Announcing the Andy Kaplan Prize and call for nominations

The retrovirology community has founded a new prize in tribute to our late colleague, Andrew Kaplan, with the goals of honoring and advancing the career of a postdoctoral researcher in our field. We seek help identifying outstanding candidates for this new award

Evidence for the acquisition of multi-drug resistance in an HIV-1 clinical isolate via human sequence transduction

AbstractInsertions in HIV-1 reverse transcriptase's fingers subdomain can enhance chain terminator excision and confer resistance to multiple nucleoside analogs. Inserts that resemble flanking sequences likely arise by local sequence duplication. However, a remarkable variety of non-repeat fingers insertions have been observed. Here, molecular epidemiology, sequence analyses and mechanistic modeling were employed to show that one Japanese isolate's RT fingers insert likely resulted from non-homologous recombination between virus and host sequences and the transductive copying of 37 nucleotides from human chromosome 17. These findings provide evidence that human sequence transduction can, at least rarely, contribute to genetic and phenotypic variation in pandemic HIV

Stability and conformation of the dimeric HIV-1 genomic RNA 5\u27UTR

During HIV-1 assembly, the viral Gag polyprotein specifically selects the dimeric RNA genome for packaging into new virions. The 5\u27 untranslated region (5\u27UTR) of the dimeric genome may adopt a conformation that is optimal for recognition by Gag. Further conformational rearrangement of the 5\u27UTR, promoted by the nucleocapsid (NC) domain of Gag, is predicted during virus maturation. Two 5\u27UTR dimer conformations, the kissing dimer (KD) and the extended dimer (ED), have been identified in vitro, which differ in the extent of intermolecular basepairing. Whether 5\u27UTRs from different HIV-1 strains with distinct sequences have access to the same dimer conformations has not been determined. Here, we applied fluorescence cross-correlation spectroscopy and single-molecule Forster resonance energy transfer imaging to demonstrate that 5\u27UTRs from two different HIV-1 subtypes form (KDs) with divergent stabilities. We further show that both 5\u27UTRs convert to a stable dimer in the presence of the viral NC protein, adopting a conformation consistent with extensive intermolecular contacts. These results support a unified model in which the genomes of diverse HIV-1 strains adopt an ED conformation

The “Connection” Between HIV Drug Resistance and RNase H

Currently, nucleoside reverse transcriptase inhibitors (NRTIs) and nonnucleoside reverse transcriptase inhibitors (NNRTIs) are two classes of antiretroviral agents that are approved for treatment of HIV-1 infection. Since both NRTIs and NNRTIs target the polymerase (pol) domain of reverse transcriptase (RT), most genotypic analysis for drug resistance is limited to the first ∼300 amino acids of RT. However, recent studies have demonstrated that mutations in the C-terminal domain of RT, specifically the connection subdomain and RNase H domain, can also increase resistance to both NRTIs and NNRTIs. In this review we will present the potential mechanisms by which mutations in the C-terminal domain of RT influence NRTI and NNRTI susceptibility, summarize the prevalence of the mutations in these regions of RT identified to date, and discuss their importance to clinical drug resistance

Mutation Rates and Intrinsic Fidelity of Retroviral Reverse Transcriptases

Retroviruses are RNA viruses that replicate through a DNA intermediate, in a process catalyzed by the viral reverse transcriptase (RT). Although cellular polymerases and host factors contribute to retroviral mutagenesis, the RT errors play a major role in retroviral mutation. RT mutations that affect the accuracy of the viral polymerase have been identified by in vitro analysis of the fidelity of DNA synthesis, by using enzymological (gel-based) and genetic assays (e.g., M13mp2 lacZ forward mutation assays). For several amino acid substitutions, these observations have been confirmed in cell culture using viral vectors. This review provides an update on studies leading to the identification of the major components of the fidelity center in retroviral RTs

Crystal structures of oligonucleotides including the integrase processing site of the Moloney murine leukemia virus

In the first step of retroviral integration, integrase cleaves the linear viral DNA within its long terminal repeat (LTR) immediately 3′ to the CA dinucleotide step, resulting in a reactive 3′ OH on one strand and a 5′ two base overhang on the complementary strand. In order to investigate the structural properties of the 3′ end processing site within the Moloney murine leukemia virus (MMLV) LTR d(TCTTTCATT), a host-guest crystallographic method was employed to determine the structures of four self-complementary 16 bp oligonucleotides including LTR sequences (underlined), d(TTTCATTGCAATGAAA), d(CTTTCATTAATGAAAG), d(TCTTTCATATGAAAGA) and d(CACAATGATCATTGTG), the guests, complexed with the N-terminal fragment of MMLV reverse transcriptase, the host. The structures of the LTR-containing oligonucleotides were compared to those of non-LTR oligonucleotides crystallized in the same lattice. Properties unique to the CA dinucleotide step within the LTR sequence, independent of its position from the end of the duplex, include a positive roll angle and negative slide value. This propensity for the CA dinucleotide step within the MMLV LTR sequence to adopt only positive roll angles is likely influenced by the more rigid, invariable 3′ and 5′ flanking TT dinucleotide steps and may be important for specific recognition and/or cleavage by the MMLV integrase

Effects of DNA and protein size on substrate cleavage by human tyrosyl-DNA phosphodiesterase 1

Tyrosyl-DNA phosphodiesterase 1 (TDP1) catalyzes the hydrolysis of phosphodiester linkages between a DNA 3′ phosphate and a tyrosine residue as well as a variety of other DNA 3′ substituents, and has been implicated in the repair of covalent complexes involving eukaryotic type IB topoisomerases. To better understand the substrate features that are recognized by TDP1, the size of either the DNA or protein component of the substrate was varied. Competition experiments and gel shift analyses comparing a series of substrates with DNA lengths increasing from 6 to 28 nucleotides indicated that, contrary to predictions based on the crystal structure of the protein, the apparent affinity for the substrate increased as the DNA length was increased over the entire range tested. It has previously been found that a substrate containing the full-length native form of human topoisomerase I protein is not cleaved by TDP1. Protein-oligonucleotide complexes containing either a 53 or 108 amino acid long topoisomerase I-derived peptide were efficiently cleaved by TDP1, but like the full length protein, a substrate containing a 333 amino acid topoisomerase I fragment was resistant to cleavage. Consistent with these results, evidence is presented that processing by the proteasome is required for TDP1 cleavage in vivo

Likely Role of APOBEC3G-Mediated G-to-A Mutations in HIV-1 Evolution and Drug Resistance

The role of APOBEC3 (A3) protein family members in inhibiting retrovirus infection and mobile element retrotransposition is well established. However, the evolutionary effects these restriction factors may have had on active retroviruses such as HIV-1 are less well understood. An HIV-1 variant that has been highly G-to-A mutated is unlikely to be transmitted due to accumulation of deleterious mutations. However, G-to-A mutated hA3G target sequences within which the mutations are the least deleterious are more likely to survive selection pressure. Thus, among hA3G targets in HIV-1, the ratio of nonsynonymous to synonymous changes will increase with virus generations, leaving a footprint of past activity. To study such footprints in HIV-1 evolution, we developed an in silico model based on calculated hA3G target probabilities derived from G-to-A mutation sequence contexts in the literature. We simulated G-to-A changes iteratively in independent sequential HIV-1 infections until a stop codon was introduced into any gene. In addition to our simulation results, we observed higher ratios of nonsynonymous to synonymous mutation at hA3G targets in extant HIV-1 genomes than in their putative ancestral genomes, compared to random controls, implying that moderate levels of A3G-mediated G-to-A mutation have been a factor in HIV-1 evolution. Results from in vitro passaging experiments of HIV-1 modified to be highly susceptible to hA3G mutagenesis verified our simulation accuracy. We also used our simulation to examine the possible role of A3G-induced mutations in the origin of drug resistance. We found that hA3G activity could have been responsible for only a small increase in mutations at known drug resistance sites and propose that concerns for increased resistance to other antiviral drugs should not prevent Vif from being considered a suitable target for development of new drugs

Expression of IMP1 Enhances Production of Murine Leukemia Virus Vector by Facilitating Viral Genomic RNA Packaging

Murine leukemia virus (MLV)-based retroviral vector is widely used for gene transfer. Efficient packaging of the genomic RNA is critical for production of high-titer virus. Here, we report that expression of the insulin-like growth factor II mRNA binding protein 1 (IMP1) enhanced the production of infectious MLV vector. Overexpression of IMP1 increased the stability of viral genomic RNA in virus producer cells and packaging of the RNA into progeny virus in a dose-dependent manner. Downregulation of IMP1 in virus producer cells resulted in reduced production of the retroviral vector. These results indicate that IMP1 plays a role in regulating the packaging of MLV genomic RNA and can be used for improving production of retroviral vectors