Macaque interferon-induced transmembrane proteins limit replication of SHIV strains in an Envelope-dependent manner
[['The macaque models of HIV-1 infection have been critical to the understanding of retroviral pathogenesis as well as for testing antiretroviral therapies and candidate vaccines for HIV-1 .', 'However , multiple species-specific host factors restrict HIV-1 replication in macaque cells [1] .', 'To overcome these restrictions chimeric SIV/HIV-1 viruses ( SHIVs ) , which encode the SIV antagonists of known restriction factors are used to infect macaques to model HIV-1 infection .', 'Existing SHIV/macaque models typically employ SHIVs that encode HIV-1 sequences from viruses that were adapted by viral passage in cell culture , and often these viruses are from chronic stages of infection .', 'However , there is evidence that the chronic stage HIV-1 variants are distinct from HIV-1 variants that are selected for transmission in humans [2 , 3] .', 'In addition , SHIVs encoding HIV-1 sequences derived directly from humans typically require further adaptation in vitro in macaque cells and/or in vivo by serial macaque-passage [1] in order to obtain pathogenic viruses that establish persistent infection in macaques .', 'These variant chimeric viruses used in the SHIV/macaque models are thus “adapted” SHIVs .', 'We have previously determined that most circulating , transmitted HIV-1 Envelope ( Env ) variants , including the transmitted/founder variants , do not use macaque CD4 entry receptor efficiently [4] and thus SHIVs generated using these Envs replicate poorly in macaque cells .', 'The adaptation of SHIV Env sequences in macaques increases replication and pathogenicity of SHIVs [5–11] but also leads to antigenic changes in Env that can limit their utility for vaccine and therapeutic approaches [12] .', 'SHIVs encoding circulating HIV-1 variants obtained directly from the newly infected patients without adaptation ( termed unadapted SHIVs ) that maintain the antigenic properties of the transmitted variants are desired as challenge viruses for vaccine and therapeutic studies .', 'However , most attempts at generating these SHIVs have failed as unadapted SHIVs replicate poorly , if at all , in macaque cells and do not establish persistent infection [13] .', 'The virus-host dynamics that contribute to the differences in replication of adapted and unadapted SHIVs in macaques are not well defined .', 'One of the main host determinants that exerts immune pressure on viruses in vivo is the type-1 interferon ( IFN ) response .', 'Upon infection , detection of viral pathogen-associated molecular patterns ( PAMPs ) by host pattern recognition receptors ( PRRs ) activates a signaling cascade that results in production of IFNα/β and other inflammatory cytokines [14] .', 'IFNα/β in turn activates autocrine or paracrine IFN-signaling pathways that results in expression of hundreds of IFN-stimulated genes ( ISGs ) –thus inducing an “antiviral state” .', 'Proteins encoded by certain ISGs such as APOBEC3s , TRIM5 , Tetherin/BST-2 , and MxB/Mx2 potently block lentiviral replication [15 , 16] and are referred to as “restriction factors” .', 'Lentiviruses have in turn evolved evasion/escape mechanisms and encode viral antagonists to counteract the restriction factors .', 'These antagonistic virus-host interactions have resulted in an evolutionary “arms race” that drives continuous rounds of selection for advantageous mutations in the restriction factor genes [17] .', 'Due to viral-host coevolution and because viruses can evolve faster than their hosts , lentiviral antagonists in turn adapt to the restriction factors encoded by their natural hosts .', 'Thus , restriction factors are less active against wild-type viruses replicating in their natural host but act as potent barriers against cross-species transmission [15 , 16] .', 'HIV-1 infection in humans and SIV infection in macaques induces a robust IFN response during acute infection in vivo [16 , 18–20] .', 'Despite the induction of an IFN response , HIV-1 and SIV replication persists in their respective natural hosts .', 'Recent studies suggest that transmitted strains of HIV-1 are more IFN-resistant than strains obtained from chronic stages of infection [21 , 22] , although this has not been seen in all studies [23 , 24] .', 'However , in the case of unadapted SHIV infection in macaques , the selection pressure may be distinct as the HIV-1 portion of the genome has been adapted in humans and SHIVs therefore may be under a different selection pressure that is more similar to a cross-species transmission event .', 'In some cases , adapted rapidly replicating , pathogenic SHIVs have been derived by rapid serial macaque-passage , performed within the first two weeks of infection , a time during which macaques mount a robust IFN response to infection [18 , 20] , potentially implicating escape from the IFN response as contributing to the adaptation process .', 'Recently , we examined the sensitivity of a panel of adapted and unadapted SHIVs to IFNα in macaque lymphocytes and found that the unadapted SHIVs were potently inhibited by IFNα despite encoding the SIV antagonists of known restriction factors [25] .', 'In contrast , SHIVs encoding adapted HIV-1 variants were largely resistant to IFNα .', 'This difference mapped to HIV-1 Env , which was also a major determinant of replication differences , with adapted SHIVs demonstrating more rapid replication kinetics than unadapted SHIVs in macaque lymphocytes .', 'These findings suggest that there are macaque-specific ISGs that restrict replication of unadapted SHIVs , and the adaptation of Env potentially plays a role in evading or antagonizing the macaque IFN response .', 'The goals of this study were to characterize the host IFN response in macaque lymphocytes and to identify IFN-induced host factor ( s ) that specifically inhibit replication of unadapted SHIVs .', 'To this end , we measure the IFN-induced gene expression in immortalized pig-tailed macaque ( Ptm ) CD4+ lymphocytes and identify previously uncharacterized interferon-induced transmembrane protein ( IFITM ) genes as candidate host factors for the observed IFN inhibition of unadapted SHIVs .', 'The IFITM family of proteins are small ( 125–135 amino acids ) transmembrane proteins with a type II transmembrane protein topology that are expressed at a basal level in multiple cell types [26 , 27] .', 'Of the five IFITM genes encoded by humans , IFITM1 , IFITM2 , and IFITM3 are induced by IFN and display broad antiviral activity against a number of enveloped viruses [28 , 29] .', 'Recent studies have shown that human IFITMs ( IFITM1 , 2 and 3 ) are incorporated into HIV-1 virions and impair viral fusion and cell-to-cell spread [30–33] .', 'Here , we demonstrate that Ptm IFITMs are packaged at higher levels in unadapted SHIVs when compared to adapted SHIVs , that depletion of IFITMs partially rescues the IFN sensitivity of unadapted SHIVs , and that depletion of basal levels of IFITMs increases replication of unadapted SHIVs in the absence of IFN .', 'These findings identify IFITMs as IFN-induced host factors that limit replication of SHIVs in macaque lymphocytes in an Env-dependent manner .'], ['In order to characterize the IFN response in macaque lymphocytes and to identify IFNα-induced host factor ( s ) that might inhibit replication of unadapted SHIVs , we measured IFNα-induced gene expression in immortalized Ptm CD4+ lymphocytes .', 'Triplicate cultures of Ptm lymphocytes were left untreated or treated with IFNα at a concentration similar to that observed in natural SIV and HIV-1 infection ( 1000 U/ml ) [18 , 20 , 34] .', 'Twenty-four hours later , RNA was isolated , and RNA-seq libraries were prepared for sequencing .', 'Comparable numbers of reads were obtained for each sample ( average ~21 . 5 million reads for untreated and ~21 . 2 million reads for IFNα-treated , respectively ) and similar percentages of reads were unambiguously mapped ( average ~65 . 3% for untreated and ~64 . 9% for IFNα-treated , respectively ) to the pig-tailed macaque genome ( M . nemestrina Mnem 1 . 0 ) .', 'A total of 198 genes were found to be significantly differentially expressed ( |logFC| ≥ 0 . 585 & FDR 5% ) upon IFNα treatment ( Fig 1 ) .', '147/198 genes were found to be significantly upregulated and 51/198 genes were found to be significantly downregulated upon IFNα treatment ( Fig 1 , S1 Table ) .', 'In order to identify candidate IFNα-induced host factor ( s ) that inhibit replication of unadapted SHIVs , we prioritized the upregulated genes and applied the following criteria .', 'Because the differences in IFNα-induced inhibition between SHIVs mapped to Env we prioritized upregulated genes with predicted subcellular localization at sites of viral entry or assembly , such as intracellular and plasma membranes where interaction with Env is plausible .', 'For this , we determined the amino acid sequences of the open reading frames ( ORFs ) of the 147 significantly upregulated genes and used TMHMM v2 . 0 [35] to predict which of these encode transmembrane helices .', 'We found that 31/147 genes were predicted to encode at least one transmembrane helix ( Fig 1 , S2 Table ) .', 'Based on these criteria of differential expression upon IFNα treatment and predicted membrane localization , we selected two previously uncharacterized Ptm genes ( LOC105494124 and LOC105494127: both predicted to be IFITM3-like genes ) and IFITM1 ( XM_011762253 ) for subsequent analysis ( Fig 1 ) .', 'Notably , IFITM1 and IFITM3 from other species have been reported as broad-spectrum , anti-viral factors that impair viral fusion [28 , 29] .', 'Previous work to determine the number and location of IFITM sequences within animal genomes focused broadly on vertebrates [36] or primates [37] .', 'As a result , variation in IFITM genes amongst closely related species remains largely undescribed .', 'Since our RNA-seq analysis showed increased expression of multiple Ptm IFITM-related genes , including some uncharacterized sequences , we wanted to understand the location , sequence , and evolutionary relationship of previously described human IFITMs and the IFNα-upregulated Ptm IFITMs ( Fig 1 ) .', 'We mapped the relative position of all IFITM genes within the canonical IFITM locus , focused on comparing the genome of three macaques ( pig-tailed , rhesus , and crab-eating ) with human .', 'The macaque IFITM loci were mapped using the human IFITM1 , IFITM3 , and IFITM5 sequences as queries of each macaque genome assembly .', 'IFITM1 and IFITM5 had a single clear hit in each genome as compared to the human locus , suggesting one-to-one orthology of these genes ( Fig 2A; top panel; purple and red ) .', 'In contrast , IFITM3 mapped to three unique locations within the IFITM locus of each macaque genome .', 'An analysis of the synteny of these sequences across macaques and humans shows that one of these sequences is syntenic with human IFITM3 ( Fig 2A; top panel; orange ) , while the other two sequences are not found in humans .', 'Of these macaque IFITM3 sequences , two encode apparently intact IFITM3 genes ( Fig 2A; top panel; green and orange ) , while the third copy has been pseudogenized with mutations that introduce numerous stop codons ( Fig 2A; top panel; gray ) .', 'Humans have a duplication of IFITM3 , named IFITM2 [36] , and a phylogenetic tree shows that all the intact and pseudogenized macaque IFITM3 genes group together with the human IFITM2 and IFITM3 sequences ( Fig 2B and S1 File ) .', 'While IFITM2 has only been described in humans , chimpanzees , and gorillas [37] , we find that macaques also have a duplication of IFITM3 within the IFITM locus .', 'This copy LOC105494124 , here called IFITM3A , retains an intron and an ORF of the same length ( ~402nt ) as the parental IFITM3 ( LOC105494127 ) .', 'In addition to the IFITM3 sequences within the IFITM locus of the macaques , we found numerous shorter sequences ( 100-400bp ) outside of the IFITM locus with sequence identity to IFITM3 greater than 80% .', 'These hits represent partial and full-length copies of IFITM genes .', 'Two of these sequences were identified in the RNA-seq analysis , denoted as IFITM3L sequences on chromosomes 16 and 9 ( Fig 2A; bottom panel; brown , pink ) .', 'These copies are both found in the intronic region of genes and have lost the intron present in IFITM sequences within the IFITM locus .', 'Based upon their phylogenetic grouping with IFITM3 , their dispersion in the genome , and their lack of introns , these sequences are likely retrocopies of IFITM3 , often referred to as pseudogenes of IFITM3 .', 'However , both of these retrocopies preserve a putative ORF similar in length to IFITM3 and we find these copies to be expressed .', 'We compared the magnitude of IFNα-dependent induction of these IFITM-genes by mapping the reads from our RNA-seq data to the Ptm genomic loci .', 'We found that the bulk of the RNA-seq read coverage for IFITM3A and IFITM1 was from uniquely mapping reads ( S1 Fig ) .', 'For IFITM3 , there were a considerable number of ambiguous reads that did not map uniquely , particularly in the first exon ( S1 Fig ) .', 'However , there was still enough coverage from the uniquely mapping reads to suggest that this locus was expressed at the RNA level .', 'In addition , we measured the levels of IFITM protein expression upon IFNα treatment in Ptm lymphocytes .', 'Out of five commercially available IFITM antibodies tested , we found two that react with Ptm IFITMs and selected the one with the highest reactivity for subsequent analysis .', 'Importantly , the selected anti-human IFITM3 antibody used in this study does not distinguish between Ptm IFITM3 and Ptm IFITM3A ( S2 Fig ) ; therefore , the immunoblots using this antibody are indicated as “IFITM3/3A” .', 'Our immunoblotting results indicate that IFITM3/3A is induced by IFNα in a dose dependent manner in Ptm lymphocytes ( Fig 2C ) .', 'Lastly , we performed immunofluorescence imaging of the Ptm IFITMs in mouse embryonic fibroblasts ( MEFs ) lacking the IFITM locus ( IFITMdel ) [38] and HEK293T cells , which also lack baseline IFITM expression [39] .', 'In both cell types , the IFITMs were observed in intracellular clusters primarily in the perinuclear region , and IFITM1 staining was also observed at the plasma membrane ( S3 Fig ) .', 'The cellular localization of Ptm IFITMs is similar to what is reported for human IFITMs [30 , 40] .', 'Incorporation of human IFITMs ( IFITM1 , 2 and 3 ) into HIV-1 virions impairs viral fusion and cell-to-cell spread [30–33] .', 'In addition , human IFITMs affect HIV-1 Env processing and virion Env incorporation [41] .', 'Thus , we explored whether Ptm IFITMs contribute to the sensitivity of SHIVs to IFNα treatment in macaque lymphocytes and whether they explain Env-dependent differences in IFNα sensitivity between adapted and unadapted SHIVs .', 'We employed a prototypical , macaque-adapted SHIV ( SHIV AD8-EO ) and a prototypical , unadapted SHIV ( SHIV Q23AE ) .', 'SHIV AD8-EO was derived by five serial-macaque passages followed by further adaption in macaque PBMCs [8] while SHIV Q23AE was generated from a HIV-1 Env from early in infection and then modified with a single amino acid substitution ( A204E ) to allow the Env to use macaque CD4 for entry [42] .', 'In order to determine whether or not the Ptm IFITMs are differentially incorporated into unadapted versus adapted SHIV virions , Ptm lymphocytes were infected with SHIV AD8-EO and SHIV Q23AE .', 'Virions were harvested nine days post-infection and an amount of virus equivalent to 10 ng of SIV p27 were immunoblotted .', 'We found that Ptm IFITM3/3A was readily detected in the unadapted SHIV Q23AE virions but not in the adapted SHIV AD8-EO ( Fig 3A ) .', 'When four times more virions ( 40 ng of SIV p27 ) were immunoblotted , IFITM3/3A could be detected , but the level of IFITM3/3A in the adapted SHIV AD8-EO virions was lower than the unadapted SHIV Q23AE ( Fig 3A ) .', 'Virions were also purified by analytical sucrose density gradient fractionation to remove secreted , soluble cellular proteins and budding cellular microvesicles .', 'Immunoblot analyses of the gradient fractions with IFITM3/3A and SIV p27 antibodies revealed co-fractionation of IFITM3/3A with SIV p27 suggesting that IFITM3/3A are incorporated in the SHIV virion ( Fig 3B ) .', 'Consistent with the results in Fig 3A , we observed that IFITM3/3A is packaged at higher levels in the unadapted SHIV Q23AE virions in comparison to the adapted SHIV AD8-EO virions .', 'Importantly , the basal and IFNα-induced levels of IFITM3/3A were similar in uninfected cells or cells infected with adapted SHIV A8-EO or the unadapted SHIV Q23AE ( Fig 3C ) .', 'These findings suggest that infection with SHIV does not affect steady state or IFNα-induced IFITM levels in Ptm lymphocytes , but rather the Ptm IFITMs are differentially incorporated into an unadapted SHIV relative to an adapted SHIV .', 'Given the differences in IFITM packaging between the two prototype SHIVs , we determined whether the differences observed in IFITM incorporation are specific to the two SHIVs used in this study or also characteristic of other adapted and unadapted SHIVs .', 'We employed a panel of eight SHIVs that we previously used to examine IFNα sensitivity in Ptm lymphocytes [25] .', 'The panel includes four SHIVs encoding HIV-1 Env variants adapted through passage in cell culture and/or macaques ( AD8-EO , AD8-OG , SF162P3 , and 1157ipd3N4 ) and four SHIVs encoding HIV-1 Env variants isolated directly from infected individuals without culture or macaque adaption , three of which represent variants from early in infection ( QF495AE , Q23AE , MG505GV , and BG505AE ) and all of which encode a single amino acid substitution ( A204E or G312V ) to allow macaque CD4 use .', 'The four unadapted SHIVs in this panel were potently inhibited by IFNα ( IC50 range 2 to 164 U/ml ) whereas the four adapted SHIVs were resistant to IFNα ( IC50 range 2454 to >5000 U/ml ) [25] .', 'We utilized these SHIVs to examine virion incorporation of IFITMs .', 'When virions equivalent to 5 ng of SIV p27 were immunoblotted , Ptm IFTIM3/3A was readily detected in the unadapted SHIV virions but not in the adapted SHIVs ( Fig 4A ) .', 'Thus , we observed that IFITM3/3A virion incorporation was linked to sensitivity to IFNα for this virus panel .', 'We did detect some IFITM3/3A incorporation in adapted SHIV virions when 3 . 6- to 9 . 6-times more virions were loaded ( Fig 4B ) suggesting that IFITM3/3A is not totally excluded from these viruses .', 'Taken together , our results suggest that Ptm IFITMs are packaged at higher levels in the unadapted SHIV virions in comparison to the adapted SHIV virions .', 'In order to determine the contribution of Ptm IFITMs in restricting SHIV replication , we generated IFITM-knockout ( KO ) cell pools .', 'We designed CRISPR guide RNA ( crRNA ) predicted to target Ptm IFITM1 , IFITM3A , and IFITM3 ( Table 1 ) .', 'crRNA that did not target any macaque genes was used as a Non-Targeting Control ( NTC ) .', 'Because of the high sequence similarity between the Ptm IFITMs , one of our crRNA targeted both IFITM1 and IFITM3/3A ( this double KO is referred to as “M1+M3” ) .', 'We generated four independent batches of IFITM-KO cell pools to examine whether IFITMs affect IFNα sensitivity of unadapted SHIV .', 'IFNα-induced IFITM protein expression was confirmed through immunoblotting of IFNα-treated KO cells .', 'The levels of IFITM3/3A in the IFNα-treated , KO cells were 5 . 9- to 12 . 5-fold lower compared to the IFNα-treated , NTC cells ( Fig 5A; left panel; 8 . 3-fold for M1+M3 KO , 12 . 5-fold for IFITM3A KO , and 5 . 9-fold for IFITM3 KO ) , and comparable to control cells that were not treated with IFNα , suggesting a partial KO and/or presence of unedited cells in the KO pool .', 'The levels of IFITM1 in the IFNα-treated , M1+M3 KO cells were lower compared to the IFNα-treated ( 100 fold ) or IFNα-untreated ( 17 fold ) control cells ( Fig 5A; right panel ) .', 'The KO cells were infected with the unadapted SHIV Q23AE or adapted SHIV AD8-EO at a multiplicity of infection ( MOI ) of 0 . 02 to allow spreading viral infection .', 'The infected cells were cultured in the presence of 1000 U/ml of IFNα over a nine-day time course .', 'Viral replication was also measured in the IFNα-untreated NTC cells to determine baseline replication .', 'In order to obtain a quantifiable measure of IFNα sensitivity , we measured the ratio of the area under the curve ( AUC ) of the replication curve in the IFNα-treated cells to the AUC of the replication curve in the untreated , NTC cells .', 'Consistent with our prior results , the prototype unadapted SHIV Q23AE exhibited a pronounced IFNα-induced inhibition of viral replication ( 6 . 7-fold at day 9 post infection ) whereas the adapted SHIV AD8-EO was largely resistant to IFNα ( AUC ratio 0 . 86 vs 0 . 96 , p = 0 . 0091 two-tailed student’s t-test ) ( Fig 5B and 5C ) .', 'Next , we compared the IFNα sensitivity of adapted and unadapted SHIV in IFITM-KO cells .', 'We observed that in comparison to the NTC cells ( AUC ratio 0 . 86 ) , KO of M1+M3 ( AUC ratio 0 . 93 , p = 0 . 0054 ) or IFITM3 ( AUC ratio 0 . 92 , p = 0 . 0156 ) , but not IFITM3A ( AUC ratio 0 . 85 ) , resulted in modest but statistically significant rescue of IFNα-induced inhibition of SHIV Q23AE replication ( Fig 5B and 5D ) .', 'In contrast , IFITM KOs had no significant effect on IFNα sensitivity of adapted SHIV AD8-EO ( Fig 5C and 5E ) .', 'Thus , the differential activity of IFITMs on the unadapted SHIVs accounts for some , but not all , of the IFNα sensitivity of these viruses .', 'We have previously observed that the IFNα sensitivity of the SHIVs positively correlates with the replication capacity of the virus [25] .', 'Thus , we hypothesized that basal levels of Ptm IFITMs have the potential to limit SHIV replication .', 'We employed the IFITM-KO cell pools described above and assessed the ability of unadapted SHIV Q23AE and adapted SHIV AD8-EO to replicate in the absence of IFNα over a nine-day time course .', 'Reduction of IFITM protein expression was confirmed through immunoblotting of KO cells .', 'The levels of IFITM3/3A in the KO cells were lower than or comparable to the NTC cells ( Fig 6A ) .', 'Since the IFITM3/3A antibody does not distinguish between IFITM3 and IFITM3A , we are unable to rule out weather the observed IFITM levels in the IFITM3 KO cells is due to the unedited cells in the KO pool or cross-detection of IFITM3A .', 'Consistent with our previous results , we observed that the unadapted SHIV Q23AE replicates slowly with peak virus levels of ~104 pg/ml of SIV p27 at six days post-infection ( Fig 6B ) .', 'In contrast , the adapted SHIV AD8-EO replicates rapidly reaching peak virus levels of >106 pg/ml of SIV p27 by six days post-infection ( Fig 6C ) .', 'In order to compare the replication kinetics of the adapted and unadapted SHIVs across the IFITM-KO cells , we determined AUC for the replication curves from Fig 6B and 6C .', 'When compared to the NTC cells ( AUC 30 . 1 ± 0 . 6 ) , a modest but statistically significant increase in SHIV Q23AE replication was observed in the M1+M3 KO cells ( AUC 32 . 1 ± 0 . 5 , p = 0 . 002 ) and the IFITM3 KO cells ( 31 . 6 ± 0 . 7 , p = 0 . 011 ) , despite comparable IFITM3/3A protein levels , but not in the IFITM3A KO cells ( AUC 29 . 9 ± 0 . 6 , Fig 6D ) .', 'In contrast , IFITM KOs had no effect on the replication capacity of adapted SHIV AD8EO ( Fig 6E ) .', 'Thus , basal levels of IFITMs selectively limit replication of unadapted SHIV in macaque lymphocytes .', 'We have observed that the unadapted SHIVs have 2 . 7- to 14 . 3-fold lower virion Env levels than adapted SHIVs and that virion Env content correlates with replication capacity and sensitivity to IFNα [25] .', 'A recent study reported that the human IFITMs impair HIV-1 infectivity by decreasing Env processing in the host cell; increasing Env shedding; and reducing virion Env incorporation [41] .', 'In order to determine the impact of Ptm IFITMs on Env processing , virion incorporation , and viral infectivity , we co-expressed SHIV AD8-EO or SHIV Q23AE and Ptm IFITM in HEK293T cells .', 'The cell and virion lysate were immunoblotted and infectivity of cell-free virions were measured on TZM-bl reporter cell line .', 'Consistent with other studies [37 , 40 , 43] , we did not observe any differential effects of Ptm IFITM expression on Env processing in the producer cells and Env incorporation into viral particles ( S4A and S4B Fig ) .', 'In addition , Ptm IFITM expression did not affect infectivity of SHIV virions produced during 48 hours in these experiments ( S4C Fig ) .', 'This is perhaps not surprising given that the effect of IFN on replication is only apparent several days after infection in a spreading infection .', 'Consistent with our previous study [25] , we did observe that the unadapted SHIV Q23AE has lower virion Env levels than adapted SHIV AD8-EO and there are differences in the migration pattern of gp160/gp120 , likely attributable to the glycosylation levels of the Env .', 'Thus , our findings that the Ptm IFITMs limit replication of unadapted SHIV Q23AE during spreading infection lend support to the previously reported observations that IFITM-mediated restriction is most potent when IFITMs are present in both the donor and the target cells [30 , 40] .'], ['Here we identify a novel example of cross-species restriction in which macaque-specific restriction factors selectively restrict replication of SHIV based on circulating , transmitted HIV-1 Env variant .', 'In this study , we employed transcriptional profiling to define the repertoire of ISGs induced by IFNα in Ptm CD4+ lymphocytes .', 'On the basis of this profiling , IFITMs , which have been implicated as anti-viral factors for a number of different viruses , including lentiviruses , were identified as a candidate restriction factors in these cells .', 'We documented IFITM gene duplications in the macaque genomes and evaluated the ability of previously uncharacterized Ptm IFITMs to restrict SHIVs .', 'We found that a prototypical , macaque-adapted SHIV is resistant to IFITM-mediated restriction , whereas a prototypical , unadapted SHIV , which encodes a circulating HIV-1 Env variant , is inhibited by Ptm IFITMs .', 'Further , we demonstrate that IFITM virion incorporation tracks with the IFNα sensitivity of the SHIVs .', 'For example , IFITMs are packaged at higher levels in IFNα-sensitive SHIVs when compared to IFNα-resistant SHIVs .', 'Overall , our results suggest that the increased replication that results from adaptation of SHIVs in macaques may in part reflect an adaptation to an IFITM-mediated restriction .', 'The evolutionary “arms race” that results from antagonistic virus-host interactions has led to many evolutionary innovations that accelerate host adaptation to a virus [17] .', 'The duplication of restriction factor genes is one such strategy through which the host is able to explore multiple evolutionary trajectories to select for advantageous mutations in restriction factor genes .', 'For example , increase in the copy number of the restriction factor could allow the host to rapidly evolve against a number of different viruses and/or collectively target a given virus through different mechanisms to constrain viral evolution .', 'Thus , it is not surprising that many restriction factor gene families , such as APOBEC3 [44 , 45] , Mx1 [46] , and TRIM5 [47 , 48] , have undergone gene duplications .', 'In our RNA-seq dataset , we found sequence reads that uniquely map to multiple locations in the IFITM gene locus in the Ptm genome that are not found in the human genome , suggesting IFITM gene duplications .', 'The duplication of IFITM genes has been previously reported for vertebrates [36] and a recent study reported recurrent duplication and divergence of IFITM3 in the primate genomes [37] .', 'An in-depth analysis of IFITM gene duplications specific to the macaque genomes was lacking , though several of the macaque IFITM3 copies we describe were included in previous analyses ( S5 Fig ) .', 'Here , we demonstrate that macaques have three copies of IFITM3 genes that map to three separate loci within the IFITM locus .', 'We found that a duplicated IFITM3 gene ( LOC105494124 , here called IFITM3A ) encodes an ORF of the same length as the parental IFITM3 gene ( LOC105494127 ) that differs by 10 . 5% at the amino acid level .', 'Moreover , IFITM3A is distinct from the human IFITM3 duplication , named IFITM2 , which is found in chimpanzees and gorillas in addition to humans [37] .', 'Interestingly , we find that all three macaque species ( pig-tailed , rhesus , and crab-eating ) contain an IFITM3 pseudogene within the IFITM locus , which is absent in humans .', 'These pseudogene sequences group with high confidence in a clade that is separate from both the IFITM1 and intact macaque IFITM3s ( Fig 2B ) .', 'Phylogenetically , we are unable to resolve whether these pseudogenes were born before or after the hominoid/Old World monkey split .', 'Though , their presence in all three macaques and absence in humans suggests they were likely born in at least the last common ancestor of these macaques , but likely after the branching of Old World monkeys and hominoids .', 'These pseudogene sequences are preceded by a long branch that presumably reflects a period of rapid but neutral evolution after birth or pseudogenization of this sequence .', 'Together , these species restricted duplications of IFITM3 ( IFITM2 in humans , chimps , gorillas , and IFITM3A , IFITM3 pseudogene , and IFITM3Ls in macaques ) suggest this gene may be recurrently duplicated during primate evolution .', 'In addition to genes within the IFITM locus , our RNA-seq analysis identified expression of two IFITM3-like retrogenes ( here called IFITM3L ) , which are present in the macaque genome on chromosome 16 and 9 .', 'These IFITM3L copies are both found in the intronic region of genes and have lost the intron present in IFITM sequences within the IFITM locus .', 'These putative IFITM retrogenes await a described function .', 'In this study , we demonstrated that Ptm IFITMs are differentially incorporated in SHIV virions with higher levels in unadapted SHIVs in comparison to adapted SHIVs .', 'It has been suggested that human IFITMs directly interact with HIV-1 Env [41] , and their restriction activity maps to Env- and Gag-dependent virion incorporation [30] .', 'Thus , it is plausible that the Envs encoded by unadapted SHIVs display greater colocalization and/or interaction with Ptm IFITMs .', 'In contrast , the Envs encoded by adapted SHIVs evade and/or antagonize this interaction .', 'Moreover , our data rule out the alternative possibility that the lower IFITM incorporation in the adapted SHIVs is due to reduced induction and/or degradation of IFITMs as similar steady-state and IFNα-induced levels of IFITM3 was observed in cells infected with adapted or unadapted SHIVs .', 'By generating IFITM-KO Ptm lymphocytes and infecting them with a prototype , macaque-adapted SHIV or a prototype , unadapted SHIV encoding HIV-1 Env isolated directly from an infected individual , we demonstrated that IFITMs partly contribute to the IFNα-induced inhibition of unadapted SHIV .', 'We found that IFITM1 and parental IFITM3 , but not the duplicated IFITM3A , determines IFNα sensitivity of unadapted SHIV .', 'Because of the high sequence similarity between the Ptm IFITMs , the CRISPR guide RNA designed to target IFITM1 also targeted IFITM3/3A .', 'However , we could not discern the identity of IFITM3s ( IFITM3 and/or IFITM3A ) that were targeted in these double “M1+M3” KO cells , as the only available anti-IFITM3 antibody does not distinguish between the two Ptm IFITM3s .', 'Thus , due to the targeting of more than one IFITM by CRISPR guide RNA and because of the cross-reactivity of IFITM antibody , we cannot determine which specific IFITM contributes to anti-viral activity or whether multiple IFITMs are playing a role in inhibiting unadapted SHIV .', 'Because there is extensive duplication of IFITMs in the macaque genome , including presence of IFITM3L genes on different chromosomes , we did not attempt combinatorial knockouts so as to avoid spurious effects due to gene-editing and recombination across multiple chromosomes .', 'Our findings that IFITM KOs only result in partial recovery of viral replication of unadapted SHIVs to levels observed in the IFNα-untreated Ptm lymphocytes , suggest that either there is functional redundancy between Ptm IFITM
['Introduction', 'Results', 'Discussion', 'Materials and methods']
['HIV-1 does not persistently infect macaques due in part to restriction by several macaque host factors .', 'This has been partially circumvented by generating chimeric SIV/HIV-1 viruses ( SHIVs ) that encode SIV antagonist of known restriction factors .', 'However , most SHIVs replicate poorly in macaques unless they are further adapted in culture and/or macaques ( adapted SHIVs ) .', 'Therefore , development of SHIVs encoding HIV-1 sequences derived directly from infected humans without adaptation ( unadapted SHIVs ) has been challenging .', 'In contrast to the adapted SHIVs , the unadapted SHIVs have lower replication kinetics in macaque lymphocytes and are sensitive to type-1 interferon ( IFN ) .', 'The HIV-1 Envelope ( Env ) in the chimeric virus determines both the reduced replication and the IFN-sensitivity differences .', 'There is limited information on macaque restriction factors that specifically limit replication of the more biologically relevant , unadapted SHIV variants .', 'In order to identify the IFN-induced host factor ( s ) that could contribute to the inhibition of SHIVs in macaque lymphocytes , we measured IFN-induced gene expression in immortalized pig-tailed macaque ( Ptm ) lymphocytes using RNA-Seq .', 'We found 147 genes that were significantly upregulated upon IFN treatment in Ptm lymphocytes and 31/147 were identified as genes that encode transmembrane helices and thus are likely present in membranes where interaction with viral Env is plausible .', 'Within this group of upregulated genes with putative membrane-localized proteins , we identified several interferon-induced transmembrane protein ( IFITM ) genes , including several previously uncharacterized Ptm IFITM3-related genes .', 'An evolutionary genomic analysis of these genes suggests the genes are IFITM3 duplications not found in humans that are both within the IFITM locus and also dispersed elsewhere in the Ptm genome .', 'We observed that Ptm IFITMs are generally packaged at higher levels in unadapted SHIVs when compared to adapted SHIVs .', 'CRISPR/Cas9-mediated knockout of Ptm IFITMs showed that depletion of IFITMs partially rescues the IFN sensitivity of unadapted SHIV .', 'Moreover , we found that the depletion of IFITMs also increased replication of unadapted SHIV in the absence of IFN treatment , suggesting that Ptm IFITMs are likely important host factors that limit replication of unadapted SHIVs .', 'In conclusion , this study shows that Ptm IFITMs selectively restrict replication of unadapted SHIVs .', 'These findings suggest that restriction factors including IFITMs vary in their potency against different SHIV variants and may play a role in selecting for viruses that adapt to species-specific restriction factors .']
['Macaque model systems are critical gatekeepers for testing HIV-1 prevention methods and for studies of HIV-1 transmission and pathogenesis .', 'HIV-1 does not persistently infect macaques due to inhibition of the virus by several macaque-specific restriction factors necessitating the use of chimeric SIV/HIV-1 viruses ( SHIVs ) .', 'Existing SHIV/macaque models typically employ SHIVs that encode HIV-1 sequences from viruses amplified in culture and further adapted in macaques ( adapted SHIVs ) .', 'Development of SHIVs encoding circulating HIV-1 variants derived directly from infected humans ( unadapted SHIVs ) , which best model HIV-1 infection in humans , has been challenging as these SHIVs replicate poorly in macaque cells .', 'While some host restrictions to HIV-1 replication in macaques have been defined , there is limited information on macaque-specific restriction factors that limit replication of circulating HIV-1 variants .', 'Here , we demonstrate that the macaque interferon-induced transmembrane proteins ( IFITMs ) selectively restrict replication of unadapted SHIVs .', 'These findings may help develop new approaches to improve the SHIV/macaque model of HIV-1 infection by rationally designing clinically-relevant SHIVs that overcome restriction by macaque-specific restriction factors .']
['blood cells', 'medicine and health sciences', 'immune cells', 'pathology and laboratory medicine', 'pathogens', 'immunology', 'microbiology', 'vertebrates', 'viral structure', 'animals', 'mammals', 'retroviruses', 'primates', 'immunodeficiency viruses', 'viruses', 'rna viruses', 'old world monkeys', 'white blood cells', 'monkeys', 'animal cells', 'proteins', 'medical microbiology', 'hiv', 'microbial pathogens', 'hiv-1', 'viral replication', 'virions', 'lymphocytes', 'siv', 'macaque', 'genetic loci', 'biochemistry', 'eukaryota', 'cell biology', 'virology', 'viral pathogens', 'interferons', 'genetics', 'biology and life sciences', 'cellular types', 'lentivirus', 'amniotes', 'organisms']