Quantitating the Multiplicity of Infection with Human Immunodeficiency Virus Type 1 Subtype C Reveals a Non-Poisson Distribution of Transmitted Variants

Currently available simian immunodeficiency virus (SIV) infectious molecular clones (IMCs) and isolates used in nonhuman primate (NHP) models of AIDS were originally derived from infected macaques during chronic infection or end stage disease and may not authentically recapitulate features of transmitted/founder (T/F) genomes that are of particular interest in transmission, pathogenesis, prevention, and treatment studies. We therefore generated and characterized T/F IMCs from genetically and biologically heterogeneous challenge stocks of SIVmac251 and SIVsmE660. Single-genome amplification (SGA) was used to identify full-length T/F genomes present in plasma during acute infection resulting from atraumatic rectal inoculation of Indian rhesus macaques with low doses of SIVmac251 or SIVsmE660. All 8 T/F clones yielded viruses that were infectious and replication competent in vitro, with replication kinetics similar to those of the widely used chronic-infection-derived IMCs SIVmac239 and SIVsmE543. Phenotypically, the new T/F virus strains exhibited a range of neutralization sensitivity profiles. Four T/F virus strains were inoculated into rhesus macaques, and each exhibited typical SIV replication kinetics. The SIVsm T/F viruses were sensitive to TRIM5␣ restriction. All T/F viruses were pathogenic in rhesus macaques, resulting in progressive CD4؉ T cell loss in gastrointestinal tissues, peripheral blood, and lymphatic tissues. The animals developed pathological immune activation; lymphoid tissue damage, including fibrosis; and clinically significant immunodeficiency leading to AIDS-defining clinical endpoints. These T/F clones represent a new molecular platform for the analysis of virus transmission and immunopathogenesis and for the generation of novel "bar-coded" challenge viruses and next-generation simianhuman immunodeficiency viruses that may advance the HIV/AIDS vaccine agenda. IMPORTANCENonhuman primate research has relied on only a few infectious molecular clones for a myriad of diverse research projects, including pathogenesis, preclinical vaccine evaluations, transmission, and host-versus-pathogen interactions. With new data suggesting a selected phenotype of the virus that causes infection (i.e., the transmitted/founder virus), we sought to generate and characterize infectious molecular clones from two widely used simian immunodeficiency virus lineages (SIVmac251 and SIVsmE660). Although the exact requirements necessary to be a T/F virus are not yet fully understood, we generated cloned viruses with all the necessary characteristic of a successful T/F virus. The cloned viruses revealed typical acute and set point viral-load dynamics with pathological immune activation, lymphoid tissue damage progressing to significant immunodeficiency, and AIDS-defining clinical endpoints in some animals. These T/F clones represent a new molecular platform for studies requiring authentic T/F viruses.

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Derivation and Characterization of Pathogenic Transmitted/Founder Molecular Clones from Simian Immunodeficiency Virus SIVsmE660 and SIVmac251 following Mucosal Infection

Lopker

Prete

Estes

et al. 2016

“…An oligo(dT) primer was used to generate cDNA from HIV-1 RNA isolated from blood plasma or CSF. SGA (29)(30)(31) was then used to amplify full-length env sequences through the 3= U3 region. After one round of SGA, amplicons were reamplified, gel purified using a QIAquick gel extraction kit (Qiagen), and cloned into the pcDNA 3.1D/ V5-His-TOPO expression vector (Invitrogen) by use of a pcDNA 3.1 directional TOPO expression kit (Invitrogen) and Max Efficiency Stbl2 competent cells (Invitrogen).…”

Section: Methodsmentioning

confidence: 99%

Quantification of Entry Phenotypes of Macrophage-Tropic HIV-1 across a Wide Range of CD4 Densities

Joseph

Arrildt

Swanstrom

et al. 2014

148

Defining a macrophage-tropic phenotype for HIV-1 to assess a role in pathogenesis is complicated by the fact that HIV-1 isolates vary continuously in their ability to enter monocyte-derived macrophages (MDMs) in vitro, and MDMs vary in their ability to support HIV-1 entry. To overcome these limitations, we identified consistent differences in entry phenotypes between five paired blood-derived, T cell-tropic HIV-1 env genes, four of which are CCR5-using (R5) and one of which is CXCR4-using (X4), and cerebrospinal fluid (CSF)-derived, R5 macrophage-tropic env genes. We performed entry assays using the CD4-and CCR5-inducible Affinofile cell line, expressing a range of CD4 levels that approximates the range from MDMs to CD4 ؉ T cells. The macrophage-tropic viruses were significantly better at infecting cells expressing low levels of CD4 than the T cell-tropic viruses from the same subjects, with the titration of CD4 providing a distinctive and quantitative phenotype. This difference in CD4 utilization was not due to macrophage-tropic viruses being CD4 independent. Furthermore, macrophage-tropic viruses did not differ from paired T cell-tropic viruses in their ability to use low levels of CCR5 (t paired ‫؍‬ ؊1.39; P ‫؍‬ 0.24) or their use of an alternative conformation of CCR5. We also infected MDMs with a panel of viruses and observed that infectivity of each virus differed across four donors and between three preparations from a single donor. We concluded that the evolutionary transition from replication in T cells to that in macrophages involves a phenotypic transition to acquire the ability to infect cells expressing low levels of CD4 and that this phenotype is more reliably measured in Affinofile cells than in macrophages. IMPORTANCEHIV-1 typically infects memory T cells by using CD4 and CCR5 to enter cells. The virus evolves to infect new cell types by changing the coreceptor from CCR5 to CXCR4 to infect naive T cells or adapting to the use of low levels of CD4 to infect macrophages. However, defining the phenotype of macrophage tropism has been difficult due to inherent variability in the use of macrophages generated in culture to support entry of HIV-1. We describe the use of Affinofile cells with inducible and variable levels of CD4 to identify a signature phenotype for macrophage-tropic HIV-1. The ability to define HIV-1 variants that have evolved an entry phenotype that allows more efficient entry into cells with low levels of CD4 sets the stage for a clearer placement of these variants in HIV-associated pathogenesis.T he HIV-1 Env protein determines the entry phenotype of the virus, typically using CD4 as the receptor and CCR5 as the coreceptor. The ability of HIV-1 to replicate in a novel cell type likely requires adaptation of the viral envelope protein to efficiently utilize the receptor and coreceptor present on that cell type. The emergence of CXCR4-using virus late in infection has long been thought to represent adaptation to infect a novel host cell (reviewed in reference 1), most likely CD...

“…This is unsurprising, as sequence similarity between genomic partners is a strict requirement for efficient recombination (7,(10)(11)(12). Given that the vast majority of HIV-1 infections are not the result of coinfections with multiple divergent viral strains but are initiated from a single virion, a model system that measures recombination between genetically similar genomes rather than inter/intrasubtypes will better approximate the quasispecies in vivo (13)(14)(15). However, little is known about recombination likely to be found within the viral quasispecies of an infected individual, because it is difficult to detect recombination between genetically similar genomes.…”

mentioning

confidence: 99%

Identifying Recombination Hot Spots in the HIV-1 Genome

Smyth

Schlub

Grimm

et al. 2014

HIV-1 infection is characterized by the rapid generation of genetic diversity that facilitates viral escape from immune selection and antiretroviral therapy. Despite recombination's crucial role in viral diversity and evolution, little is known about the genomic factors that influence recombination between highly similar genomes. In this study, we use a minimally modified fulllength HIV-1 genome and high-throughput sequence analysis to study recombination in gag and pol in T cells. We find that recombination is favored at a number of recombination hot spots, where recombination occurs six times more frequently than at corresponding cold spots. Interestingly, these hot spots occur near important features of the HIV-1 genome but do not occur at sites immediately around protease inhibitor or reverse transcriptase inhibitor drug resistance mutations. We show that the recombination hot and cold spots are consistent across five blood donors and are independent of coreceptor-mediated entry. Finally, we check common experimental confounders and find that these are not driving the location of recombination hot spots. This is the first study to identify the location of recombination hot spots between two similar viral genomes with great statistical power and under conditions that closely reflect natural recombination events among HIV-1 quasispecies. IMPORTANCEThe ability of HIV-1 to evade the immune system and antiretroviral therapy depends on genetic diversity within the viral quasispecies. Retroviral recombination is an important mechanism that helps to generate and maintain this genetic diversity, but little is known about how recombination rates vary within the HIV-1 genome. We measured recombination rates in gag and pol and identified recombination hot and cold spots, demonstrating that recombination is not random but depends on the underlying gene sequence. The strength and location of these recombination hot and cold spots can be used to improve models of viral dynamics and evolution, which will be useful for the design of robust antiretroviral therapies.