HIV-TB coinfection: exploring HIV-2 restriction in macrophages by interferon-induced effectors, GBP5 and SP110
Kyabaggu, Denis Senkandwa
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HIV and TB are among top causes of mortality and morbidity globally. Incident TB cases in 2015 were approximately 10.5 million, with 11% having HIV. Having HIV is also the biggest risk factor for developing TB disease. Clinical studies show HIV-2/TB coinfection cases progress to disease slower than HIV-1/TB. However, the underlying cellular, immunological, and molecular host-pathogen interactions accounting for these observations remain unclear. The host immune response to both viral and mycobacterial infection of macrophages is partly dependent on type I interferon, which in turn induces expression of various interferon-stimulated genes (ISGs) to control the pathogens. GBP5, a GTPase with a role in activating the inflammasome; and Sp110, a nuclear body protein, are among various ISGs whose expression levels are elevated during individual viral and bacterial infections in macrophages. GBP5 seems to restrict HIV-1 replication in immune cells, while Sp110 is known to restrict MTB proliferation in macrophages, but is now thought to also promote HIV-1 replication. We hypothesized differences in the effect of these proteins on the infectivity of the two HIV subtypes in macrophages. We also hypothesized that the viral protein Vpr is associated with the host expression levels of GBP5 and Sp110. We used small hairpin RNA to knock out GBP5 and Sp110 from THP-1 human monocytoidcell line, differentiated using PMA into a macrophage phenotype;and infected these cells with GFP-expressing HIV-2 ROD 9ΔenvΔnefstrain pseudotyped with VSV-G envelope for single cycle infection. Percentages of macrophages infected with GFP-expressing viruses were measured by Flow cytometry. To determine effect of GBP5 and Sp110 onreplication of the two HIV subtypes, HIV-1 and HIV-2, we measured virus production in supernatants of productively infected THP-1 macrophages by titering cell-free supernatants on TZMBl-reporter cells at day 3 and 6 post infection. There was a marginal increase, and a dramatic decrease, of HIV-2ΔenvΔnef GFP+ virus infectivity inall THP-1 macrophage conditions in the absence of viral Vpr, and Vpx, respectively. Knocking downmacrophage Sp110 gene expression reduced single cycle (VSV-G pseudotyped) HIV-2 ROD 9ΔenvΔnefGFP+wild type and Vpr mutant virus infectivity but promoted Vpx mutant.There was a reduction in number of infectious HIV-1 particles released from Sp110 knockdown compared to normal macrophages over 6 days post infection; no difference in HIV-1 virus production between GBP5 knock down and normal macrophages in one experiment; and a decrease in HIV-1 virus production from GBP5 knockout compared to normal macrophages 3 and 6 days post infection in a follow up experiment.Interestingly, reduced Sp110 expression corresponded to increased HIV-2 production from macrophages in one experiment but no difference in viral production between normal and Sp110 knock down macrophages by day 3 post infection in the follow up experiment. There was no HIV-2 virus production from the Sp110 knock down cells on day 6 in the follow up experiment and virus spread was reduced in GBP5 knock down, relative to normal macrophages. Interestingly, reduced Sp110 expression corresponded to increased HIV-2 production from macrophages in one experiment, but there was no difference in viral production between normal and Sp110 knock down macrophages by day 3 post infection in the follow up experiment. There was no virus production from the Sp110 knock down cells on day 6 in the follow up experiment. HIV-2 virus production was also reduced in GBP5 knock down, relative to normal, productively infected THP1/PMA macrophages. Our results support the argument that HIV-2, just like HIV-1, possibly utilizes Vpr to prevent sensing of virus infection in macrophages, thus allowing viral replication without inducing interferon stimulation, and subsequent viral restriction. Our results also suggest that the host macrophage restriction factor Sp110 may indeed enhance VSV-G pseudotyped GFP+ HIV-2 and HIV-2delVpr infectivity but restrict HIV-2 Vpx mutant. This could imply presence of a yet unknown association between another HIV-2 viral accessory protein, Vpx, and the macrophage restriction factor, Sp110 known to counter invading intracellular bacteria. We report much lower productive infectious virus release of HIV-2, compared to HIV-1, from macrophages. Overall, our results suggest that Sp110 may transiently restrict HIV-2 infection of macrophages, but is eventually manipulated by the virus to promote viral replication.