Structure and Function of Lentiviral Genomic and Messenger RNA

The positive sense lentiviral RNA genome is packaged within the virus as a dimer of two single strands. The RNA of primate lentiviruses human immunodeficiency virus (HIV-1) and simian immunodeficiency virus (SIVmac239) are distantly related and the secondary structures of these viral RNAs share many known biological functions. Using selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE), I present an analysis of the secondary structure of ex virio genomic SIVmac239 RNA in relation to that of HIV-1 as well as an investigation into the secondary structure of the various in vitro mRNA species of HIV-1 resolved using the SHAPE technique. First, I describe a SHAPE-derived model of SIVmac239 genomic RNA structure. When compared to that of HIV-1, I find very few conserved structural regions outside the previously studied functional structures. I observe that this is due to the flexible nature of the adenosine-rich lentiviral genome. The structures that are conserved are located in regions with high guanosine concentration, forming more stable pairing interactions. These results suggest that lentiviral genomic RNA structure is flexible and metastable unless held by stronger pairing interactions that seem to persist through the course of viral evolution. The lentiviral genomic RNA structures that I have studied do share a few common base pairs, including a small stem-loop at the site of the first splice acceptor (SA1). In the second part, I describe the effect of mutating this structure on viral replication and on the splicing profile of the viral mRNA. To further investigate viral splicing regulation, I determined the SHAPE-derived structures of the most abundant mRNA variants for all of the protein products of HIV-1. Results reveal local interactions that form at regulatory regions in the viral transcripts. Because RNA is an important feature throughout the lentiviral replication cycle, a greater understanding into the role of RNA in various aspects of viral replication will increase comprehension toward the complex biology of infection. This analysis provides insight into evolutionary conservation of RNA structures that play functional roles and may be possible targets for novel factors as part of a broad spectrum of viral inhibitory agents.Doctor of Philosoph

Characterizing HIV-1 Splicing by Using Next-Generation Sequencing

ABSTRACT Full-length human immunodeficiency virus type 1 (HIV-1) RNA serves as the genome or as an mRNA, or this RNA undergoes splicing using four donors and 10 acceptors to create over 50 physiologically relevant transcripts in two size classes (1.8 kb and 4 kb). We developed an assay using Primer ID-tagged deep sequencing to quantify HIV-1 splicing. Using the lab strain NL4-3, we found that A5 ( env / nef ) is the most commonly used acceptor (about 50%) and A3 ( tat ) the least used (about 3%). Two small exons are made when a splice to acceptor A1 or A2 is followed by activation of donor D2 or D3, and the high-level use of D2 and D3 dramatically reduces the amount of vif and vpr transcripts. We observed distinct patterns of temperature sensitivity of splicing to acceptors A1 and A2. In addition, disruption of a conserved structure proximal to A1 caused a 10-fold reduction in all transcripts that utilized A1. Analysis of a panel of subtype B transmitted/founder viruses showed that splicing patterns are conserved, but with surprising variability of usage. A subtype C isolate was similar, while a simian immunodeficiency virus (SIV) isolate showed significant differences. We also observed transsplicing from a downstream donor on one transcript to an upstream acceptor on a different transcript, which we detected in 0.3% of 1.8-kb RNA reads. There were several examples of splicing suppression when the env intron was retained in the 4-kb size class. These results demonstrate the utility of this assay and identify new examples of HIV-1 splicing regulation. IMPORTANCE During HIV-1 replication, over 50 conserved spliced RNA variants are generated. The splicing assay described here uses new developments in deep-sequencing technology combined with Primer ID-tagged cDNA primers to efficiently quantify HIV-1 splicing at a depth that allows even low-frequency splice variants to be monitored. We have used this assay to examine several features of HIV-1 splicing and to identify new examples of different mechanisms of regulation of these splicing patterns. This splicing assay can be used to explore in detail how HIV-1 splicing is regulated and, with moderate throughput, could be used to screen for structural elements, small molecules, and host factors that alter these relatively conserved splicing patterns

Comparison of SIV and HIV-1 Genomic RNA Structures Reveals Impact of Sequence Evolution on Conserved and Non-Conserved Structural Motifs

RNA secondary structure plays a central role in the replication and metabolism of all RNA viruses, including retroviruses like HIV-1. However, structures with known function represent only a fraction of the secondary structure reported for HIV-1NL4-3. One tool to assess the importance of RNA structures is to examine their conservation over evolutionary time. To this end, we used SHAPE to model the secondary structure of a second primate lentiviral genome, SIVmac239, which shares only 50% sequence identity at the nucleotide level with HIV-1NL4-3. Only about half of the paired nucleotides are paired in both genomic RNAs and, across the genome, just 71 base pairs form with the same pairing partner in both genomes. On average the RNA secondary structure is thus evolving at a much faster rate than the sequence. Structure at the Gag-Pro-Pol frameshift site is maintained but in a significantly altered form, while the impact of selection for maintaining a protein binding interaction can be seen in the conservation of pairing partners in the small RRE stems where Rev binds. Structures that are conserved between SIVmac239 and HIV-1NL4-3 also occur at the 5′ polyadenylation sequence, in the plus strand primer sites, PPT and cPPT, and in the stem-loop structure that includes the first splice acceptor site. The two genomes are adenosine-rich and cytidine-poor. The structured regions are enriched in guanosines, while unpaired regions are enriched in adenosines, and functionaly important structures have stronger base pairing than nonconserved structures. We conclude that much of the secondary structure is the result of fortuitous pairing in a metastable state that reforms during sequence evolution. However, secondary structure elements with important function are stabilized by higher guanosine content that allows regions of structure to persist as sequence evolution proceeds, and, within the confines of selective pressure, allows structures to evolve

Interplay between Single Resistance-Associated Mutations in the HIV-1 Protease and Viral Infectivity, Protease Activity, and Inhibitor Sensitivity

ABSTRACT Resistance-associated mutations in the HIV-1 protease modify viral fitness through changes in the catalytic activity and altered binding affinity for substrates and inhibitors. In this report, we examine the effects of 31 mutations at 26 amino acid positions in protease to determine their impact on infectivity and protease inhibitor sensitivity. We found that primary resistance mutations individually decrease fitness and generally increase sensitivity to protease inhibitors, indicating that reduced virion-associated protease activity reduces virion infectivity and the reduced level of per virion protease activity is then more easily titrated by a protease inhibitor. Conversely, mutations at more variable positions (compensatory mutations) confer low-level decreases in sensitivity to all protease inhibitors with little effect on infectivity. We found significant differences in the observed effect on infectivity with a pseudotype virus assay that requires the protease to cleave the cytoplasmic tail of the amphotropic murine leukemia virus (MuLV) Env protein. Additionally, we were able to mimic the fitness loss associated with resistance mutations by directly reducing the level of virion-associated protease activity. Virions containing 50% of a D25A mutant protease were 3- to 5-fold more sensitive to protease inhibitors. This level of reduction in protease activity also resulted in a 2-fold increase in sensitivity to nonnucleoside inhibitors of reverse transcriptase and a similar increase in sensitivity to zidovudine (AZT), indicating a pleiotropic effect associated with reduced protease activity. These results highlight the interplay between enzyme activity, viral fitness, and inhibitor mechanism and sensitivity in the closed system of the viral replication complex

Characterizing HIV-1 Splicing by Using Next-Generation Sequencing

Full-length human immunodeficiency virus type 1 (HIV-1) RNA serves as the genome or as an mRNA, or this RNA undergoes splicing using four donors and 10 acceptors to create over 50 physiologically relevant transcripts in two size classes (1.8 kb and 4 kb). We developed an assay using Primer ID-tagged deep sequencing to quantify HIV-1 splicing. Using the lab strain NL4-3, we found that A5 (env/nef) is the most commonly used acceptor (about 50%) and A3 (tat) the least used (about 3%). Two small exons are made when a splice to acceptor A1 or A2 is followed by activation of donor D2 or D3, and the high-level use of D2 and D3 dramatically reduces the amount of vif and vpr transcripts. We observed distinct patterns of temperature sensitivity of splicing to acceptors A1 and A2. In addition, disruption of a conserved structure proximal to A1 caused a 10-fold reduction in all transcripts that utilized A1. Analysis of a panel of subtype B transmitted/founder viruses showed that splicing patterns are conserved, but with surprising variability of usage. A subtype C isolate was similar, while a simian immunodeficiency virus (SIV) isolate showed significant differences. We also observed transsplicing from a downstream donor on one transcript to an upstream acceptor on a different transcript, which we detected in 0.3% of 1.8-kb RNA reads. There were several examples of splicing suppression when the env intron was retained in the 4-kb size class. These results demonstrate the utility of this assay and identify new examples of HIV-1 splicing regulation. IMPORTANCE During HIV-1 replication, over 50 conserved spliced RNA variants are generated. The splicing assay described here uses new developments in deep-sequencing technology combined with Primer ID-tagged cDNA primers to efficiently quantify HIV-1 splicing at a depth that allows even low-frequency splice variants to be monitored. We have used this assay to examine several features of HIV-1 splicing and to identify new examples of different mechanisms of regulation of these splicing patterns. This splicing assay can be used to explore in detail how HIV-1 splicing is regulated and, with moderate throughput, could be used to screen for structural elements, small molecules, and host factors that alter these relatively conserved splicing patterns

Harm avoidance and mobility during middle childhood and adolescence among Hadza foragers

Cross-cultural sex differences in mobility and harm avoidance have been widely reported, often emphasizing fitness benefits of long-distance travel for males and high costs for females. Data emerging from adults in small-scale societies, however, are challenging the assumption that female mobility is restricted during reproduction. Such findings warrant further exploration of the ontogeny of mobility. Here, using a combination of machine-learning, mixed-effects linear regression, and GIS mapping, we analyze range size, daily distance traveled, and harm avoidance among Hadza foragers during middle childhood and adolescence. Distance traveled increased with age and, while male adolescents had the longest daily ranges, average daily distance traveled by each sex was similar. We found few age- or sex-related patterns in harm-avoidant responses and a high degree of individual variation. When queried on the same issues, children and their parents were often in alignment as to expectations pertaining to harm avoidance, and siblings tended to behave in similar ways. To the extent that sex differences in mobility did emerge, they were associated with ecological differences in physical threats associated with sex-specific foraging behaviors. Further, we found no strong association between harm avoidance and mobility. Young Hadza foragers of both sexes are highly mobile, regardless of how harm avoidant they are. Taken together, our findings indicate that the causal arrows between harm avoidance and mobility must be evaluated in ecologically specific frameworks where cultural expectations of juvenile mobility can be contextualized

Racial Disparities in Brachytherapy Treatment among Women with Cervical and Endometrial Cancer in the United States

Brachytherapy improves clinical outcomes among women diagnosed with cervical and endometrial cancers. Recent evidence demonstrates that declining brachytherapy boosts for women with cervical cancer were associated with higher mortality. In this retrospective cohort study, women diagnosed with endometrial or cervical cancer in the United States between 2004 and 2017 were selected from the National Cancer Database for evaluation. Women ≥18 years of age were included for high intermediate risk (PORTEC-2 and GOG-99 definition) or FIGO Stage II-IVA endometrial cancers and FIGO Stage IA-IVA—non-surgically treated cervical cancers. The aims were to (1) evaluate brachytherapy treatment practice patterns for cervical and endometrial cancers in the United States; (2) calculate rates of brachytherapy treatment by race; and (3) determine factors associated with not receiving brachytherapy. Treatment practice patterns were evaluated over time and by race. Multivariable logistic regression assessed predictors of brachytherapy. The data show increasing rates of brachytherapy for endometrial cancers. Compared to non-Hispanic White women; Native Hawaiian and other Pacific Islander (NHPI) women with endometrial cancer and Black women with cervical cancer were significantly less likely to receive brachytherapy. For both NHPI and Black women, treatment at community cancer centers was associated with a decreased likelihood of brachytherapy. The data suggest racial disparities among Black women with cervical cancer and NHPI women with endometrial cancer and emphasize an unmet need for brachytherapy access within community hospitals

Codon alignment and predicted pairing partners in the RRE region of SIVmac239 and HIV-1_NL4-3.

<p>(A) Predicted structures within the RRE are shown for SIVmac239 (left) and HIV-1 (middle). Codons of stem I are in brackets with their corresponding amino acids labeled and numbered from the first codon of stem I. Blue brackets indicate an area of conserved pairing partners; green brackets indicate an area of shifted pairing partners in stem I. The HIV-1_NL4-3 structure with forced SIVmac239 pairing is also shown with sequence variations that occur in SIVmac239 indicated while those that change the amino acid sequence are in green (right). Blue lines indicate base pairs that are exactly conserved between the two viruses. (B) The sequences of SIVmac239 (top) and HIV-1_NL4-3 (bottom) aligned horizontally. Curved lines indicate base-pairing partners. Gray boxes indicate regions of amino acid alignment. Roman numerals indicate helices discussed in the text.</p

SHAPE analysis of the polypurine tracts of SIVmac239 and HIV-1_NL4-3 and base composition of both genomes in structured and unstructured regions.

<p>(A) RNA structure models for the cPPT and PPT of SIVmac239 and HIV-1_NL4-3. Nucleotides involved in the polypurine tracts are colored according to their SHAPE reactivity values as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003294#ppat-1003294-g001" target="_blank">Figure 1</a>. Other nucleotides are light gray. Nucleotides not shown are indicated by hatched lines. (B) Histograms of SHAPE reactivity values, integrated and normalized, along the span of the polypurine tracts. HIV-1_NL4-3 reactivity values are displayed in a lighter color scale. (C) Histogram of percentage of each individual nucleotide compared to the percentage of each in the entire genome. For each individual nucleotide, SIVmac239 (green) is on the left and HIV-1_NL4-3 (blue) is on the right. The percent paired for each nucleotide is indicated by hatched lines. (D) Histogram of percentage of each nucleotide in the genome compared to the percentage in highly structured regions of known function (5′-UTR and RRE). SIVmac239 (green) is on the left and HIV-1_NL4-3 (blue) is on the right.</p

Codon alignment and predicted pairing partners in the stem-loop surrounding SA1 of SIVmac239 and HIV-1_NL4-3.

<p>(A) Structures for the conserved stem in SIVmac239 (left) and HIV-1_NL4-3 (right). Blue lines indicate the base pairs that are exactly conserved between the two viruses. (B) The sequences of SIVmac239 (top) and HIV-1_NL4-3 are aligned horizontally. Curved lines indicate base-pairing partners. Gray boxes indicate regions of amino acid alignment. Bold letters represent the bases that are involved in the conserved pairing interactions.</p