Comparative Analysis of Differential Cellular Transcriptome and Proteome Regulation by HIV-1 and HIV-2 Pseudovirions in the Early Phase of Infection

In spite of the similar structural and genomic organization of human immunodeficiency viruses type 1 and 2 (HIV-1 and HIV-2), striking differences exist between them in terms of replication dynamics and clinical manifestation of infection. Although the pathomechanism of HIV-1 infection is well characterized, relatively few data are available regarding HIV-2 viral replication and its interaction with host–cell proteins during the early phase of infection. We utilized proteo-transcriptomic analyses to determine differential genome expression and proteomic changes induced by transduction with HIV-1/2 pseudovirions during 8, 12 and 26 h time-points in HEK-293T cells. We show that alteration in the cellular milieu was indeed different between the two pseudovirions. The significantly higher number of genes altered by HIV-2 in the first two time-points suggests a more diverse yet subtle effect on the host cell, preparing the infected cell for integration and latency. On the other hand, GO analysis showed that, while HIV-1 induced cellular oxidative stress and had a greater effect on cellular metabolism, HIV-2 mostly affected genes involved in cell adhesion, extracellular matrix organization or cellular differentiation. Proteomics analysis revealed that HIV-2 significantly downregulated the expression of proteins involved in mRNA processing and translation. Meanwhile, HIV-1 influenced the cellular level of translation initiation factors and chaperones. Our study provides insight into the understudied replication cycle of HIV-2 and enriches our knowledge about the use of HIV-based lentiviral vectors in general.


Introduction
Despite significant advances in anti-retroviral therapy (ARV), the acquired immunodeficiency syndrome (AIDS) caused by the human immunodeficiency viruses 1 and 2 (HIV-1 and 2) continues to pose a global health challenge.Both viruses are prominent members of the lentivirinae class of the retroviridae family.While HIV-1 is responsible for the majority of infections, HIV-2 remains mostly confined to countries in West Africa, although recent studies have shown that the virus is gaining a foothold in the United States, India and Europe [1].Even though both viruses share the same mode of transmission, and can both result in AIDS, the dynamics of infection and clinical progression are strikingly different.Infection with HIV-1 is known to be more pathogenic, with faster progression to AIDS and higher plasma viral load.On the other hand, infection with HIV-2 is characterized by lower transmissibility, slower decline of CD4 positive T cells and overall, a higher chance for nonprogression to AIDS [2,3].Both viruses share a similar genomic architecture and protein composition.The genome of HIV consists of two copies of positive, single-stranded RNA coding for the main structural capsid and matrix proteins and viral enzymes.Moreover, the genome codes for the regulatory proteins, which are required for efficient viral replication, in addition to auxiliary proteins.The HIV-2 genome does not contain the viral protein u (Vpu) gene but instead contains viral protein x (Vpx), which is also found in the simian immunodeficiency virus of sooty mangabey (SIV/sm) lineages [4][5][6].
The HIV life cycle, like that of all retroviruses, can be divided into an early and late phase.The early phase begins with the binding and entry of the viral particle to a target cell.Approximately 1-2 h post-entry, reverse transcription begins inside the viral core [7].The pre-integration complex (PIC) enters the nucleus through a complex interplay between viral and host proteins [8].The ability of HIV to enter the nucleus of non-dividing cells via its accessory proteins makes the virus a perfect base for gene delivery vector systems.It is proven that HIV-based vectors can effortlessly deliver genes into several cell types, including hematopoietic stem cells [9].Several host proteins associated with the PIC aid the viral genome during integration.LEDGF/p75 is a transcriptional regulator, which is shown to tether the viral genome to the transcriptionally active chromatin sites [10,11].
The end of proviral genome integration marks the transition from the early to late phase in the life cycle of HIV, during which transcription ensues, and new viral particles are assembled [12].For the transcription of viral RNA, Tat binds to the trans-acting response element (TAR) region of the viral genome and recruits cellular factors, such as proteinpositive transcription elongation factor b (P-TEFb), comprising cyclin-dependent kinase 9 (CDK9) and cyclin T1 (CCNT1), allowing for the synthesis of full-length viral RNA and different mRNA products [13].
Multiple studies have focused on the proteomic and transcriptomic changes of cells from latent HIV infection or cells from non-progressing patients.It was previously shown that HIV can be controlled by the infected cells through transcription and cell cycle regulators, such as TGF-β signaling [14,15].Also, in latently infected CD4+ T-cells, genes related to apoptosis, cell cycle and human leukocyte antigen class II (HLA-II) were upregulated [16].Moreover, the upregulation of an inflammatory state is also observed in latently infected immune cells [17].
We have previously carried out a comparative analysis between HIV-1 and HIV-2 in the immediate early phase of infection; however, to the best of our knowledge, we are not aware of similar studies on the entire early phase.Previous works focusing on HIV-1 viral replication revealed that proteins related to translation, transcription and DNA condensation were differentially expressed in the early phase [18].Moreover, the most abundant proteomic changes were detected at 24 h post-infection [19].Also, differential regulation of metabolic, apoptotic and mitochondrial proteins was observed in HIV-1infected T cells [20].
Many studies have characterized the impact of HIV-1 infection on target cells; in contrast, data covering virus-host interactions in the case of HIV-2 are lacking.We previously showed significant differences in the virus-host interaction between the two viruses as early as 2 h after transduction in HEK-293T cells.
In this study, we focused our efforts on the analysis of later time-points at 8, 12 and 26 h post-transduction in the same cell line.

Transcriptomics 2.1.1. Analysis of Transcriptomic Changes at Different Time-Points Following HIV-1 and -2 Transduction
Compared to the mock transduced cells, HIV-1 managed to significantly alter the expression of 158 genes eight hours post-transduction.Of the significantly altered tran-2.1.1.Analysis of Transcriptomic Changes at Different Time-Points Following HIV-1 and -2 Transduction Compared to the mock transduced cells, HIV-1 managed to significantly alter the expression of 158 genes eight hours post-transduction.Of the significantly altered transcripts, 103 coded for proteins, 30 were non-coding RNA and 5 coded for mitochondrial RNA products.Furthermore, 16 pseudogenes and 4 uncategorized gene products were also detected.At 12 h post-transduction, we detected 114 genes, which were regulated differentially by HIV-1.Out of these 114 genes, 70 were protein coding, 5 were products of mitochondrial RNA, 33 coded for non-coding RNAs, 2 for pseudogenes and 4 were the products of unknown genes.The most prominent alteration in the transcriptome of HIV-1 infected cells was detected at the 26 h time-point, wherein a total of 380 gene products were altered, compared to the mock control.Out of the 380 genes, 290 coded for proteins, 70 for non-coding RNA, 15 for pseudogenes and 5 were unknown gene products.
On the other hand, at the 8 h time-point, HIV-2 managed to alter the expression of 283 genes compared with the mock-transduced cells, of which 142 coded for proteins, 58 for non-coding RNA, 3 for mitochondrial RNA and 77 for pseudogenes, and 3 were the products of unknown genes.At the 12 h time-point, the expression of 299 genes were significantly altered by HIV-2, out of which 153 were protein coding, 50 were non-coding RNA, 1 coding for mitochondrial RNA and 93 were products of pseudogenes, while 2 were coding for unknown genes.26 h post-transduction, 182 genes were differentially expressed, of which 70 were protein coding, 29 were non-coding RNA transcripts, 4 coding for mitochondrial RNA, 78 were products of pseudogenes, and 1 was an unknown gene product.The altered differential gene expression pattern of the transduced cells at the different time-points is presented in Figures 1 and 2, and the full list of the detected significant transcripts can be found in Tables S1 and S2.

Differentially Induced Genes by HIV-1
The top 10 DEGs that were protein-coding from HIV-1-transduced HEK cells at different time-points were selected and presented in Figure 3.

Differentially Induced Genes by HIV-1
The top 10 DEGs that were protein-coding from HIV-1-transduced HEK cells at different time-points were selected and presented in Figure 3.

Differentially Induced Genes by HIV-2
We selected and presented the top 10 up-and downregulated protein-coding genes at 8, 12 and 26 h following transduction with HIV-2 in Figure 4.

Gene Ontology Analysis of the Significantly Altered Transcripts across the Different Time-Points
To further enrich the data of the detected transcripts, gene ontology (GO) analysis was performed with the most significant, differentially expressed (p < 0.05, log2FC > 1) protein-coding genes at the 8, 12 and 26 h time-points from both HIV-1 and HIV-2 transduced cells, respectively, compared to mock-transduced cells.Analysis revealed further differences between HIV-1 and HIV-2 transduced cells compared to the mock control.
At 8 h, HIV-1 managed to regulate the expression of genes involved in the cell cycle, positive regulation of metabolic processes and positive regulation of the RNA metabolic process.At the same time-point, HIV-2 regulated the cellular level of genes, which are part of cell adhesion, extracellular matrix organization and tissue development (Figure 5).At the 12 h time-point, there were no significantly affected GO terms in the case of HIV-1 transduced cells.However, HIV-2 managed to alter the expression of genes related to cell differentiation, cell adhesion and cell migration (Figure 6).

Gene Ontology Analysis of the Significantly Altered Transcripts across the Different Time-Points
To further enrich the data of the detected transcripts, gene ontology (GO) analysis was performed with the most significant, differentially expressed (p < 0.05, log 2 FC > 1) proteincoding genes at the 8, 12 and 26 h time-points from both HIV-1 and HIV-2 transduced cells, respectively, compared to mock-transduced cells.Analysis revealed further differences between HIV-1 and HIV-2 transduced cells compared to the mock control.
At 8 h, HIV-1 managed to regulate the expression of genes involved in the cell cycle, positive regulation of metabolic processes and positive regulation of the RNA metabolic process.At the same time-point, HIV-2 regulated the cellular level of genes, which are part of cell adhesion, extracellular matrix organization and tissue development (Figure 5).At the 12 h time-point, there were no significantly affected GO terms in the case of HIV-1 transduced cells.However, HIV-2 managed to alter the expression of genes related to cell differentiation, cell adhesion and cell migration (Figure 6).Twenty-six hours post-transduction, HIV-1 altered the regulation of genes involved in response to oxidative stress, response to toxic substances and homeostatic processes.Meanwhile, HIV-2 affected genes that are part of the negative regulation of cell motility, negative regulation of cell migration and peptide cross-linking (Figure 7).A full list of the detected GO terms can be found in Tables S5 and S6.

Proteomics Analysis
The altered differential protein expression pattern of the transduced cells at the 12 and 26 h time-points is presented in Figures 8 and 9. Proteins that were differentially regulated in all of the three observed time-points were selected and presented in Figure 10, and the full list of the detected proteins can be found in Table S7.
The altered differential protein expression pattern of the transduced cells at the 12 and 26 h time-points is presented in Figures 8 and 9. Proteins that were differentially regulated in all of the three observed time-points were selected and presented in Figure 10, and the full list of the detected proteins can be found in Table S7.

Gene Ontology Analysis of the Significantly Altered Proteins
GO term enrichment analysis of the significantly altered proteins was carried out for HIV-1 and HIV-2 transduced samples at 12 and 26 h post-transduction (Figure 11).At 12 h, compared with the mock-transduced cells, HIV-1 altered the expression of proteins involved in mRNA splicing via spliceosome, ATP metabolic processes and ubiquitin protein ligase binding.In contrast, HIV-2 strongly affected members of the negative regulation of macromolecule biosynthetic process, regulation of cellular response to stress and regulation of localization.At 26 h, HIV-1 managed to alter the expression of proteins involved in response to virus, viral genome replication and RNA polymerase II transcription regulator complex.On the other hand, HIV-2 altered proteins are part of the viral genome replication, regulation of viral genome replication, and spliceosomal snRNP complex.The full list of the detected GO terms can be found in Table S8.
To further enrich the relations between the DEPs and the GO terms, we selected six GO terms from HIV-1 and HIV-2-transduced samples at 12 and 26 h.The relationship between the DEPs and selected GO terms, as well as the logFC of the DEPs, are shown in Figure 12.
stress and regulation of localization.At 26 h, HIV-1 managed to alter the expression of proteins involved in response to virus, viral genome replication and RNA polymerase II transcription regulator complex.On the other hand, HIV-2 altered proteins are part of the viral genome replication, regulation of viral genome replication, and spliceosomal snRNP complex.The full list of the detected GO terms can be found in Table S8.To further enrich the relations between the DEPs and the GO terms, we selected six GO terms from HIV-1 and HIV-2-transduced samples at 12 and 26 h.The relationship between the DEPs and selected GO terms, as well as the logFC of the DEPs, are shown in Figure 12.In regard to HIV-2, at 12 h post-transduction, the selected terms included regulation of cellular response to stress, negative regulation of macromolecule biosynthetic process, cytosol, anchoring junction, RNA binding and hydrolase activity.At 26 h post-transduction, the selected terms were response to endoplasmic reticulum stress, response to virus, spliceosomal complex, RNA polymerase II transcription regulator complex, ubiquitin protein ligase binding, translation regulator activity, nucleic acid binding for HIV-1.In the case of HIV-2, the terms comprised negative regulation of translation, negative regulation of DNA-templated transcription, chromatin, spliceosomal snRNP complex, translation regulator activity, nucleic acid binding and transcription corepressor activity.The color intensities of the rectangles reflect the logarithm of the fold change values, representing the extent of changes in corresponding protein levels relative to the control.Notably, at 26 h post-transduction, the majority of Figure 12.Protein association analysis.This figure illustrates the association between six selected GO terms and differentially regulated proteins (DRPs) by HIV-1 (left) and HIV-2 (right) at 12 (top) and 26 (bottom) hours post-transduction.The chosen GO terms encompassed mRNA splicing via spliceosome, cellular response to stress, extracellular exosome, focal adhesion, protein binding and molecular function inhibitor activity for HIV-1.In regard to HIV-2, at 12 h post-transduction, the selected terms included regulation of cellular response to stress, negative regulation of macromolecule biosynthetic process, cytosol, anchoring junction, RNA binding and hydrolase activity.At 26 h posttransduction, the selected terms were response to endoplasmic reticulum stress, response to virus, spliceosomal complex, RNA polymerase II transcription regulator complex, ubiquitin protein ligase binding, translation regulator activity, nucleic acid binding for HIV-1.In the case of HIV-2, the terms comprised negative regulation of translation, negative regulation of DNA-templated transcription, chromatin, spliceosomal snRNP complex, translation regulator activity, nucleic acid binding and transcription corepressor activity.The color intensities of the rectangles reflect the logarithm of the fold change values, representing the extent of changes in corresponding protein levels relative to the control.Notably, at 26 h post-transduction, the majority of DRPs are observed in the HIV-2 vs. Mock comparison.However, some DRPs were selectively identified from the HIV-2 vs. HIV-1 comparison, exemplified by instances such as HSPA1A being upregulated compared with HIV-1 (shown here) but downregulated compared with mock.

Discussion
Lentivirus-based vectors are widely utilized in research and clinical settings.For safe application, it is important to delineate the changes induced by these vectors in the host cell.HIV-1 and HIV-2 share many similarities, including routes of transmission and clinical outcomes of infection.However, there are striking differences between the two viruses in terms of replication dynamics and clinical course of infection.In comparison to HIV-1, HIV-2 is characterized by an acute "surge" in viral production right after infection; thereafter, a prolonged latency stage ensues, characterized by lower viral replication and a slower rate of disease progression [1,21,22].Factors involved in the replication dynamics and prolonged latency of HIV-2 remain mostly unknown.While there are adequate data about the proteo-transcriptomic changes induced by HIV-1, data regarding HIV-2 are lacking.
Given the wide divergence in receptor utilization between the two virions, we used VSV-G for pseudotyping in this study, which was shown to utilize the low-density lipoprotein (LDL) receptor as a target [23].This was carried out in order to bypass the receptorinduced proteo-transcriptomic changes, facilitating the transduction of a wide range of cells.HEK-293T cells, on the other hand, lack restriction factors and antiviral immune response and express the large T antigen, making them widely utilized for lentiviral production, allowing for effective transfection efficiency and, thereafter, robust vector production.
We utilized RNAseq and proteomic analysis to determine changes in the cellular transcriptome and proteome in the early-and early-late phases of the HIV life cycle, focusing in this study on 8, 12 and 26 h time-points post-transduction.HEK-293T cells transduced with HIV-1 showed a dynamic change in the number of DEGs, changing from 158 at 8 h to 114 at 12 h and, finally, to 380 at 26 h post-transduction.Compared with HIV-1, at the 8 h time-point, HIV-2 differentially regulated the expression of 283 genes, significantly higher than HIV-1 at the same time-point.At 12 h, HIV-2 managed to alter the expression of 299 genes, and the number dropped to 182 at 26 h.The higher number of genes altered by HIV-2 in the first two time-points suggests a more diverse effect on the host cell, followed by a stagnant state at 26 h, compared with HIV-1, which steadily altered the cellular transcriptome (Figure 1).At 8 h, amongst the most upregulated protein-coding genes by HIV-1 was the A-kinase anchor protein 9 (AKAP9).The AKAP family of proteins has been described as contributors to the cAMP-mediated signaling.Indeed, different health conditions, such as chronic heart failure, cancers and immune deficiencies, were associated with alterations in the expression of AKAP [24].Dysregulation of the cAMP levels has been associated with T-cell dysfunction, which is beneficial to HIV, as it aids the virus in evading immune response [25].Moreover, RelB was downregulated by HIV-1 at the 8 h time-point, and it was shown that viral components, such as HIV-1 Vpr, bovine foamy virus (BFV) transactivator (Btas) and human T cell leukemia virus (HTLV1) Tax1 can interact with RelB to facilitate virus replication [26].Additionally, silencing of RelB can induce a stop at the G1 phase in which HIV gene expression is promoted.Interestingly, the HIV-1 Tat protein can also induce G1 arrest and is capable of interacting with RelB to promote viral gene expression [26][27][28].Moreover, HIV-1 generated G1-like state in infected macrophages can promote HIV-1 replication by bypassing SAMHD1-mediated restriction [29].Taken together, it is conceivable that HIV-1 Tat protein may induce G1 arrest through the downregulation of RelB expression while using the available RelB protein to promote bypassing cellular restrictions in the early phase and promote gene expression in the late phase of infection.
Additionally, expression of genes coding for the group and coiled-coil domain containing 2 (GCC2) were upregulated 8 h after transduction with HIV-1.GCC2 was implicated in the Nef-mediated downregulation of MHCI, thereby protecting the infected cell from CD8 + T-cell-mediated killing [30].
The cyclin-dependent kinase inhibitor 1 A (CDKN1A) was downregulated after eight hours following transduction with HIV-1.It was previously shown that CDKN1A was able to inhibit the integration of HIV-1 into hematopoietic stem cells by forming complexes with the viral integrase [31].It was also revealed that HIV-1 Vpr can activate the cellular expression of CDKNA1A and initiate cell cycle arrest in macrophages and T cells, thus aiding the integration of the PIC [32,33].All in all, the regulation of CDKN1As can positively influence the life cycle of HIV, especially the reverse transcription and integration events, although this regulation might be tied to other cellular factors and could vary according to the type of the infected cell.
At 12 h post-transduction, HIV-1 enhanced the expression of COX20, a member of the Cytochrome C oxidase complex and an essential part of the mitochondrial respiratory chain complex IV [34].The p2 peptide, which is released during HIV-1 uncoating in the early phase of the viral life cycle, can activate cytochrome C oxidase and increase ATP production to enhance HIV-1 reverse transcription and nuclear import [35].Later on, during viral replication, the HIV-1 Tat protein inhibits the activity of cytochrome C oxidase, which leads to Tat-mediated apoptosis [36].
Also, calcium/calmodulin-dependent protein kinase 1D (CAMK1D) was downregulated by HIV-1.The activity of CAMK1D was found to be negatively regulated by HIV-1 infection, which was in correlation with our results, wherein we observed decreased mRNA levels [37].It was also shown that siRNA knockdown of CAMK1D can inhibit HIV-1 infection [38].
As genome integration begins roughly 15 h post-infection, upregulation of proteincoding genes involved in the maintenance of DNA structure is to be expected near that time-point [39].Indeed, our data showed that genes of proteins involved in the DNA break repair, such as BRCA2 and NBN, were augmented 12 h post-transduction [40,41].
Finally, after 26 h of transduction, several protein-coding genes involved in the oxidative stress response were upregulated by HIV-1.HIV infection generates reactive oxygen species, resulting in the deregulation of oxidative stress pathways and induction of mitochondrial dysfunction [42].Genes coding for hem-oxygenase 1 (HMOX1), oxidative stress-induced growth inhibitor 1 (OSGIN1) and NAD(P)H quinone dehydrogenase 1 (NQO1) were upregulated by HIV-1, surmounting active defense against the generated oxidative stress.
In our previous publication, we described that changes generated by transduction with HIV-2 pseudovirions were significantly different from those generated by HIV-1 [43].Indeed, this pattern was also noticed in later time-points as well.
At the eight-hour time-point, HIV-2 managed to differentially alter the inhibitor of DNA binding protein 1, HLH protein (ID1) and 3 (ID3), compared with the mock control.ID proteins are a class of helix-loop-helix transcription factors that are involved in cell proliferation, cell cycle regulation and cell migration [44].Additionally, decorin (DCN)-a protein involved in intracellular communication and cell proliferation-was continuously upregulated by HIV-2 in all of our observed time-points.Overexpression of DCN was shown to induce apoptosis through caspase-3 and cause cell cycle arrest at the G0-G1 phase by upregulating the expression of p21 [45,46].
HIV-2 also managed to upregulate the expression of S100A6, a calcium-binding protein involved in cell proliferation, cytoskeletal functions and cellular response to stress.Elevated S100A6 levels were shown to induce cell proliferation and migration in cancer cells [47,48].The differential regulation of genes involved in cell proliferation is important for the integration of viral DNA.Moreover, the control of cell proliferation is important for the expansion of the viral reservoir and is a key part of HIV's effect on the cell [49,50].
Amongst the differentially expressed genes by HIV-2 at 12 h post-transduction were genes-products of which are involved in lipid homeostasis.Patients tend to develop lipid homeostatic abnormalities called lipodystrophy due to HIV infection and subsequent therapy [51,52].mRNA of fatty acid binding protein 4 (FABP4)-which is a strong biomarker of HIV, generated metabolic syndrome and lipodystrophy-was significantly upregulated by HIV-2 but not by HIV-1 even at later time-points [53].An important finding was the downregulation of ATP binding cassette subfamily G member 1 (ABCG1), a key transporter in cholesterol efflux.It was revealed in a study that HIV-1 Nef is capable of inhibiting the efflux of cholesterol through downregulation of the ATP binding cassette subfamily a member 1 (ABCA1) transporter but has little effect on the ABCG1.Impairment in the cholesterol homeostasis of the transduced cells is an important part of HIV replication, as sufficient cholesterol is required for the production of nascent viral particles; additionally, inhibition of the cholesterol efflux promotes inflammatory macrophage differentiation by inducing TLR signalization [54,55].As HIV-2 did not alter ABCA1, even at later time-points, and only altered ABCG1 at the 12 and 26 h, it appears that HIV-2 does not affect the cellular cholesterol level.Moreover, it is possible that the induction of inflammation at an early stage of HIV infection through dysregulation of the cellular cholesterol level in the early phase of the viral life cycle can initiate an early immune response against HIV-2-infected cells.Since HIV-1 was able to alter the expression of ABCA1, ATP binding cassette subfamily a member 2 (ABCA2), and even ABCG1 at the 26 h time-point, we can speculate that HIV-1 has a more noticeable effect over the cellular cholesterol transport compared with HIV-2 (Table S1).
In all of our observed time-points, HIV-2 resulted in the upregulation of transcripts coding for proteins associated with the extracellular and intracellular matrix, such as keratins (KRT4, 5, 7) and collagens (COL1A2, COL6A3).Meanwhile, HIV-1 only managed to alter the expression of collagens (COL11A1) but not keratins.There is no available information on keratins playing any role in the life cycle of the virus; however, HIV-1 was shown to induce the remodeling of the extracellular matrix through the impairment of metalloproteinases [56].Dynamic changes in the extracellular matrix are necessary for cellular processes, such as cell proliferation, tissue repair and the procession of biomolecules, including chemokines, growth factors and cytokines.Indeed, for a successful immune response against pathogens, remodeling of the extracellular matrix is needed, and alteration in the composition of the matrix can limit the ability of the immune cells to effectively combat infections [57].Moreover, remodeling of the intracellular matrix is required for the efficient transport and assembly of viral particles [58,59].Both HIV-1 and 2 managed to alter the expression of EGR1, a transcription factor involved in Tat-dependent HIV gene expression.EGR1 is important in the therapeutic strategy "kick and kill", wherein latently infected cells are treated with latency reversal agents in order to reactivate latent HIV reservoirs.EGR1 was upregulated following treatment with various agents, and it was shown to directly interact with the HIV-1 promoter to induce viral transcription [60,61].
An interesting finding was the regulation of different pseudogenes by both HIV-1 and HIV-2 in all of the observed time-points.Previously, pseudogenes were described as functionless DNA created by mutations, frame-shifts and gene duplication events.However, in the past few years, their role in cellular homeostasis has been reimagined as it was discovered that pseudogenes can regulate the expression of their parent genes [62].Moreover, evidence suggests that self-derived mRNAs play an important role in the regulation of immune response against viruses and tumors.For example, 15S and 5S ribosomal RNAderived pseudogenes can increase the expression of proinflammatory cytokines [63].Data derived from HIV-1 transduced cells shows little alteration in the number of pseudogenes compared with mock transduced control; however, the number of differentially regulated pseudogenes was significantly higher in HIV-2-transduced cells compared with HIV-1.A total of 77 pseudogenes were differently regulated at 8 h and 93 at 12 h post-transduction; meanwhile, 78 pseuodogenic mRNA products were detected at 26 h.Amongst the HIV-2 altered pseudogenes, eukaryotic translation elongation factor alpha 1 (eEF1a1), protein disulfide isomerase family A member 3 (PDIA3) and nucleophosmin 1 (NPM1)-derived pseudogenic mRNA were detected.eEF1a1 has been described as cofactors for reverse transcription required for the stability of the reverse transcriptase complex [64,65].During gp120 and CD4 interaction, disulfide isomerases initiate disulfide bond rearrangement in gp120, which aids the viral surface glycoprotein in its fusion with the cell membrane [66,67].NPM1 is known to interact with HIV-1 Tat, and infection with HIV-1 induces acetylation of NPM1.This process is critical for the nuclear localization of Tat and for Tat-mediated transcription [68].Pseudogenes were described as negative regulators of gene expres-sion; it is plausible that the observed differential regulation of the expression of multiple pseudogenes by HIV-2 might contribute to its distinct pathophysiology.
GO analysis revealed further differences between HIV-1 and HIV-2-transduced cells.At 8 h, HIV-1 had a greater effect on cellular metabolism according to the significant GO terms observed, which included the positive regulation of metabolic processes, regulation of primary metabolic processes and positive regulation of RNA metabolic processes, among others.Meanwhile, HIV-2 mostly affects genes responsible for cell adhesion, extracellular matrix organization or keratinocyte differentiation.Increased metabolic activity promotes HIV-1 viral infectivity and replication, and previous studies have shown that inhibition of glycolysis would result in the inhibition of reverse transcription [69,70].Rather surprisingly, we did not detect any significant GO term at 12 h post-infection in HIV-1-transduced cells.The number of differentially expressed genes was the lowest at 12 h compared with the 8 and 26 h time-points in HIV-1-infected HEK-293T cells.At the same time-point, HIV-2 managed to affect the genes involved in cell differentiation, animal organ development and system development.At 26 h post-transduction, HIV-1 managed to upregulate the expression of genes that are part of the response to oxidative stress, cellular response to toxic substances and detoxification.Compared with HIV-1, HIV-2 did not impact the cellular environment in an impactful way, only affecting genes that are related to the negative regulation of cell motility, negative regulation of cell migration and skin development.
Following the transcriptomic analysis, we utilized mass spectrometry to gain more information about changes in the cellular proteome generated by HIV in transduced cells.We found that the majority of the differentially regulated proteins were related to chaperon function, proteasome and mRNA processing.Our data showed that both HIV-1 and HIV-2 regulated the expression of 17 and 117 different proteins at 12 and 26 h post-transduction, respectively.
At 12 h, Zyx, PA2G4, hnRNPK and HSPA1A, among others, were significantly downregulated by both HIV-1 and HIV-2.Zyxin plays an important role in focal adhesion and actin polymerization; additionally, it was also found to be involved in many intracellular signaling pathways [71].PA2G4, on the other hand, is a DNA/RNA binding protein that is involved in diverse cellular functions, most notably cell growth, apoptosis and differentiation.Previous studies have shown that this protein is downregulated by HIV-1 Vpr, potentiating G2 arrest and apoptosis in U87MG cells [72].
The hnRNP family of proteins mediates diverse functions in the cells, mostly involved in mRNA transcription, splicing, export, stability and translation.hnRNPQ, which was downregulated only by HIV-1, was shown to be involved in the proviral transcription of HIV-1 through interactions with viral protein Rev. It was also found to play an important role in RNA replication, splicing and mRNA stability of Hepatitis C virus [73].
Splicing factor SRSF2 and TRIM28 were also differently regulated by both pseudovirions at 26 h.SRSF2 was implicated in the downregulation of late steps of HIV-1 replication by mediating the splicing of tat and rev genes [74].TRIM28, on the other hand, was shown to promote the latency of HIV-1 through the inhibition of positive transcription elongation factor (P-TEFb) and SUMOylating CDK9 along with SUMO4 [75].hnRNPU, hnRNPK, hnRNPA2/B1, hnRNPQ, together with HSPA1A, DDX3X, SRSF1, NCL and TRIM28, were observed in complex with HIV mRNA, and with viral proteins in staufen1 ribonuclear complexes [76].
In contrast to HIV-1, HIV-2 downregulated NCAPH and PRRC2A, two proteins that are known to play an important role in cell proliferation and migration [77,78].Retroviruses and, indeed, DNA and RNA viruses, in general, are known to modulate the cell cycle and cellular proliferation in order to facilitate viral replication and survival [79].However, why only HIV-2 influenced these two key players at the 12 h time-point deserves further exploration.At the 26 h time-point, HIV-2 differentially regulated a slightly higher number of proteins compared with HIV-1.In comparison to HIV-1, HIV-2 upregulated members of the HSP70 (1A, 5 and 9), CCT3 and TFRC, to name a few.As a chaperone protein, HSP70 functions include mediating folding, translocation and degradation of target protein.In the context of HIV infection, it was shown that HSP70 exerts a dose-dependent inhibition on HIV-1 in CD4+ T cells [80].Sugiyama et al. also reported that HSP70 inhibits the ubiquitination and degradation of APOBEC3G by Vif [81].Moreover, by stimulating the binding of the HIV-1 matrix to karyopherin alpha, HSP70 is implicated in the nuclear import of PIC of HIV-1 [82].CCT3, on the other hand, was shown to assist Gag polyprotein, enhancing the infectivity of Mason-Pfizer monkey virus (M-PMV) [83,84].
TFRC is a membrane glycoprotein that mediates the uptake of the transferrin-iron complex through receptor-mediated endocytosis [85].It was shown that HIV infection leads to increased cellular iron levels, which enhances HIV replication [86].HIV-1 Nef was shown to mediate the reduction of TFRC's recycling rate, leading to its accumulation in early endosomes and, therefore, reduced expression at the cell surface in a T cell line [87].However, this finding was not corroborated in another study, wherein Nef proteins of HIV-1 and its simian precursors did not affect the level of surface TFRC.However, Nef proteins of other lentiviruses reduced the internalization of the receptor in the myeloid and T cell line [88].The differential regulation of TFRC might be dependent on the phase of the viral life-cycle and the type of the infected cell.The upregulation of TFRC by HIV-2 is indeed an interesting finding.Recently, TFRC was identified as a candidate entry protein for the Influenza A virus [89], and given the wide spectrum of receptor utilization by HIV-2, it may as well be utilized by the virus, although further studies are needed to confirm this hypothesis.
Compared to HIV-1, HIV-2 managed to downregulate the expression of RANBP1, which deserves significant attention.This protein was found to contain a nuclear export signal (NES) domain that is similar in structure and function to that found in HIV Rev; therefore, it is hypothesized that it shares a step in the post-transcriptional pathway with Rev [90].Its downregulation by HIV-2 raises questions about whether it could be implicated in the suppression of the transport of proviral transcripts.
To further outline the differences between the two pseudovirions, the GO term analysis was carried out on significantly altered proteins in transduced cells.Very few GO terms were apparent at 12 h in the case of both viruses.At 26 h, however, there were several GO terms shared between HIV-1 and HIV-2, such as regulation of alternative mRNA splicing via the spliceosome, regulation of DNA-templated transcription elongation and post-transcriptional regulation of gene expression (Table S8).While there were many shared terms between the two, many differences were also observed at that time-point.GO terms, such as cellular response to unfolded protein, regulation of gene expression and positive regulation of apoptotic processes, were detected in the case of HIV-1, while negative regulation of DNA metabolic processes, negative regulation of protein ubiquitination and positive regulation of translation were characteristic of HIV-2.
It is important to note that some proteins identified in the proteomic analysis did not correlate with the findings from the transcriptomic data.Transcriptomic changes are not always reflected at the level of proteins, given the substantial role of post-transcriptional and post-translational regulation governing protein production; also, given the methodology applied here and the relatively long intervals between the time-points [91], a correlation between transcriptome and proteome at the same time-point was unlikely.However, we could follow the regulation trends of many proteins across the time-points in the case of both pseudovirions, and many significant differences were observed (Table S2).
In conclusion, the analysis of the proteomic and transcriptomic data from the early phase of HIV-1 and HIV-2-based pseudovirion-transduced HEK-293T cells indicates that the cellular response varies significantly between the two.Our analysis was focused on changes in HEK-293T cells, not primary immune cells, but we hope that the data gathered can be of aid in understanding the pathomechanistic aspect of transduction with lentivectors.Moreover, to our knowledge, studies of the effects of HIV transduction in the early phase of infection are indeed lacking, especially in the case of HIV-2.We hope that our study may provide insight into the understudied replication cycle of HIV-2 and enrich our knowledge about the use of HIV-based lentiviral vectors as a whole.

Transduction of HEK-293T Cells for Proteomic Analysis
A day before transduction, HEK-293T cells were seeded in T-25 flask (0.7-1 × 10 6 cells/flask) in 5 mL of DMEM supplemented with 10% FBS, 1% L-glutamine and 1% penicillinstreptomycin.The next day, the medium was discarded, and the cells were transduced with 15 ng RT-equivalent of HIV-1/HIV-2 or "mock" pseudovirions in serum and antibiotic-free media complemented with 8 µg/mL polybrene.Cells were thereafter incubated at 37 • C, 5 % CO 2 for eight, 12 and 26 h.Next, the media was discarded, and cells were mechanically detached and suspended in PBS.After brief centrifugation, the pellet was stored at −20 • C until further analysis.

GeLC-MS/MS Analyis
Lysis of the HIV-1, HIV-2 or mock transduced cells was performed in 100 µL lysis buffer (50 mM Tris pH 8.3, 1 mM EDTA, 17 mM β-mercaptoethanol, 0.5% (v/v) Triton-X100) using three freeze-thaw cycles.Bradford method was used to determine the protein concentration, and 100 µg of protein in each case was subjected to in-gel digestion followed by liquid chromatography-tandem mass spectrometry (GeLC-MS/MS) analysis [100].Briefly, samples were run into a 5% SDS-polyacrylamide gel using a 100 V current for 20 min.The proteins were stained with PageBlue Protein Staining solution (Thermo Scientific, Waltham, MA, USA), and the stained gel slice was excised, separated into three equal portions and subjected to in-gel trypsin digestion.Reduction was performed with 20 mM dithiothreitol (Bio-Rad, Hercules, CA, USA) for 1 h at 56 • C, followed by alkylation with 55 mM iodoacetamide (Bio-Rad, Hercules, CA, USA) for 45 min at room temperature in the dark.Overnight trypsin digestion was performed at 37 • C using stabilized MS-grade TPCK-treated bovine trypsin (ABSciex, Framingham, MA, USA).The digested peptides were extracted and dried in a speed-vac (Thermo Scientific, Waltham, MA, USA).The peptides were re-dissolved in 33 µL 1% formic acid (VWR Ltd., Radnor, PA, USA) before LC-MS/MS analysis.The peptide concentration of the samples was determined using the BCA method.Prior to mass spectrometry analyses, the samples were spiked with equal amounts of indexed retention time (iRT) peptide mixtures (Biognosys, Schlieren, Switzerland), and the samples were analyzed in duplicate.
Prior to the mass spectrometric analysis, peptides were separated in a 180 min water/acetonitrile gradient using an Easy nLC 1200 nano UPLC (Thermo Scientific, Waltham, MA, USA).The peptide mixtures were desalted in an ACQUITY UPLC Symmetry C18 trap column (20 mm × 180 µm, 5 µm particle size, 100 Å pore size, Waters, Milford, MA, USA), followed by separation in Acclaim PepMap RSLC C18 analytical columns (150 mm × 50 µm × 2 µm particle size, 100 Å pore size, Thermo Scientific, Waltham, MA, USA).Chromatographic separation was performed using a gradient of 5-7% solvent B over 5 min, followed by a rise to 15% of solvent B over 50 min and then to 35% solvent B over 60 min.Thereafter, solvent B was increased to 40% over 28 min and then to 85% over 5 min, followed by a 10 min rise to 85% of solvent B, after which the system returned to 5% solvent B in 1 min for a 16 min hold-on.Solvent A was 0.1% formic acid in LC water (Sigma, St. Louis, MO, USA); solvent B was 95% acetonitrile (Sigma, St. Louis, MO, USA) containing 0.1% formic acid.The flow rate was set to 300 nL/min.Data-dependent analyses were carried out on an Orbitrap Fusion mass spectrometer (Thermo Scientific, Waltham, MA, USA).The 14 most abundant multiply charged positive ions were selected from each survey MS scan using a scan range of 350-1600 m/z for MS/MS analyses (Orbitrap analyzer resolution: 60,000, AGC target: 4.0 × 10 5 , acquired in profile mode).Collision-induced dissociation (CID) fragmentation was performed in the linear ion trap with 35% normalized collision energy (AGC target: 2.0 × 10 3 , acquired in centroid mode).Dynamic exclusion was enabled during the cycles (exclusion time: 45 s).

Data analysis of Mass Spectrometry
The acquired LC-MS/MS data were used for protein identification the help of MaxQuant 1.6.2.10 software [101] searching against the Human SwissProt database (release: 2020.02,20,394 sequence entries), the HIV-1 and HIV-2 SwissProt databases (release: 2020.02,381 sequence entries for HIV-1 and 109 sequence entries for HIV-2), and against the contaminants database provided by the MaxQuant software ver.1.6.2.10.Cys carbamidomethylation, Met oxidation and N-terminal acetylation were set as variable modifications.A maximum of 2 missed cleavage sites were allowed.Results were imported into Scaffold 4.8.9 software (ProteomeSoftware Inc., Portland, OR, USA).Proteins were accepted with at least 3 identified peptides using 1% protein false discovery rate (FDR) and 0.1% peptide FDR.For label-free quantification, the normalized total precursor intensities were used, and quantitative values of the identified proteins were normalized to the quantitative values of the iRT mixture.

Proteomic Data Analysis
The list of identified proteins with intensity values was exported from Scaffold and further processed in the R environment (v4.3.1)[96].Normalized values were calculated accounting for variations in overall sample protein concentrations (determined post-digestion by the BCA method), injection volumes and the sum of detected protein intensities per sample.We employed a series of mixed-effects ANOVA models, one for each protein, to identify the statistically significant differences in protein abundances between the investigated groups.Sample and measurement replicates were treated as random effects, while the different transduced cell groups were modeled as fixed effects [102].We ran the computations utilizing the emmeans (v1.8.8) and lme4 (1.1.34)packages [103].After the linear model fitting, post-hoc tests were applied to determine the p-values of group differences and significant results with an FDR < 0.05 criteria were retained.The protein coding gene annotation was retrieved from the daily updated 'gene_info', 'gene2go' files available on the NCBI FTP site (https://ftp.ncbi.nlm.nih.gov/gene/DATA/,accessed on 15 November 2023) and reorganized by in-house shell scripts.We carried out enrichment analysis for gene ontology (GO) with the topGO R package (v2.52.0) using a custom-generated gene-to-GOs mapping file [104].The protein universe consisted of the entire set of proteins (807) detected in all 26 h post-transduction samples; significant proteins were filtered according to the FDR < 0.05 threshold.In the topGO algorithm, we chose the default 'weight01' method with the Kolmogorov-Smirnov statistical test and selected the enriched GO terms having FDR-adjusted p-values < 0.05.Data visualization was performed with ggplot2 (v3.4.3), ggrepel (v0.9.3), ggpubr (v0.6.0),GOplot (v1.0.2) [105], ggnewscale (v0.4.9),VennDiagram (v1.7.3)R packages.
Funding: This work was supported by the Hungarian Scientific Research Fund (NKFI 125238) to J.T and by the Thematic Excellence Programme (TKP2020-IKA-04) of the Ministry for Innovation and Technology in Hungary and GINOP-2.3.3-15-2016-00020.Project no.TKP2021-EGA-20 (Biotechnology) has been implemented with the support provided from the National Research, Development and Innovation Fund of Hungary, financed under the TKP2021-EGA funding scheme.The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Figure 1 .
Figure 1.Venn diagram showing the differential gene expression of protein-coding genes between HIV-1 and mock or HIV-2 and mock-transduced samples in all of the observed time points.log2 fold change (LFC) > 1 adj.p-value < 0.05.

Figure 1 .
Figure 1.Venn diagram showing the differential gene expression of protein-coding genes between HIV-1 and mock or HIV-2 and mock-transduced samples in all of the observed time points.log2 fold change (LFC) > 1 adj.p-value < 0.05.

27 Figure 2 .
Figure 2. Volcano plot showing the altered genes from HIV-1 (top) and HIV-2 (bottom) transduced HEK293T cells compared with the mock control throughout the different time-points.The log2 fold change (log2FC) of the detected genes is shown on the x-axis, while the log10 adjusted p-value of each gene is present on the y-axis.Number of dots represent the detected genes from each sample, while the color corresponds to the significantly up (red) or downregulated (blue) genes.Grey dots represent the non-significant transcripts.

Figure 2 .
Figure 2. Volcano plot showing the altered genes from HIV-1 (top) and HIV-2 (bottom) transduced HEK293T cells compared with the mock control throughout the different time-points.The log2 fold change (log2FC) of the detected genes is shown on the x-axis, while the log10 adjusted p-value of each gene is present on the y-axis.Number of dots represent the detected genes from each sample, while the color corresponds to the significantly up (red) or downregulated (blue) genes.Grey dots represent the non-significant transcripts.

Figure 3 .
Figure 3. Top 10 up-and downregulated DEGs at the 8 (A), 12 (B) and 26 (C) hour time-points following transduction with HIV-1.log2FC of the up-and downregulated transcripts coding for proteins in HIV-1 transduced HEK-293T cells compared to mock transduction.Red bars show genes that are regulated similarly in all observed time-points compared with mock-transduced samples, while grey bars correspond to the significantly altered transcripts that were regulated only in the given time-point.

Figure 3 .
Figure 3. Top 10 up-and downregulated DEGs at the 8 (A), 12 (B) and 26 (C) hour time-points following transduction with HIV-1.log2FC of the up-and downregulated transcripts coding for proteins in HIV-1 transduced HEK-293T cells compared to mock transduction.Red bars show genes that are regulated similarly in all observed time-points compared with mock-transduced samples, while grey bars correspond to the significantly altered transcripts that were regulated only in the given time-point.

Figure 4 .
Figure 4. Top 10 up-and downregulated DEGs at the 8 (A), 12 (B) and 26 (C) hours time-points following transduction with HIV-2.log2FC of the up-and downregulated transcripts coding for proteins in HIV-2 transduced HEK-293T cells, compared to mock transduction.Red bars show genes that are regulated similarly in all observed time points, while grey bars correspond to the significantly altered transcripts that were regulated only in the given time-point.

Figure 4 .
Figure 4. Top 10 up-and downregulated DEGs at the 8 (A), 12 (B) and 26 (C) hours time-points following transduction with HIV-2.log2FC of the up-and downregulated transcripts coding for proteins in HIV-2 transduced HEK-293T cells, compared to mock transduction.Red bars show genes that are regulated similarly in all observed time points, while grey bars correspond to the significantly altered transcripts that were regulated only in the given time-point.

Figure 5 .
Figure 5. Gene ontology (GO) analysis of DEGs in HIV-1 (left) and HIV-2 (right) transduced cells at 8 h time-point.Top genes classified according to significant enrichment terms.Color intensity corresponds to the significance of each term.Counts represent the number of differentially expressed genes associated with the listed gene ontology ID.

Figure 5 .
Figure 5. Gene ontology (GO) analysis of DEGs in HIV-1 (left) and HIV-2 (right) transduced cells at 8 h time-point.Top genes classified according to significant enrichment terms.Color intensity corresponds to the significance of each term.Counts represent the number of differentially expressed genes associated with the listed gene ontology ID.

27 Figure 6 .
Figure 6.Gene ontology (GO) analysis of DEGs in HIV-2-transduced cells at 12 h time-point.Top genes classified according to significant enrichment terms.Color intensity corresponds to the significance of each term.Counts represent the number of differentially expressed genes associated with the listed gene ontology ID.

Figure 6 .
Figure 6.Gene ontology (GO) analysis of DEGs in HIV-2-transduced cells at 12 h time-point.Top genes classified according to significant enrichment terms.Color intensity corresponds to the significance of each term.Counts represent the number of differentially expressed genes associated with the listed gene ontology ID.

27 Figure 7 .
Figure 7. Gene ontology (GO) analysis of DEGs in HIV-1 (left) and HIV-2 (right) transduced cells at the 26 h time-point.Top genes classified according to significant enrichment terms.Color intensity corresponds to the significance of each term.Counts represent the number of differentially expressed genes associated with the listed gene ontology ID.

Figure 7 .
Figure 7. Gene ontology (GO) analysis of DEGs in HIV-1 (left) and HIV-2 (right) transduced cells at the 26 h time-point.Top genes classified according to significant enrichment terms.Color intensity corresponds to the significance of each term.Counts represent the number of differentially expressed genes associated with the listed gene ontology ID.

Figure 9 .
Figure 9. Volcano plot illustrating the changes in protein abundance in HIV-1 and HIV-2 transduced cells at 12 and 26 h post-transduction.The x-axis displays the log2 fold change (log2FC) of detected proteins, while the y-axis shows the log10 adjusted p-value for each protein.Each dot represents a detected protein, with color indicating significant upregulation (light orange) or downregulation (green, blue), and grey dots representing non-significantly regulated proteins.Note that only the top ten significantly regulated proteins, either up or downregulated, are labeled in the plot for each category.

Figure 9 .
Figure 9. Volcano plot illustrating the changes in protein abundance in HIV-1 and HIV-2 transduced cells at 12 and 26 h post-transduction.The x-axis displays the log2 fold change (log2FC) of detected proteins, while the y-axis shows the log10 adjusted p-value for each protein.Each dot represents a detected protein, with color indicating significant upregulation (light orange) or downregulation (green, blue), and grey dots representing non-significantly regulated proteins.Note that only the top ten significantly regulated proteins, either up or downregulated, are labeled in the plot for each category.

Figure 9 .
Figure 9. Volcano plot illustrating the changes in protein abundance in HIV-1 and HIV-2 transduced cells at 12 and 26 h post-transduction.The x-axis displays the log2 fold change (log2FC) of detected proteins, while the y-axis shows the log10 adjusted p-value for each protein.Each dot represents a detected protein, with color indicating significant upregulation (light orange) or downregulation (green, blue), and grey dots representing non-significantly regulated proteins.Note that only the top ten significantly regulated proteins, either up or downregulated, are labeled in the plot for each category.

Figure 10 .
Figure 10.Violin plots depicting the intensity distribution of differentially regulated proteins across all observed time-points for HIV-1 (blue) and HIV-2 (light orange) transduced cells.The x-axis represents the distinct time-points, while the y-axis corresponds to the protein's intensity.

Figure 10 .
Figure 10.Violin plots depicting the intensity distribution of differentially regulated proteins across all observed time-points for HIV-1 (blue) and HIV-2 (light orange) transduced cells.The x-axis represents the distinct time-points, while the y-axis corresponds to the protein's intensity.

Figure 11 .
Figure 11.Classification of gene ontology (GO) terms of the enriched significant proteins from HIV-1 (left) and 2 (right) infected cells 12 (top) and 26 (bottom) hours post-transduction compared with the mock.Color intensity refers to the adjusted p-value of each term, while the x-axis is the percentages of significant proteins in GO term per all differentially expressed proteins (DEPs).The names of the enriched GO terms are on the y-axis.

Figure 11 .
Figure 11.Classification of gene ontology (GO) terms of the enriched significant proteins from HIV-1 (left) and 2 (right) infected cells 12 (top) and 26 (bottom) hours post-transduction compared with the mock.Color intensity refers to the adjusted p-value of each term, while the x-axis is the percentages of significant proteins in GO term per all differentially expressed proteins (DEPs).The names of the enriched GO terms are on the y-axis.

Figure 12 .
Figure 12.Protein association analysis.This figure illustrates the association between six selected GO terms and differentially regulated proteins (DRPs) by HIV-1 (left) and HIV-2 (right) at 12 (top) and 26 (bottom) hours post-transduction.The chosen GO terms encompassed mRNA splicing via spliceosome, cellular response to stress, extracellular exosome, focal adhesion, protein binding and molecular function inhibitor activity for HIV-1.In regard to HIV-2, at 12 h post-transduction, the selected terms included regulation of cellular response to stress, negative regulation of macromolecule biosynthetic process, cytosol, anchoring junction, RNA binding and hydrolase activity.At 26 h post-transduction, the selected terms were response to endoplasmic reticulum stress, response to virus, spliceosomal complex, RNA polymerase II transcription regulator complex, ubiquitin protein ligase binding, translation regulator activity, nucleic acid binding for HIV-1.In the case of HIV-2, the terms comprised negative regulation of translation, negative regulation of DNA-templated transcription, chromatin, spliceosomal snRNP complex, translation regulator activity, nucleic acid binding and transcription corepressor activity.The color intensities of the rectangles reflect the logarithm of the fold change values, representing the extent of changes in corresponding protein levels relative to the control.Notably, at 26 h post-transduction, the majority of