXB2(His-AC-gac) virus which selectively use tRNAHisto initiate reverse transcription. The results of these studies suggest that the U5 and PBS regions in the viral RNA genome are important determinants for HXB2(His-AC) viruses in the selective use of tRNAHisto initiate reverse transcription
Preferences for the selection of unique tRNA primers revealed from analysis of HIV-1 replication in peripheral blood mononuclear cells
BACKGROUND: All human immunodeficiency virus (HIV-1) uses a host tRNA(Lys,3 )as the primer for reverse transcription. The tRNA(Lys,3 )is bound to a region on the HIV-1 genome, the primer-binding site (PBS), that is complementary to the 18 terminal nucleotides of tRNA(Lys,3). How HIV-1 selects the tRNA from the intracellular milieu is unresolved. RESULTS: HIV-1 tRNA primer selection has been investigated using viruses in which the primer-binding site (PBS) and a sequence within U5 were altered so as to be complementary to tRNA(Met), tRNA(Pro )or tRNA(Ile). Analysis of the replication of these viruses in human peripheral blood mononuclear cells (PBMC) revealed preferences for the selection of certain tRNAs. HIV-1 with the PBS altered to be complementary to tRNA(Met), with and without the additional mutation in U5 to be complementary to the anticodon of tRNA(Met), stably maintains the PBS complementary to tRNA(Met )following extended in vitro culture in PBMC. In contrast, viruses with either the PBS or PBS and U5 mutated to be complementary to tRNA(Ile )were unstable during in vitro replication in PBMC and reverted to utilize tRNA(Lys,3). Viruses with the PBS altered to be complementary to tRNA(Pro )replicated in PBMC but reverted to use tRNA(Lys,3); viruses with mutations in both the U5 and PBS complementary to tRNA(Pro )maintained this PBS, yet replicated poorly in PBMC. CONCLUSION: The results of these studies demonstrate that HIV-1 has preferences for selection of certain tRNAs for high-level replication in PBMC
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