The HIV 5' Gag Region Displays a Specific Nucleotide Bias Regulating Viral Splicing and Infectivity.

Alternative splicing and the expression of intron-containing mRNAs is one hallmark of HIV gene expression. To facilitate the otherwise hampered nuclear export of non-fully processed mRNAs, HIV encodes the Rev protein, which recognizes its intronic response element and fuels the HIV RNAs into the CRM-1-dependent nuclear protein export pathway. Both alternative splicing and Rev-dependency are regulated by the primary HIV RNA sequence. Here, we show that these processes are extremely sensitive to sequence alterations in the 5'coding region of the HIV genomic RNA. Increasing the GC content by insertion of either GFP or silent mutations activates a cryptic splice donor site in gag, entirely deregulates the viral splicing pattern, and lowers infectivity. Interestingly, an adaptation of the inserted GFP sequence toward an HIV-like nucleotide bias reversed these phenotypes completely. Of note, the adaptation yielded completely different primary sequences although encoding the same amino acids. Thus, the phenotypes solely depend on the nucleotide composition of the two GFP versions. This is a strong indication of an HIV-specific mRNP code in the 5' gag region wherein the primary RNA sequence bias creates motifs for RNA-binding proteins and controls the fate of the HIV-RNA in terms of viral gene expression and infectivity.

CRNKL1 Is a Highly Selective Regulator of Intron-Retaining HIV-1 and Cellular mRNAs.

The HIV-1 Rev protein is a nuclear export factor for unspliced and incompletely spliced HIV-1 RNAs. Without Rev, these intron-retaining RNAs are trapped in the nucleus. A genome-wide screen identified nine proteins of the spliceosome, which all enhanced expression from the HIV-1 unspliced RNA after CRISPR/Cas knockdown. Depletion of DHX38, WDR70, and four proteins of the Prp19-associated complex (ISY1, BUD31, XAB2, and CRNKL1) resulted in a more than 20-fold enhancement of unspliced HIV-1 RNA levels in the cytoplasm. Targeting of CRNKL1, DHX38, and BUD31 affected nuclear export efficiencies of the HIV-1 unspliced RNA to a much larger extent than splicing. Transcriptomic analyses further revealed that CRNKL1 also suppresses cytoplasmic levels of a subset of cellular mRNAs, including some with selectively retained introns. Thus, CRNKL1-dependent nuclear retention is a novel cellular mechanism for the regulation of cytoplasmic levels of intron-retaining HIV-1 mRNAs, which HIV-1 may have harnessed to direct its complex splicing pattern.IMPORTANCE To regulate its complex splicing pattern, HIV-1 uses the adaptor protein Rev to shuttle unspliced or partially spliced mRNA from the nucleus to the cytoplasm. In the absence of Rev, these RNAs are retained in the nucleus, but it is unclear why. Here we identify cellular proteins whose depletion enhances cytoplasmic levels of the HIV-1 unspliced RNA. Depletion of one of them, CRNKL1, also increases cytoplasmic levels of a subset of intron-retaining cellular mRNA, suggesting that CRNKL1-dependent nuclear retention may be a basic cellular mechanism exploited by HIV-1.

Generation of T follicular helper cells in vitro: requirement for B-cell receptor cross-linking and cognate B- and T-cell interaction.

The minimum requirements for in vitro modelling of natural CD4+ T-cell differentiation into T follicular helper (Tfh) cells are still under investigation. We co-cultured wild-type and T-cell receptor (TCR) transgenic CD4+ T cells from naive mice with dendritic cells and B-cell receptor (BCR) transgenic B cells in the presence of HIV-derived virus-like particles containing matched B-cell and T-cell epitopes. This co-culturing induced co-expression of Tfh-master regulator transcription factor BCL-6 and CXCR5 in up to 10% of the wild-type and up to 40% of the TCR-transgenic CD4+ T cells. Phenotypic markers, production of interleukin-21 and isotype switching of the B cells to IgG1 further indicated a helper function of the induced Tfh cells in vitro. Dendritic cells supported the generation of functional Tfh cells, but were unable to induce them without cognate B cells. Hence, our study presents a robust experimental system for efficient generation of functionally active Tfh cells in vitro and confirms the importance of cognate B- and T-cell cross-talk for the Tfh differentiation process.

Teriflunomide and monomethylfumarate target HIV-induced neuroinflammation and neurotoxicity.

HIV-associated neurocognitive disorders (HAND) affect about 50% of infected patients despite combined antiretroviral therapy (cART). Ongoing compartmentalized inflammation mediated by microglia which are activated by HIV-infected monocytes has been postulated to contribute to neurotoxicity independent from viral replication. Here, we investigated effects of teriflunomide and monomethylfumarate on monocyte/microglial activation and neurotoxicity. Human monocytoid cells (U937) transduced with a minimal HIV-Vector were co-cultured with human microglial cells (HMC3). Secretion of pro-inflammatory/neurotoxic cytokines (CXCL10, CCL5, and CCL2: p < 0.001; IL-6: p < 0.01) by co-cultures was strongly increased compared to microglia in contact with HIV-particles alone. Upon treatment with teriflunomide, cytokine secretion was decreased (CXCL10, 3-fold; CCL2, 2.5-fold; IL-6, 2.2-fold; p < 0.001) and monomethylfumarate treatment led to 2.9-fold lower CXCL10 secretion (p < 0.001). Reduced toxicity of co-culture conditioned media on human fetal neurons by teriflunomide (29%, p < 0.01) and monomethylfumarate (27%, p < 0.05) indicated functional relevance. Modulation of innate immune functions by teriflunomide and monomethylfumarate may target neurotoxic inflammation in the context of HAND.

Cytoplasmic HIV-RNA in monocytes determines microglial activation and neuronal cell death in HIV-associated neurodegeneration.

Despite highly active antiretroviral therapy, HIV-associated neurocognitive disorders (HAND) are still highly prevalent. Direct neurotoxicity of microglia activated by HIV-infected monocytes independent from viral replication may account for this observation. To investigate underlying molecular and viral determinants, human monocytoid cells (U937) transduced with HIV-particles were co-cultured with primary human microglia or astrocytes. Using genetically-engineered HIV-particles key steps of infection were examined. Levels of pro-inflammatory/neurotoxic cytokines were investigated in co-culture supernatants by flow cytometry. Neurotoxicity mediated by the supernatants was analysed using primary cortical rat neurons. To corroborate our findings, cytokine profiles in cerebrospinal fluid (CSF) of neuropsychologically asymptomatic HIV positive (HIV(+)) patients (n=45) were correlated with neurofilament H (NfH) as surrogate of neuronal/axonal degeneration. In contrast to direct exposure of HIV to microglia, only the presence of HIV-transduced monocytoid cells strongly activated human microglia as evidenced by enhanced secretion of CXCL10, CCL5, CCL2, and IL-6 (1.3-7.1-fold; p<0.01) leading to two-fold increased neurotoxicity (p<0.001). In direct comparison, astrocyte activation by HIV-transduced monocytoid cells was limited. Using different mutant HIV-particles we show that the presence of cytoplasmic HIV-RNA in monocytoid cells is the viral determinant for this unique microglial activation pattern and subsequent neuronal cell death; reverse transcription and expression of viral genes were not essential. In CSF of presymptomatic HIV(+) patients, CXCL10, CCL5 and IL-6 were correlated with NfH as surrogate marker of neurodegeneration as well as CSF-pleocytosis. In conclusion, cytosolic viral RNA in monocytes is mandatory for subsequent microglial activation and neurotoxicity; activated astrocytes may augment neuroinflammation. In addition, neuroinflammation and neurodegeneration occur even in preclinical HIV(+) patients and are associated with cytokines regulated in vitro. Our data may aid in the development of biomarkers and glia-directed therapeutic approaches of HAND.

HIV-derived lentiviral particles promote T-cell independent activation and differentiation of naïve cognate conventional B2-cells in vitro.

In animal models, lentiviral particles (LP) were shown to be promising HIV vaccine candidates. Since little is known about the direct impact of LP on antigen-specific B cells, we incorporated Hen Egg Lysozyme (HEL) into LP (HEL-LP) derived from HIV to study their effect on HEL-specific, B cell receptor-transgenic B-cells (HEL(+)B-cells) in vitro. We observed preferential binding of HEL-LP to HEL(+)B-cells and their efficient internalization. HEL-LP were able to effectively cross-link B-cell receptors as indicated by the loss of surface CD62L. In the absence of CD4(+) T-cells, other activation events induced by LP in cognate naïve B-cells included increased expression of activation and co-stimulatory molecules as well as an enhanced proliferative response. Additionally, the B-cell phenotype shifted toward a germinal center pattern with further differentiation into memory and IgG3- and IgA-producing cells. The observed CD4(+) T-cell independent activation and differentiation may be due to LP-induced expression of CD40L by a subset of cognate B-cells. Thus, even in the absence of CD4(+) T-cells LP provide strong direct activation signals to cognate naïve B-cells, which may contribute to the strong humoral immune responses observed after LP immunization.

The HIV-1 Rev protein enhances encapsidation of unspliced and spliced, RRE-containing lentiviral vector RNA.

BACKGROUND: During the RNA encapsidation process of human immunodeficiency virus (HIV) viral genomic, unspliced RNA (gRNA) is preferentially incorporated into assembling virions. However, a certain amount of spliced viral transcripts can also be detected in viral particles. Recently, we observed that nuclear export of HIV and lentiviral vector gRNA by Rev is required for efficient encapsidation. Since singly-spliced HIV transcripts also contain the Rev-response element (RRE), we investigated if the encapsidation efficiency of RRE-containing spliced HIV-vector transcripts is also increased by the viral Rev protein. FINDINGS: Starting with a lentiviral vector imitating the splicing pattern of HIV, we constructed vectors that express an unspliced transcript either identical in sequence to the singly-spliced or the fully-spliced RNA of the parental construct. After transfection of the different lentiviral vectors cytoplasmic and virion-associated RNA levels and vector titers were determined in the presence and absence of Rev. Rev enhanced the infectious titer of vectors containing an RRE 6 to 37-fold. Furthermore, Rev strongly increased encapsidation efficiencies of all RRE-containing transcripts up to 200-fold. However, a good correlation between encapsidation efficiency and lentiviral vector titer could only be observed for the gRNA. The infectious titer of the vector encoding the fully-spliced RNA without RRE as well as the encapsidation efficiency of all transcripts lacking the RRE was not influenced by Rev. Interestingly, the splicing process itself did not seem to interfere with packaging, since the encapsidation efficiencies of the same RNA expressed either by splicing or as an unspliced transcript did not differ significantly. CONCLUSIONS: Rev-mediated nuclear export enhances the encapsidation efficiency of RRE-containing lentiviral vector RNAs independently of whether they have been spliced or not.

Targeting antibody responses to the membrane proximal external region of the envelope glycoprotein of human immunodeficiency virus.

Although human immunodeficiency type 1 (HIV-1) infection induces strong antibody responses to the viral envelope glycoprotein (Env) only a few of these antibodies possess the capacity to neutralize a broad range of strains. The induction of such antibodies represents an important goal in the development of a preventive vaccine against the infection. Among the broadly neutralizing monoclonal antibodies discovered so far, three (2F5, Z13 and 4E10) target the short and hidden membrane proximal external region (MPER) of the gp41 transmembrane protein. Antibody responses to MPER are rarely observed in HIV-infected individuals or after immunization with Env immunogens. To initiate antibody responses to MPER in its membrane-embedded native conformation, we generated expression plasmids encoding the membrane-anchored ectodomain of gp41 with N-terminal deletions of various sizes. Following transfection of these plasmids, the MPER domains are displayed on the cell surface and incorporated into HIV virus like particles (VLP). Transfected cells displaying MPER mutants bound as efficiently to both 2F5 and 4E10 as cells transfected with a plasmid encoding full-length Env. Mice immunized with VLPs containing the MPER mutants produced MPER-specific antibodies, the levels of which could be increased by the trimerization of the displayed proteins as well as by a DNA prime-VLP boost immunization strategy. Although 2F5 competed for binding to MPER with antibodies in sera of some of the immunized mice, neutralizing activity could not be detected. Whether this is due to inefficient binding of the induced antibodies to MPER in the context of wild type Env or whether the overall MPER-specific antibody response induced by the MPER display mutants is too low to reveal neutralizing activity, remains to be determined.

Cytoplasmic utilization of human immunodeficiency virus type 1 genomic RNA is not dependent on a nuclear interaction with gag.

In some retroviruses, such as Rous sarcoma virus and prototype foamy virus, Gag proteins are known to shuttle between the nucleus and the cytoplasm and are implicated in nuclear export of the viral genomic unspliced RNA (gRNA) for subsequent encapsidation. A similar function has been proposed for human immunodeficiency virus type 1 (HIV-1) Gag based on the identification of nuclear localization and export signals. However, the ability of HIV-1 Gag to transit through the nucleus has never been confirmed. In addition, the lentiviral Rev protein promotes efficient nuclear gRNA export, and previous reports indicate a cytoplasmic interaction between Gag and gRNA. Therefore, functional effects of HIV-1 Gag on gRNA and its usage were explored. Expression of gag in the absence of Rev was not able to increase cytoplasmic gRNA levels of subgenomic, proviral, or lentiviral vector constructs, and gene expression from genomic reporter plasmids could not be induced by Gag provided in trans. Furthermore, Gag lacking the reported nuclear localization and export signals was still able to mediate an efficient packaging process. Although small amounts of Gag were detectable in the nuclei of transfected cells, a Crm1-dependent nuclear export signal in Gag could not be confirmed. Thus, our study does not provide any evidence for a nuclear function of HIV-1 Gag. The encapsidation process of HIV-1 therefore clearly differs from that of Rous sarcoma virus and prototype foamy virus.

The human immunodeficiency virus type 1 Rev protein: ménage à trois during the early phase of the lentiviral replication cycle.

The Rev protein of human immunodeficiency viruses (HIV) has long been recognized to be essential for the late phase of the virus replication cycle, due to its strong enhancement of expression of viral structural proteins. Surprisingly, a number of recent papers have demonstrated that Rev can also interfere with integration of the reverse-transcribed cDNA into the host-cell genome. This seems to be due to Rev's binding to integrase and LEDGF/p75, an important cellular cofactor of HIV-1 integration. As Rev is presumably expressed at sufficiently high levels only after the encoding genome has already integrated, the main function of Rev during the early phase might be to reduce genotoxicity due to excessive integration events after superinfection of the same cell by subsequent viruses. Other potential consequences for HIV-1 replication and evolution after co-infection of the same cell with two viruses are discussed.

Nuclear RNA export and packaging functions of HIV-1 Rev revisited.

Although the viral Rev protein is necessary for HIV replication, its main function in the viral replication cycle has been controversial. Reinvestigating the effect of Rev on the HIV-1 RNA distribution in various cell lines and primary cells revealed that Rev enhanced cytoplasmic levels of the unspliced HIV-1 RNA, mostly 3- to 12-fold, while encapsidation of the RNA and viral infectivity could be stimulated >1,000-fold. Although this clearly questions the general notion that the nuclear export of viral RNAs is the major function of Rev, mechanistically encapsidation seems to be linked to nuclear export, since the tethering of the nuclear export factor TAP to the HIV-1 RNA also enhanced encapsidation. Interference with the formation of an inhibitory ribonucleoprotein complex in the nucleus could lead to enhanced accessibility of the cytoplasmic HIV-1 RNA for translation and encapsidation. This might explain why Rev and tethered TAP exert the same pattern of pleiotropic effects.

Rev proteins of human and simian immunodeficiency virus enhance RNA encapsidation.

The main function attributed to the Rev proteins of immunodeficiency viruses is the shuttling of viral RNAs containing the Rev responsive element (RRE) via the CRM-1 export pathway from the nucleus to the cytoplasm. This restricts expression of structural proteins to the late phase of the lentiviral replication cycle. Using Rev-independent gag-pol expression plasmids of HIV-1 and simian immunodeficiency virus and lentiviral vector constructs, we have observed that HIV-1 and simian immunodeficiency virus Rev enhanced RNA encapsidation 20- to 70-fold, correlating well with the effect of Rev on vector titers. In contrast, cytoplasmic vector RNA levels were only marginally affected by Rev. Binding of Rev to the RRE or to a heterologous RNA element was required for Rev-mediated enhancement of RNA encapsidation. In addition to specific interactions of nucleocapsid with the packaging signal at the 5' end of the genome, the Rev/RRE system provides a second mechanism contributing to preferential encapsidation of genomic lentiviral RNA.