Interferon-α subtype treatment induces the repression of SRSF1 in HIV-1 target cells and affects HIV-1 post integration steps

Efficient replication of HIV-1 depends on balanced levels of host cell components, including cellular splicing factors. Type I interferons (IFN-I), playing a crucial role in the innate immune defense against viral infections, are well known to induce the transcription of IFN-stimulated genes (ISGs) including potent host restriction factors. Not so well known is, that IFN-repressed genes (IRepGs) also affect viral infections by downregulating host dependency factors that are essential for viral replication. So far, knowledge about IRepGs involved in HIV-1 infection is very limited. Here, we demonstrate that expression levels of the serine/arginine-rich splicing factor 1 (SRSF1) were repressed upon treatment with IFNα subtypes in HIV-1 susceptible cell lines as well as primary cells. Furthermore, we could demonstrate in two independent patient cohorts that HIV-1 infection and the concomitant inflammation during the acute and chronic phase, resulted in the strong induction of ISGs, but at the same time significantly repressed SRSF1. 4sU-labeling of newly transcribed mRNAs revealed that IFN-mediated repression of SRSF1 originated from a transcriptional shutdown. Experimental downregulation as well as overexpression of SRSF1 expression levels resulted in crucial changes in HIV-1 LTR-transcription, alternative splice site usage and virus production. While lower SRSF1 levels resulted in low vif mRNA levels and thus severely reduced viral infectivity, higher levels of SRSF1 impaired LTR-Tat-activity and HIV-1 particle production. Our data highlight the so far undescribed role of SRSF1 acting as an IFN-repressed cellular dependency factor decisively regulating HIV-1 post integration steps. Author Summary IFN-I play a central role in the innate immune defense against viral infections by regulating the expression of interferon stimulated genes (ISGs) and interferon repressed genes (IRepGs). The stimulation of host restriction factors and the reduction of host dependency factors decisively affects the efficiency of HIV-1 replication. After the stable integration of the provirus into the host chromosome, HIV-1 exploits the host cell transcription and splicing machinery for its replication. A network of conserved splice sites and splicing regulatory elements maintain balanced levels of viral transcripts essential for virus production and immune evasion. We demonstrate the so far undescribed role of the splicing factor SRSF1 as an IRepG crucially involved in HIV-1 RNA processing. In HIV-1 infected individuals, we observed inversely proportional expression of high ISG15 and low SRSF1 levels, which were restored in ART treated patients. We could demonstrate, that IFN-I stimulation of HIV-1 target cells resulted in a significant repression of SRSF1 RNA and protein levels. Since low SRSF1 expression decisively reduced HIV-1 vif mRNA levels, a severe impairment of viral replication was observed in APOBEC3G expressing cells. As overexpression negatively affected HIV-1 LTR transcription and virus production, balanced levels of SRSF1 are indispensable for efficient replication.


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The human immunodeficiency virus type 1 (HIV-1) depends on cellular components of the host, 67 which are crucial for efficient replication and thus termed host dependency factors (1). Once indispensable for viral replication. Thus, alternative splicing and exploitation of the full range of the 72 cellular splicing code is required to produce balanced levels of all essential viral mRNAs (2, 3).

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balanced levels of SRSF1 are crucial for efficient HIV-1 replication, as both higher and lower levels 126 led to severe impairments at the level of LTR transcription, alternative splicing or virus production.

SRSF1 is significantly downregulated in HIV-1 infected patients
130 In a previous RNA-sequencing based study we were able to demonstrate that levels of specific infected patients when compared to healthy individuals (Fig 1a). Furthermore, SRSF2, SRSF5 138 and SRSF8 transcript levels were also lower in this cohort, albeit the difference was not significant 139 (Fig 1a). Surprisingly, we observed that gene expression of SRSF is generally restored in patients 140 under ART treatment and transcript levels of SRSF1 were even significantly higher when 141 compared to healthy donors (Fig 1b). Even under ART-treatment, SRSF3 and SRSF10 mRNA 142 expression levels were still lower in chronically HIV-1 infected patients in contrast to healthy 143 individuals (Fig 1b). Transcript levels of SRSF2 and SRSF7 in ART-treated patients were 144 comparable to the levels observed in the healthy control group. (Fig 1b). A marginal but significant 145 difference in SRSF expression levels in healthy individuals and HIV-1 infected ART-treated 146 patients was also observed for SRSF9, however the total amount of transcripts was low abundant 147 (Fig 1b). In all patient groups, SRSF12 transcript levels were only slightly above the limit of 148 detection (Fig 1a-b).

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Since SRSF1 was the most significant of the differentially expressed genes in the patient groups 150 and also described to be crucially involved in 40), we analyzed the

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HIV-1 infected patients under ART-treatment showed reduced levels of ISG15 when compared to 160 untreated HIV-1 infected individuals albeit still higher than healthy individuals (Fig 1c). The ISG15 161 expression data of our representative cohort were in line with previous observations of HIV-1 162 infection stimulating IFN induction and thus ISGs (41,42). In order to investigate, whether 163 repression of SRSF1 correlates with ISG induction in HIV-1 infected patients, we performed an 164 SRSF1-specific RT-qPCR. For acutely and chronically HIV-1 infected patients, as well as ART-165 treated patients, lower levels of SRSF1 mRNA were detected in contrast to healthy donors (Fig   166  1d). In chronically infected patients, SRSF1 levels were downregulated in the majority of the 167 patient derived samples. However, in some patients SRSF1 mRNA levels were increased in 168 contrast to the healthy control group (Fig 1d), a finding which can be deduced from the fact that 169 chronically HIV-1 infected patients generally represent a heterogeneous cohort. ART-treated 170 patients represented a more homogeneous group in comparison to acutely or chronically infected 171 patients showing significantly decreased SRSF1 mRNA levels (Fig 1d). In general, high induction 172 of ISG15 was concomitant with strong repression of SRSF1 in single individuals. Thus, we 173 discovered a possible interrelation between the downregulation of SRSF1 and the stimulation of

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vast majority of the tested IFNα subtypes showed a positive correlation between ISG15 induction 197 and SRSF1 repression (Fig 2c)

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In differentiated macrophage-like THP-1 cells, we observed a strong 100-to 1000-fold induction 212 of ISG15 mRNA expression levels after 4 h of treatment with both IFNα2 and IFNα14 (Fig 3a).

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Treatment with IFNα2 resulted in a 13-fold downregulation of SRSF1 after 12 h while expression 214 levels were restored 24 h post treatment (Fig 3b). Treatment with IFNα14 also resulted in a 13-215 fold downregulation of SRSF1 after 24 h and a long-lasting effect with a still 6-fold downregulation 216 after 48 h (Fig 3c). Overall IFNα14 induced a stronger and more long-lasting repression than 217 IFNα2 (Fig 3b-c).

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In Jurkat T-cells, IFN-treatment with both IFNα subtypes led to a strong 10-to 100-fold induction 219 of ISG15 mRNA expressions levels after 4 h (Fig 3d) albeit less pronounced when compared to 220 the THP-1 cells (Fig 3a). A time-dependent significant downregulation of SRSF1 mRNA levels 221 could be observed after treatment with both IFNα2 and IFNα14 (Fig 3e-f). Significant 222 downregulation of SRSF1 in Jurkat T-cells already occurred after 4 to 8 h of treatment and was 223 much less pronounced than in THP-1 cells with an only about 2-fold reduction for both IFNα 224 subtypes (Fig 3e-f). In conclusion, inversely to ISG15 expression, SRSF1 was downregulated in HIV-1 target cells, in 226 particular macrophage-like THP-1 cells, upon IFN-I stimulation and thus represents an IRepG.

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In order to further analyze whether the IFN-induced reduction of SRSF1 can also be observed on 228 the protein level, we performed Western Blot analysis of IFN-treated THP-1 cells. Both treatments 229 with IFNα2 and IFNα14 resulted in a decrease in SRSF1 protein levels 36-42 h post treatment 230 (Fig 3g). While treatment with IFNα2 only led to a weak repression, treatment with IFNα14 resulted 231 in a strong downregulation of SRSF1 protein levels (Fig 3g), which was in accordance to the 232 results on mRNA expression levels (Fig 3b-c). When compared to the mRNA levels, SRSF1 233 protein levels decrease with a time shift of 12 to 24 h, which might be explained by the half-life of 234 persisting mRNA and protein levels (Fig 3h).

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In order to assess whether these findings can be translated to primary human cells, we analyzed 236 gene expression of SRSF1 after treatment with IFNα14 in primary human monocyte-derived 237 macrophages (MDMs). A strong 50-to 500-fold induction was observed for ISG15 mRNA 238 expression levels after 4 h of treatment (Fig 3i). Concomitantly, a time-dependent repression of 239 SRSF1 was detected with a significantly >2-fold downregulation of SRSF1 mRNA levels after 8 h 240 (Fig 3j). Although less pronounced than in the cell culture system, IFN-mediated repression of 241 SRSF1 mRNA expression could thus be confirmed in primary human cells.

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To assess whether the downregulation of SRSF1 was IFN-I specific, we included IFNγ as the only 243 member of the type II IFN family (45). Since IFNγ binds to the IFNγ receptor (IFNGR) and activates 244 a distinct signaling pathway (46), the IFN-regulatory factor 1 (IRF1) was chosen as a control of 245 IFN-II specific activation of the gamma interferon activation site (GAS) regulated promotor (47).

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We used THP-1 macrophage-like cells since they showed the strongest repression of SRSF1 247 upon IFN-I treatment (Fig 3b, c, e and f).

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Treatment with IFNγ led to a strong 100-fold induction of IRF1 after 4 h (Fig 3k), but only a weak

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(1.3-fold) reduction in SRSF1 mRNA expression levels was detected after 8 h of IFNγ-treatment 250 (Fig 3l). However, the overall changes in mRNA expression were negligible when compared to 251 the effect of IFN-I treatment (Fig 3b-c). Additionally, a time-dependent increase in SRSF1 mRNA 252 expression was observed after IFNγ-treatment between 12 h to 48 h, resulting in significantly 253 elevated levels at 24 h and 48 h (Fig 3l). Overall, the repression of SRSF1 seems to be a more and 440-fold compared to untreated, respectively) (Fig 4a). In contrast, the levels of newly 267 transcribed SRSF1 mRNA were severely reduced after 8 h, with a reduction in relative mRNA 268 expression levels of around 10-fold when compared to the control. After 24 h, SRSF1 mRNA 269 expression levels recovered but relative mRNA expression levels was still reduced by 2-fold when 270 compared to the control (Fig 4b). This data indicates that IFN-mediated SRSF1 downregulation 271 most likely occurs on the transcriptional level.

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SRSF1 repression was more pronounced in IFNα2-treated uninfected cells compared to HIV-1 280 infected cells (Fig 4c). IFNα14, which induced a long-lasting and 13-fold downregulation in non-281 infected cells, led to an overall weaker SRSF1 mRNA repression of about 6-fold after 12 h in HIV-1 282 infected THP-1 cells (Fig 4d). Significantly higher SRSF1 expression levels were measured in

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Differentiated macrophage-like THP-1 cells were infected with the R5-tropic HIV-1 laboratory 294 strain NL4-3 (AD8) or mock infected 16 h before treatment with R848 or IFNα14. The obtained 295 results from the treatment of HIV-1 infected or mock infected THP-1 cells with IFNα14 were 296 described in the previous section (Fig 4d). Treatment with R848 led to a significant repression of 297 SRSF1 mRNA levels in uninfected cells after 8 h and 24 h (2.5-and 3-fold respectively) (Fig 4e).

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After 8 h, SRSF1 mRNA levels were repressed by 5-fold while after 24 h even a 6-fold 299 downregulation was detected in HIV-1 infected THP-1 cells (Fig 4e). Total viral mRNA levels were 300 measured to investigate the impact of IFNα14 or R848 on viral replication. RT-qPCR was performed using a primer pair amplifying a sequence in Exon 7, which is present in all viral mRNA 302 transcripts (Fig 5). The amount of total viral RNA was reduced roughly by 6-fold after 8 h upon 303 treatment with IFNα14, while after 24 h total viral mRNA levels were comparable to the untreated 304 control (Fig 4f). Upon treatment with R848, a 14-fold reduction of total viral mRNA expression 305 levels was detected after 8 h. After 24 h, the expression levels were still repressed by roughly 2-306 fold (Fig 4f). In conclusion, this data indicates that signaling pathways triggered by sensing via

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Knockdown efficiency was verified via One-Step RT-qPCR, with siRNA inducing a knockdown of 319 the SRSF1 gene expression of >80 % when compared to the negative control siRNA (Fig 6a).

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Intracellular total viral mRNA levels, measured via Exon 1 or 7 containing mRNAs which are 321 present in all viral mRNA isoforms, were slightly elevated upon SRSF1 knockdown, albeit only 322 significant for Exon 1 (Fig 6b).

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Next, we analyzed the viral splicing pattern via semi-quantitative RT-PCR focusing on viral intron-324 less 2 kb-, intron-containing 4 kb-and tat specific mRNA-classes. SRSF1 knockdown resulted in 325 significant alterations in the viral splicing pattern of all mRNA classes (Fig 6c). These alterations 326 could also be confirmed quantitatively by RT-qPCR using transcript specific primer pairs (Fig 5,  Fig 6d- Table 1). The mRNAs of vif and vpr, the former of which is particularly crucial for efficient 328 viral replication (55, 56), were significantly downregulated by 3-and 1.4-fold (Fig 6d). Since HIV-329 1 depends on the viral protein Vif to counteract APOBEC3G (A3G)-mediated antiviral activity of 330 the host cell, this loss in vif mRNA might severely affect viral replication (14,56,57). While mRNA 331 levels of tat1 were not altered, both tat2 and tat3 mRNAs were repressed by 4-and 2-fold 332 respectively (Fig 6e). Generally, the frequency of non-coding leader exons 2/3-including 333 transcripts was significantly repressed by the factor of 5 and 2, respectively (Fig 6f). Since the 334 levels of multiply spliced mRNAs (spliced from D4-A7) were slightly but significantly decreased by 335 1.25-fold, while levels of unspliced viral mRNAs (unspliced Intron 1) was significant increased by 336 1.4-fold (Fig 6g), knockdown of SRSF1 obviously shifts the ratio towards unspliced mRNAs.

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338 Table 1: Primers used for RT-PCR and RT-qPCR. replication capacity. Therefore, we performed replication kinetics in A3G-deficient CEM-SS cells

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replication of both NL4-3 vif and NL4-3 G I3 -2 mut was strongly delayed indicating a less efficient 354 viral replication capacity (Fig 6j). This data was in agreement with previously published data (58) 355 suggesting that reduced levels of vif mRNA, as triggered by low SRSF1 amounts, strongly impair

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In conclusion, knockdown of SRSF1 disturbed the fine balance in the ratio of all were harvested and analyzed as described above.

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Relative mRNA expression levels of SRSF1 were enhanced by multiple orders of magnitude (Fig   367  7a) and protein expression and nuclear localization was confirmed using immune fluorescence 368 microscopy ( S3 Fig). As determined by the Exon 1 and 7 containing mRNAs, overexpressing 369 SRSF1 resulted in a significant decrease in total viral mRNA levels (2-fold) (Fig 7b). To further  (Fig 7c).

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Overexpression of SRSF1 resulted in significant changes in the viral splicing pattern of all HIV-1 379 mRNA classes (Fig 7d). Alterations in the expression of HIV-1 specific mRNAs were also 380 quantitatively confirmed by RT-qPCR using transcript specific primer pairs (Fig 7e-h). Levels of 381 vif and vpr mRNA were increased by more than 10-fold (Fig 7e) while tat1 mRNA expression was 382 reduced by 3-fold and tat2 and tat3 mRNAs were upregulated by roughly 2-and 4-fold respectively 383 (Fig 7f). In contrast to tat-specific mRNA-isoforms, the frequency of overall exon 2 inclusion was 384 not altered upon SRSF1 overexpression. Exon 3 inclusion was reduced by roughly 3-fold (Fig 7g).

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The levels of multiply spliced mRNAs were not altered, while levels of unspliced viral mRNAs, 386 measured via Intron 1-containing mRNAs, were significantly decreased by 1.6-fold (Fig 7h).

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Next, we performed RT-qPCR analysis with virus extracted from the cellular supernatant and 388 found a decrease in viral copies, albeit not significant (Fig 7i). As determined by ELISA, the levels 389 of p24 capsid were significantly lower when compared to mock transfected cells (Fig 7j). TZM-bl 390 reporter cells were used to monitor infectivity of virus containing cellular supernatant harvested 391 from transfected cells, revealing a significantly lower luciferase activity upon elevated SRSF1 392 levels (Fig 7k). This reduced infectivity was confirmed by X-Gal staining of TZM-bl cells infected 393 with virus containing supernatants (Fig 7l).

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Thus, overexpression of SRSF1 negatively affected Tat-LTR transcription and alternative splicing.

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Although vif mRNA levels, crucial for efficient viral replication, were significantly increased, both 396 virus production and infectivity were significantly impaired. leading to protein degradation are unlikely. A prolonged SRSF1 downregulation is detrimental for 423 a variety of cellular mechanisms and to guarantee balanced levels, SRSF1 was shown to maintain 424 homeostasis through negative splicing feedback (29, 78). This autoregulatory mechanism will 425 most likely also be responsible for the rapid upregulation that occurs immediately after the trough 426 level of SRSF1 is reached.

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Since expression levels of SRSF1 were repressed to a much higher extent in THP-1 macrophages 428 than in Jurkat T-cells, the magnitude of SRSF1 repression seems to underlie cell type specific 429 characteristics. Importantly, we were also able to confirm our cell culture derived results using 430 primary cells.

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During our initial screen, we investigated differences in the expression levels of SRSF transcripts 432 between healthy and HIV-1-infected individuals. Upon HIV-1 infection, specific SRSF transcript 433 levels and in particular SRSF1 were significantly lower in LPMCs and PBMCs when compared to 434 healthy individuals (Fig 1). Since we have shown that IFN treatment has a direct effect on the patients, we observed that this difference could be reversed. A slight, non-significant increase was 442 even observed, hence, currently we cannot exclude the possibility of ART-treatment having an 443 influence on the transcript levels of SRSF1 or SRSF in general. Interestingly, it has been shown 444 that more than 4000 genes are differentially expressed upon ART and that the IFN-induced JAK-

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However, whether this effect appears to be due to a decrease in inflammation or a direct effect of 447 the administered substances needs further investigation.

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In agreement with our data, SRSF1 in high concentrations was shown to block Tat-mediated LTR 521 transcription by competing with the viral protein Tat for an overlapping binding sequence within 522 the trans-activation response element (TAR) region. However, in the absence of Tat, SRSF1 523 increased the basal levels of HIV-1 transcription (40).

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An interesting question that remains is whether drug targeting of SRSF1 would result in viral 525 inhibition. The drug IDC16 was shown to block the replication of X4 and R5 tropic viruses, as well 526 as clinical isolates via direct interaction with SRSF1 (95). The indole derivative can significantly 527 influence splice enhancer activity of SRSF1 and impair splicing of HIV-1 pre-mRNA, thereby 528 preventing the formation of multiple spliced mRNA isoforms and the expression of the early 529 proteins Tat, Rev and Nef. However, because of the numerous influences on essential cellular 530 processes, it is unlikely that such a drug will be used to treat HIV-1 infections.

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In summary, our work shows that IFNs, in addition to the induction of antiviral genes, can also 532 downregulate host factors which has a decisive influence on the early HIV-1 replication.

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IFNα subtypes were produced as previously described (7), IFNγ was purchased from PBL assay  Table   566 1. ACTB or GAPDH were used as loading control for normalization. For qualitative analysis of response element (ISRE), was used to determine the activity of the different IFNα-subtypes (7).

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Cells were seeded at 1.5 x 10 5 cells per well in 12-well-plates and incubated overnight. If not indicated differently, all experiments were repeated in three independent replicates.

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Statistical significance compared to untreated control was determined using unpaired student's t-

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GAPDH was used as loading control. Unpaired t-tests were calculated to determine whether the difference 972 between the group of samples reached the level of statistical significance (* p<0.05, ** p<0.01 and *** 973 p<0.001).