Investigation of initiation of reverse transcription in retroviruses using vectors with two primer -binding sites

Initiation of reverse transcription in retroviruses occurs on a specific place in viral genome, called primer-binding site (PBS). Cellular tRNA is annealed to the complementary PBS and is used as a primer. We constructed murine leukemia virus (MLV)-based vectors containing two PBSs, one in normal position and another in the middle of the vector. Green fluorescence protein (GFP) gene was inserted between the two PBSs. The internal PBS was derived from a spleen necrosis virus (SNV), which uses the same tRNA-Pro as a primer. We have shown that the internal PBS was functional for initiation of reverse transcription. Replication of vectors containing two functional PBSs is expected to result in deletion of sequences between the two PBSs if initiations on these two sites occur simultaneously. The deletion was detected in 20--30% of infected cells by the GFP-negative phenotype and by direct Southern blotting analysis of DNA of infected cells. This indicated that a single virion has the capacity to initiate reverse transcription more than once. In order to fund the efficiency of initiation of a single PBS we constructed a mathematical model, which provided a relationship between several parameters of retroviral replication and the outcome of reverse transcription of vectors with two PBSs. Using this model we found that efficiency of initiation at MLV and SNV PBS is 35--50%. Next, we constructed human immunodeficiency virus type 1 (HIV-1)-based vectors with two PBSs by inserting human immunodeficiency virus type 2 (HIV-2) PBS in the middle. Again, the internal PBS was found functional and simultaneous initiations were occurring in 22--24% of all infections. This system was used to analyze the effects of mutations within HIV-2 PBS region on the efficiency of initiation. By using wild type HIV-1 PBS (present in the same vector) as internal control we were able to get accurate quantitative measurements of initiation efficiency at mutant HIV-2 PBS. Our results indicated that U5-IR stem of HIV-2 plays a very important role in initiation. Also, additional interactions between tRNA and viral genome, proposed for HIV-1, were found to be important for HIV-2 initiation, indicating that these two viruses use similar mechanisms to achieve specific and efficient initiation of reverse transcription

Frequent dual initiation of reverse transcription in murine leukemia virus-based vectors containing two primer-binding sites

AbstractRetroviruses package two copies of viral RNA into each virion. Although each RNA contains a primer-binding site for initiation of DNA synthesis, it is unknown whether reverse transcription is initiated on both RNAs. To determine whether a single virion is capable of initiating reverse transcription more than once, we constructed a murine leukemia virus-based vector containing a second primer-binding site (PBS) derived from spleen necrosis virus and inserted the green fluorescent protein gene (GFP) between the two PBSs. Initiation of reverse transcription at either PBS results in a provirus that expresses GFP. However, initiation at both PBSs can result in the deletion of GFP, which can be detected by flow cytometry and Southern blotting analysis. Approximately 22–29% of the proviruses formed deleted the GFP in a single replication cycle, indicating the minimum proportion of virions that initiated reverse transcription on both PBSs. These results show that a significant proportion of MLV-based vectors containing two PBSs have the capacity to initiate reverse transcription more than once

Genetic Drift of HIV Populations in Culture

Populations of Human Immunodeficiency Virus type 1 (HIV-1) undergo a surprisingly large amount of genetic drift in infected patients despite very large population sizes, which are predicted to be mostly deterministic. Several models have been proposed to explain this phenomenon, but all of them implicitly assume that the process of virus replication itself does not contribute to genetic drift. We developed an assay to measure the amount of genetic drift for HIV populations replicating in cell culture. The assay relies on creation of HIV populations of known size and measurements of variation in frequency of a neutral allele. Using this assay, we show that HIV undergoes approximately ten times more genetic drift than would be expected from its population size, which we defined as the number of infected cells in the culture. We showed that a large portion of the increase in genetic drift is due to non-synchronous infection of target cells. When infections are synchronized, genetic drift for the virus is only 3-fold higher than expected from its population size. Thus, the stochastic nature of biological processes involved in viral replication contributes to increased genetic drift in HIV populations. We propose that appreciation of these effects will allow better understanding of the evolutionary forces acting on HIV in infected patients

Frequent Dual Initiation in Human Immunodeficiency Virus-Based Vectors Containing Two Primer-Binding Sites: a Quantitative In Vivo Assay for Function of Initiation Complexes

We previously demonstrated that murine leukemia virus (MLV)-based vectors containing two primer-binding sites (PBSs) have the capacity to initiate reverse transcription more than once (Y. A. Voronin and V. K. Pathak, Virology 312:281-294, 2003). To determine whether human immunodeficiency virus (HIV)-based vectors also have the capacity to initiate reverse transcription twice, we constructed an HIV type 1 (HIV-1)-based vector containing the HIV-1 PBS, a green fluorescent protein reporter gene (GFP), and a second PBS derived from HIV-2 3′ of GFP. Simultaneous initiation of reverse transcription at both the 5′ HIV-1 PBS and 3′ HIV-2 PBS was predicted to result in deletion of GFP. As in the MLV-based vectors, GFP was deleted in approximately 25% of all proviruses, indicating frequent dual initiation in HIV-based vectors containing two PBSs. Quantitative real-time PCR analysis of early reverse transcription products indicated that HIV-1 reverse transcriptase efficiently used the HIV-2 PBS. To investigate tRNA primer-RNA template interactions in vivo, we introduced several mutations in the HIV-2 U5 region. The effects of these mutations on the efficiency of reverse transcription initiation were measured by quantitative real-time PCR analysis of early reverse transcription products, with initiation at the HIV-1 PBS used as an internal control. Disruption of the lower and upper parts of the U5-inverted repeat stem reduced the efficiency of initiation 20- and 6-fold, respectively. In addition, disruption of the proposed interactions between viral RNA and tRNA(Lys3) thymidine-pseudouridine-cytidine and anticodon loops decreased the efficiency of initiation seven- and sixfold, respectively. These results demonstrate the relative influence of various RNA-RNA interactions on the efficiency of initiation in vivo. Furthermore, the two-PBS vector system provides a sensitive and quantitative in vivo assay for analysis of RNA-RNA and protein-RNA interactions that can influence the efficiency of reverse transcription initiation

HIVR4P 2016, Partnering for Prevention: Conference Summary and Highlights

Abstract HIV Research for Prevention: AIDS Vaccine, Microbicide, and ARV-based Prevention Science (HIVR4P) was built on a growing consensus that effective HIV prevention requires a combination of approaches and that understanding, analyzing, and debating the cross-cutting issues that impact prevention research are all essential to combat the global HIV/AIDS epidemic. To that end, the biennial HIVR4P conference is dedicated to all biomedical HIV prevention research approaches, including HIV vaccines, microbicides, pre-exposure prophylaxis, and treatment as prevention. The HIVR4P 2016 conference was held in Chicago, Illinois (USA), on October 17–21, and included more than 700 scientific presentations and 21 satellite sessions covering the latest and most promising advances across the HIV prevention research field. The theme “Partnering for Prevention” represented the conference's commitment to breaking down silos between research disciplines as well as between researchers, program developers, care providers, advocates, communities, and funders. Delegates spanning 42 countries attended the conference. One-third of those in attendance were early career investigators, which reflects a firm commitment to emerging researchers and ultimately to the goal of developing a sustainable scientific enterprise well into the future. This article presents a concise summary of highlights from the conference. For a more detailed account, one may find full abstracts, daily summaries, and webcasts on the conference website at hivr4p.org

HIVR4P 2016, Partnering for Prevention: Conference Summary and Highlights.

HIV Research for Prevention: AIDS Vaccine, Microbicide, and ARV-based Prevention Science (HIVR4P) was built on a growing consensus that effective HIV prevention requires a combination of approaches and that understanding, analyzing, and debating the cross-cutting issues that impact prevention research are all essential to combat the global HIV/AIDS epidemic. To that end, the biennial HIVR4P conference is dedicated to all biomedical HIV prevention research approaches, including HIV vaccines, microbicides, pre-exposure prophylaxis, and treatment as prevention. The HIVR4P 2016 conference was held in Chicago, Illinois (USA), on October 17-21, and included more than 700 scientific presentations and 21 satellite sessions covering the latest and most promising advances across the HIV prevention research field. The theme "Partnering for Prevention" represented the conference's commitment to breaking down silos between research disciplines as well as between researchers, program developers, care providers, advocates, communities, and funders. Delegates spanning 42 countries attended the conference. One-third of those in attendance were early career investigators, which reflects a firm commitment to emerging researchers and ultimately to the goal of developing a sustainable scientific enterprise well into the future. This article presents a concise summary of highlights from the conference. For a more detailed account, one may find full abstracts, daily summaries, and webcasts on the conference website at hivr4p.org