Science Spotlight

Working towards better HIV-1 preclinical models

From the Overbaugh lab, Human Biology Division

Modeling HIV infection in macaques is challenging for several reasons including the transient nature of the HIV infection itself. Indeed, HIV-1 does not persist in macaques due to species-specific restriction factors. In response to infection, inflammatory cytokines such as Interferon-a (IFNa) are secreted and activate IFN stimulated genes (ISG). Some of these genes encode potent blockers of viral replication, the restriction factors. Chimeric SIV-HIV-1 viruses (SHIVs) are HIV-1 viruses encoding SIV antagonists of known restriction factors and are capable of infecting the simian hosts for a longer period of time. As Dr. Amit Sharma, from the Overbaugh lab (Human Biology Division), highlights, “infection of macaques with SHIVs is the most favored model system for preclinical HIV research.” A common way that the HIV field has generated SHIVs is to adapt them in macaques, but it is unclear what precesses allow for this adaptation.  In addition, although these adapted SHIVs are characterized by increased replication and pathogenicity, progressive selection of viruses in macaques can lead to antigen changes in the Envelope (Env) protein, the protein that is responsible for targeted viral entry in the host cells. Dr. Sharma, who just recently sarted his own lab at The Ohio State University explains:  “Existing pathogenic SHIVs are a highly selected subset of viruses and do not represent the large genetic diversity of globally circulating HIV variants. Development of SHIVs encoding circulating HIV variants, which best model HIV infection in humans, has been challenging as these SHIVs replicate poorly in macaque cells.” As a consequence, understanding the differences in replication of adapted versus unadapted SHIVs is of primary importance. Dr. Julie Overbaugh’s lab decided to tackle this challenge and recently published their work in PLoS Pathogens.

Sharma, first author of the study, and colleagues first investigated the putative restriction factors encoded by ISGs in macaque lymphocytes. Pigtailed macaque (Ptm) CD4+ lymphocytes were treated with IFNa and RNA sequencing was performed in collaboration with the Fred Hutch genomics core. Among the 147 upregulated genes, the authors focused on those encoding transmembrane proteins that can potentially interact with Env and interfere with viral persistency. Among the 31 genes corresponding to these criteria, the researchers noticed two previously uncharacterized genes IFITM1 and IFITM3, known to be antiviral factors that impair viral fusion in other species. Whereas IFITM1 is conserved between macaques and human, IFITM3 is exclusive to macaque and therefore constituted an interesting candidate for a macaque specific restriction factor.

As the IFITM proteins are known to be incorporated in virions to impair viral entry and spread, the authors hypothesized that the incorporation of IFITM3 could be different in adapted versus unadapted SHIVs. To test this hypothesis, they infected Ptm lymphocytes with either adapted or unadapted virions, treated the infected cells with IFNa, and assessed by western blotting the level of IFITM3 protein in virions that budded out from the host cells. Strikingly, although unadapted virions contained a great amount of IFITM3, the protein was almost absent from adapted virions, while infected cells expressed the same amount of IFITM3 in response to IFNa in both conditions. The researchers reasoned that if the incorporation of IFITM3 was responsible for the limited replication of unadapted SHIVs, depleting IFITM3 from the host cells would improve the replication of unadapted SHIVs, but not of adapted SHIVs. They genetically engineered Ptm cells with the CRISPR technology to deplete IFITM3 from these cells and observed that after several days of infection, the unadapted virus titer was higher in absence of IFITM3 compared to the control conditions, whereas the titer of adapted viruses was independent of IFITM3 expression.

Virus replication curves of unadapted (blue) and adapted (orange) SHIVs in Ptm lymphocytes. IFNa inhibits replication of unadapted viruses in non-targeting control (NTC) cells (dark blue dashed line vs dark blue filled line), but not of unadapted viruses in cells depleted for IFITM3 or IFITM1 + IFITM3 (M1 + M3). IFNa does not impact adapted viral replication (orange dashed line NTC vs orange filled line NTC) and IFITM3 expression has no effect on it.
Virus replication curves of unadapted (blue) and adapted (orange) SHIVs in Ptm lymphocytes. IFNa inhibits replication of unadapted viruses in non-targeting control (NTC) cells (dark blue dashed line vs dark blue filled line), but not of unadapted viruses in cells depleted for IFITM3 or IFITM1 + IFITM3 (M1 + M3). IFNa does not impact adapted viral replication (orange dashed line NTC vs orange filled line NTC) and IFITM3 expression has no effect on it. Illustration from publication.

Sharma et al. conclude from this study, which was conducted in collaboration with the Emerman lab, that they “identified macaque IFITM proteins as important host factors that restrict replication of SHIVs encoding circulating HIV variants in macaque cells.” Given that IFITM’s restriction factor function is believed to be due to its interaction with Env, one readily testable hypothesis is that adapted SHIVs acquire Env variants that do not interact with IFITM. This work is still under investigation. Also, Sharma is confident that this work opens the door to new SHIVs engineering: : “Our findings provide a way to rationally designing clinically-relevant SHIVs to improve the SHIV/macaque model of HIV infection.”

Mechanism of adaptation of SHIVs in macaques. Unadapted virions are characterized by a low number of virion Env, high inclusion of IFITMs when budding from the host cells, leading to slower replication and preventing persistence. In the other hand, adapted virions present a high level of virus Env, low inclusion of IFITMs, leading to high replication and persistence. However, antigen changes occur during this process. Illustration provided by Dr. Sharma.
Mechanism of adaptation of SHIVs in macaques. Unadapted virions are characterized by a low number of virion Env, high inclusion of IFITMs when budding from the host cells, leading to slower replication and preventing persistence. In the other hand, adapted virions present a high level of virus Env, low inclusion of IFITMs, leading to high replication and persistence. However, antigen changes occur during this process. Illustration provided by Dr. Sharma. Illustration provided by Dr. Sharma.

This work was supported by the National Institutes of Health.

Fred Hutch/UW Cancer Consortium members Drs. Overbaugh and Emerman contributed to this research.

Sharma A., McLaughlin RN. Jr, Basom RS., Kikawa C, OhAinle M, Yount JS., Emerman M., Overbaugh. Macaque interferon-induced transmembrane proteins limit replication of SHIV strains in an Envelope-dependent manner. PLoS Pathog 15 (15(7): e1007925. https://doi.org/10.1371/journal.ppat.1007925