A Variety of Mouse PYHIN Proteins Restrict Murine and Human Retroviruses.

PYHIN proteins are only found in mammals and play key roles in the defense against bacterial and viral pathogens. The corresponding gene locus shows variable deletion and expansion ranging from 0 genes in bats, over 1 in cows, and 4 in humans to a maximum of 13 in mice. While initially thought to act as cytosolic immune sensors that recognize foreign DNA, increasing evidence suggests that PYHIN proteins also inhibit viral pathogens by more direct mechanisms. Here, we examined the ability of all 13 murine PYHIN proteins to inhibit HIV-1 and murine leukemia virus (MLV). We show that overexpression of p203, p204, p205, p208, p209, p210, p211, and p212 strongly inhibits production of infectious HIV-1; p202, p207, and p213 had no significant effects, while p206 and p214 showed intermediate phenotypes. The inhibitory effects on infectious HIV-1 production correlated significantly with the suppression of reporter gene expression by a proviral Moloney MLV-eGFP construct and HIV-1 and Friend MLV LTR luciferase reporter constructs. Altogether, our data show that the antiretroviral activity of PYHIN proteins is conserved between men and mice and further support the key role of nuclear PYHIN proteins in innate antiviral immunity.

The Inhibition of Gag-Pol Expression by the Restriction Factor Shiftless Is Dispensable for the Restriction of HIV-1 Infection.

The interferon-induced host cell protein Shiftless (SFL) inhibits -1 programmed ribosomal frameshifting (-1PRF) required for the expression of HIV-1 Gal-Pol and the formation of infectious HIV-1 particles. However, the specific regions in SFL required for antiviral activity and the mechanism by which SFL inhibits -1PRF remain unclear. Employing alanine scanning mutagenesis, we found that basic amino acids in the predicted zinc ribbon motif of SFL are essential for the suppression of Gag-Pol expression but dispensable for anti-HIV-1 activity. We have shown that SFL inhibits the expression of the murine leukemia virus (MLV) Gag-Pol polyprotein and the formation of infectious MLV particles, although Gag-Pol expression of MLV is independent of -1PRF but requires readthrough of a stop codon. These findings indicate that SFL might inhibit HIV-1 infection by more than one mechanism and that SFL might target programmed translational readthrough as well as -1PRF signals, both of which are regulated by mRNA secondary structure elements.

Murine leukemia virus infection of non-dividing dendritic cells is dependent on nucleoporins.

Retroviral reverse transcription starts within the capsid and uncoating and reverse transcription are mutually dependent. There is still debate regarding the timing and cellular location of HIV's uncoating and reverse transcription and whether it occurs solely in the cytoplasm, nucleus or both. HIV can infect non-dividing cells because there is active transport of the preintegration complex (PIC) across the nuclear membrane, but Murine Leukemia Virus (MLV) is thought to depend on cell division for replication and whether MLV uncoating and reverse transcription is solely cytoplasmic has not been studied. Here, we used NIH3T3 and primary mouse dendritic cells to determine where the different stages of reverse transcription occur and whether cell division is needed for nuclear entry. Our data strongly suggest that in both NIH3T3 cells and dendritic cells (DCs), the initial step of reverse transcription occurs in the cytoplasm. However, we detected MLV RNA/DNA hybrid intermediates in the nucleus of dividing NIH3T3 cells and non-dividing DCs, suggesting that reverse transcription can continue after nuclear entry. We also confirmed that the MLV PIC requires cell division to enter the nucleus of NIH3T3 cells. In contrast, we show that MLV can infect non-dividing primary DCs, although integration of MLV DNA in DCs still required the viral p12 protein. Knockdown of several nuclear pore proteins dramatically reduced the appearance of integrated MLV DNA in DCs but not NIH3T3 cells. Additionally, MLV capsid associated with the nuclear pore proteins NUP358 and NUP62 during infection. These findings suggest that simple retroviruses, like the complex retrovirus HIV, gain nuclear entry by traversing the nuclear pore complex in non-mitotic cells.

Murine leukemia virus (MLV) P50 protein induces cell transformation via transcriptional regulatory function.

BACKGROUND: The murine leukemia virus (MLV) has been a powerful model of pathogenesis for the discovery of genes involved in cancer. Its splice donor (SD')-associated retroelement (SDARE) is important for infectivity and tumorigenesis, but the mechanism remains poorly characterized. Here, we show for the first time that P50 protein, which is produced from SDARE, acts as an accessory protein that transregulates transcription and induces cell transformation. RESULTS: By infecting cells with MLV particles containing SDARE transcript alone (lacking genomic RNA), we show that SDARE can spread to neighbouring cells as shown by the presence of P50 in infected cells. Furthermore, a role for P50 in cell transformation was demonstrated by CCK8, TUNEL and anchorage-independent growth assays. We identified the integrase domain of P50 as being responsible for transregulation of the MLV promoter using luciferase assay and RTqPCR with P50 deleted mutants. Transcriptomic analysis furthermore revealed that the expression of hundreds of cellular RNAs involved in cancerogenesis were deregulated in the presence of P50, suggesting that P50 induces carcinogenic processes via its transcriptional regulatory function. CONCLUSION: We propose a novel SDARE-mediated mode of propagation of the P50 accessory protein in surrounding cells. Moreover, due to its transforming properties, P50 expression could lead to a cellular and tissue microenvironment that is conducive to cancer development.

Genetic Differences between 129S Substrains Affect Antiretroviral Immune Responses.

Inbred mouse lines vary in their ability to mount protective antiretroviral immune responses, and even closely related strains can exhibit opposing phenotypes upon retroviral infection. Here, we found that 129S mice inherit a previously unknown mechanism for the production of anti-murine leukemia virus (MLV) antibodies and control of infection. The resistant phenotype in 129S1 mice is controlled by two dominant loci that are independent from known MLV resistance genes. We also show that production of anti-MLV antibodies in 129S7 mice, but not 129S1 mice, is independent of interferon gamma signaling. Thus, our data indicate that 129S mice inherit an unknown mechanism for control of MLV infection and demonstrate that there is genetic variability in 129S substrains that affects their ability to mount antiviral immune responses. IMPORTANCE Understanding the genetic basis for production of protective antiviral immune responses is crucial for the development of novel vaccines and adjuvants. Additionally, characterizing the genetic and phenotypic variability in inbred mice has implications for the selection of strains for targeted mutagenesis, choice of controls, and for broader understanding of the requirements for protective immunity.

BET-Independent Murine Leukemia Virus Integration Is Retargeted In Vivo and Selects Distinct Genomic Elements for Lymphomagenesis.

Moloney murine leukemia virus (MLV) infects BALB/c mice and induces T-cell lymphoma in mice. Retroviral integration is mediated by the interaction of the MLV integrase (IN) with members of the bromodomain and extraterminal motif (BET) protein family (BRD2, BRD3, and BRD4). The introduction of the W390A mutation into MLV IN abolishes the BET interaction. Here, we compared the replication of W390A MLV to that of wild-type (WT) MLV in adult BALB/c mice to study the role of BET proteins in replication, integration, and tumorigenesis in vivo. Comparing WT and W390A MLV infections revealed similar viral loads in the blood, thymus, and spleen cells. Interestingly, W390A MLV integration was retargeted away from GC-enriched genomic regions. However, both WT MLV- and W390A MLV-infected mice developed T-cell lymphoma after similar latencies represented by an enlarged thymus and spleen and multiorgan tumor infiltration. Integration site sequencing from splenic tumor cells revealed clonal expansion in all WT MLV- and W390A MLV-infected mice. However, the integration profiles of W390A MLV and WT MLV differed significantly. Integrations were enriched in enhancers and promoters, but compared to the WT, W390A MLV integrated less frequently into enhancers and more frequently into oncogene bodies such as Notch1 and Ppp1r16b. We conclude that host factors direct MLV in vivo integration site selection. Although BET proteins target WT MLV integration preferentially toward enhancers and promoters, insertional lymphomagenesis can occur independently from BET, likely due to the intrinsically strong enhancer/promoter of the MLV long terminal repeat (LTR). IMPORTANCE In this study, we have shown that the in vivo replication of murine leukemia virus happens independently of BET proteins, which are key host determinants involved in retroviral integration site selection. This finding opens a new research line in the discovery of alternative viral or host factors that may complement the dominant host factor. In addition, our results show that BET-independent murine leukemia virus uncouples insertional mutagenesis from gene enhancers, although lymphomagenesis still occurs despite the lack of an interaction with BET proteins. Our findings also have implications for the engineering of BET-independent MLV-based vectors for gene therapy, which may not be a safe alternative.

Retroviral RNA Processing.

This review is an accompaniment to a Special Issue on "Retroviral RNA Processing". It discusses post-transcriptional regulation of retroviruses, ranging from the ancient foamy viruses to more modern viruses, such as HIV-1, HTLV-1, Rous sarcoma virus, murine leukemia virus, mouse mammary tumor virus, and Mason-Pfizer monkey virus. This review is not comprehensive. However, it tries to address some of the major questions in the field with examples of how different retroviruses express their genes. It is amazing that a single primary RNA transcript can have so many possible fates: genomic RNA, unspliced mRNA, and up to 50 different alternatively spliced mRNAs. This review will discuss the sorting of RNAs for packaging or translation, RNA nuclear export mechanisms, splicing, translation, RNA modifications, and avoidance of nonsense-mediated RNA decay.

Detection and differentiation of murine leukemia virus (MLV) and murine stem cell virus (MSCV) and therefrom derived nucleic acids.

Murine leukemia virus (MLV) and murine stem cell virus (MSCV) and derived retroviral vectors are widely used to study retrovirus biology and as tools for gene delivery. The method described here represents a quantitative real time PCR (qPCR) with hydrolysis probe that can be applied within classical qPCR as well as in digital droplet PCR (ddPCR). The method targets a 60 bp long fragment located within the U5 region of the MLV/MSCV genome sequence. For the here described method a LOD95% of 25 copies per PCR reaction (DNA) and 80 copies per PCR reaction (RNA) was determined, and PCR efficiencies of 92.5 % and 98.5 %, respectively, were observed. This method enables the fast and simple titration of viral genomic RNA present in retroviral vector stocks for accurate and consistent transduction experiments. Furthermore, it enables the detection of proviral and transfer plasmid derived DNA sequences and can be modified to differentiate between retroviral RNA and DNA.

Silencing of Unintegrated Retroviral DNAs.

Retroviral infection delivers an RNA genome into the cytoplasm that serves as the template for the synthesis of a linear double-stranded DNA copy by the viral reverse transcriptase. Within the nucleus this linear DNA gives rise to extrachromosomal circular forms, and in a key step of the life cycle is inserted into the host genome to form the integrated provirus. The unintegrated DNA forms, like those of DNAs entering cells by other means, are rapidly loaded with nucleosomes and heavily silenced by epigenetic histone modifications. This review summarizes our present understanding of the silencing machinery for the DNAs of the mouse leukemia viruses and human immunodeficiency virus type 1. We consider the potential impact of the silencing on virus replication, on the sensing of the virus by the innate immune system, and on the formation of latent proviruses. We also speculate on the changeover to high expression from the integrated proviruses in permissive cell types, and briefly consider the silencing of proviruses even after integration in embryonic stem cells and other developmentally primitive cell types.

Patterns of Coevolutionary Adaptations across Time and Space in Mouse Gammaretroviruses and Three Restrictive Host Factors.

The classical laboratory mouse strains are genetic mosaics of three Mus musculus subspecies that occupy distinct regions of Eurasia. These strains and subspecies carry infectious and endogenous mouse leukemia viruses (MLVs) that can be pathogenic and mutagenic. MLVs evolved in concert with restrictive host factors with some under positive selection, including the XPR1 receptor for xenotropic/polytropic MLVs (X/P-MLVs) and the post-entry restriction factor Fv1. Since positive selection marks host-pathogen genetic conflicts, we examined MLVs for counter-adaptations at sites that interact with XPR1, Fv1, and the CAT1 receptor for ecotropic MLVs (E-MLVs). Results describe different co-adaptive evolutionary paths within the ranges occupied by these virus-infected subspecies. The interface of CAT1, and the otherwise variable E-MLV envelopes, is highly conserved; antiviral protection is afforded by the Fv4 restriction factor. XPR1 and X/P-MLVs variants show coordinate geographic distributions, with receptor critical sites in envelope, under positive selection but with little variation in envelope and XPR1 in mice carrying P-ERVs. The major Fv1 target in the viral capsid is under positive selection, and the distribution of Fv1 alleles is subspecies-correlated. These data document adaptive, spatial and temporal, co-evolutionary trajectories at the critical interfaces of MLVs and the host factors that restrict their replication.

Modulation of Viral Programmed Ribosomal Frameshifting and Stop Codon Readthrough by the Host Restriction Factor Shiftless.

The product of the interferon-stimulated gene C19orf66, Shiftless (SHFL), restricts human immunodeficiency virus replication through downregulation of the efficiency of the viral gag/pol frameshifting signal. In this study, we demonstrate that bacterially expressed, purified SHFL can decrease the efficiency of programmed ribosomal frameshifting in vitro at a variety of sites, including the RNA pseudoknot-dependent signals of the coronaviruses IBV, SARS-CoV and SARS-CoV-2, and the protein-dependent stimulators of the cardioviruses EMCV and TMEV. SHFL also reduced the efficiency of stop-codon readthrough at the murine leukemia virus gag/pol signal. Using size-exclusion chromatography, we confirm the binding of the purified protein to mammalian ribosomes in vitro. Finally, through electrophoretic mobility shift assays and mutational analysis, we show that expressed SHFL has strong RNA binding activity that is necessary for full activity in the inhibition of frameshifting, but shows no clear specificity for stimulatory RNA structures.

Establishment of a novel stable human suspension packaging cell line producing ecotropic retroviral MLV(PVC-211) vectors efficiently transducing murine hematopoietic stem and progenitor cells.

Retroviral vectors derived from murine leukemia virus (MLV) are amongst the most frequently utilized vectors in gene therapy approaches such as the genetic modification of hematopoietic cells. Currently, vector particles are mostly produced employing adherent viral packaging cell lines (VPCs) rendering the scale up of production laborious, and thus cost-intensive. Here, we describe the rapid establishment of a human suspension 293-F cell line derived ecotropic MLV VPC. Using transposon vector technology, a packaging and envelope expression cassette as well as a transfer vector facilitated the establishment of a stable VPC yielding high titers of up to 5.2 × 106 transducing units/mL (TU/mL). Vectors were concentrated using ultrafiltration devices and upon one freeze-thaw-cycle still routinely yielded titers of > 1 × 106 TU/mL. Formation of replication-competent retroviruses was not detected. However and as a first generation transfer vector was used in this proof-of-concept (POC) study, gag gene sequences were transduced into target cells within a range of 1-10 copies per 1000 genomes indicating the homologous recombination of packaging construct elements with the transfer vector. High yield VPC vector productivity was stable over a couple of months and unintended integration of the transposase gene was not observed. Ecotropic MLV vector particles were demonstrated to efficiently transduce primary murine hematopoietic stem and progenitor cells. This novel concept should foster the future establishment of suspension VPCs.

Aromatic Side Chain at Position 412 of SERINC5 Exerts Restriction Activity toward HIV-1 and Other Retroviruses.

The host transmembrane protein SERINC5 is incorporated into viral particles and restricts infection by certain retroviruses. However, what motif of SERINC5 mediates this process remains elusive. By conducting mutagenesis analyses, we found that the substitution of phenylalanine with alanine at position 412 (F412A) resulted in a >75-fold reduction in SERINC5's restriction function. The F412A substitution also resulted in the loss of SERINC5's function to sensitize HIV-1 neutralization by antibodies recognizing the envelope's membrane proximal region. A series of biochemical analyses revealed that F412A showed steady-state protein expression, localization at the cellular membrane, and incorporation into secreted virus particles to a greater extent than in the wild type. Furthermore, introduction of several amino acid mutations at this position revealed that the aromatic side chains, including phenylalanine, tyrosine, and tryptophan, were required to maintain SERINC5 functions to impair the virus-cell fusion process and virion infectivity. Moreover, the wild-type SERINC5 restricted infection of lentiviruses pseudotyped with envelopes of murine leukemia viruses, simian immunodeficiency virus, and HIV-2, and F412A abrogated this function. Taken together, our results highlight the importance of the aromatic side chain at SERINC5 position 412 to maintain its restriction function against diverse retrovirus envelopes. IMPORTANCE The host protein SERINC5 is incorporated into progeny virions of certain retroviruses and restricts the infectivity of these viruses or sensitizes the envelope glycoprotein to a class of neutralizing antibodies. However, how and which part of SERINC5 engages with the diverse array of retroviral envelopes and exerts its antiretroviral functions remain elusive. During mutagenesis analyses, we eventually found that the single substitution of phenylalanine with alanine, but not with tyrosine or tryptophan, at position 412 (F412A) resulted in the loss of SERINC5's functions toward diverse retroviruses, whereas F412A showed steady-state protein expression, localization at the cellular membrane, and incorporation into progeny virions to a greater extent than the wild type. Results suggest that the aromatic side chain at position 412 of SERINC5 plays a critical role in mediating antiviral functions toward various retroviruses, thus providing additional important information regarding host and retrovirus interaction.

Gm40600 promotes CD4+ T-cell responses by interacting with Ahnak.

It is important to characterize novel proteins involved in T- and B-cell responses. Our previous study demonstrated that a novel protein, Mus musculus Gm40600, reduced the proliferation of Mus musculus plasmablast (PB)-like SP 2/0 cells and B-cell responses induced in vitro by LPS. In the present study, we revealed that Gm40600 directly promoted CD4+ T-cell responses to indirectly up-regulate B-cell responses. Importantly, we found that CD4+ T-cell responses, including T-cell activation and differentiation and cytokine production, were increased in Gm40600 transgenic (Tg) mice and were reduced in Gm40600 knockout (KO) mice. Finally, we demonstrated that Gm40600 promoted the Ahnak-mediated calcium signalling pathway by interacting with Ahnak to maintain a cytoplasmic lateral location of Ahnak in CD4+ T cells. Collectively, our data suggest that Gm40600 promotes CD4+ T-cell activation to up-regulate the B-cell response via interacting with Ahnak to promote the calcium signalling pathway. The results suggest that targeting Gm40600 may be a means to control CD4+ T-cell-related diseases.

Characterization of a new case of XMLV (Bxv1) contamination in the human cell line Hep2 (clone 2B).

The use of misidentified cell lines contaminated by other cell lines and/or microorganisms has generated much confusion in the scientific literature. Detailed characterization of such contaminations is therefore crucial to avoid misinterpretation and ensure robustness and reproducibility of research. Here we use DNA-seq data produced in our lab to first confirm that the Hep2 (clone 2B) cell line (Sigma-Aldrich catalog number: 85011412-1VL) is indistinguishable from the HeLa cell line by mapping integrations of the human papillomavirus 18 (HPV18) at their expected loci on chromosome 8. We then show that the cell line is also contaminated by a xenotropic murine leukemia virus (XMLV) that is nearly identical to the mouse Bxv1 provirus and we characterize one Bxv1 provirus, located in the second intron of the pseudouridylate synthase 1 (PUS1) gene. Using an RNA-seq dataset, we confirm the high expression of the E6 and E7 HPV18 oncogenes, show that the entire Bxv1 genome is moderately expressed, and retrieve a Bxv1 splicing event favouring expression of the env gene. Hep2 (clone 2B) is the fourth human cell line so far known to be contaminated by the Bxv1 XMLV. This contamination has to be taken into account when using the cell line in future experiments.

Immunotherapy of CT26 murine tumors is characterized by an oligoclonal response of tissue-resident memory T cells against the AH1 rejection antigen.

Mice bearing CT26 tumors can be cured by administration of L19-mIL12 or F8-mTNF, two antibody fusion proteins which selectively deliver their cytokine payload to the tumor. In both settings, cancer cures crucially depended on CD8+ T cells and the AH1 peptide (derived from the gp70 protein of the murine leukemia virus) acted as the main tumor-rejection antigen, with ∼50% of CD8+ T cells in the neoplastic mass being AH1-specific after therapy. In order to characterize the clonality of the T cell response, its phenotype, and activation status, we isolated CD8+ T cells from tumors and secondary lymphoid organs and submitted them to T cell receptor (TCR) and total mRNA sequencing. We found an extremely diverse repertoire of more than 40 000 unique TCR sequences, but the ten most abundant TCRs accounted for >60% of CD8+ T-cell clones in the tumor. AH1-specific TCRs were consistently found among the most abundant sequences. AH1-specific T cells in the tumor had a tissue-resident memory phenotype. Treatment with L19-mIL12 led to overexpression of IL-12 receptor and of markers of cell activation and proliferation. These data suggest that the antitumor response driven by antibody-cytokine fusions proceeds through an oligoclonal expansion and activation of tumor-infiltrating CD8+ T cells.

5-Methylcytosine RNA Modifications Promote Retrovirus Replication in an ALYREF Reader Protein-Dependent Manner.

RNA modifications play diverse roles in regulating RNA function, and viruses co-opt these pathways for their own benefit. While recent studies have highlighted the importance of N6-methyladenosine (m6A)-the most abundant mRNA modification-in regulating retrovirus replication, the identification and function of other RNA modifications in viral biology have been largely unexplored. Here, we characterized the RNA modifications present in a model retrovirus, murine leukemia virus (MLV), using mass spectrometry and sequencing. We found that 5-methylcytosine (m5C) is highly enriched in viral genomic RNA relative to uninfected cellular mRNAs, and we mapped at single-nucleotide resolution the m5C sites, which are located in multiple clusters throughout the MLV genome. Further, we showed that the m5C reader protein ALYREF plays an important role in regulating MLV replication. Together, our results provide a complete m5C profile in a virus and its function in a eukaryotic mRNA.IMPORTANCE Over 130 modifications have been identified in cellular RNAs, which play critical roles in many cellular processes, from modulating RNA stability to altering translation efficiency. One such modification, 5-methylcytosine, is relatively abundant in mammalian mRNAs, but its precise location and function are not well understood. In this study, we identified unexpectedly high levels of m5C in the murine leukemia virus RNA, precisely mapped its location, and showed that ALYREF, a reader protein that specifically recognizes m5C, regulates viral production. Together, our findings provide a high-resolution atlas of m5C in murine leukemia virus and reveal a functional role of m5C in viral replication.

Construction of a replication-competent retroviral vector for expression of the VSV-G envelope glycoprotein for cancer gene therapy.

Gibbon ape leukemia virus (GALV) can infect a wide variety of cells but fails to infect most cells derived from laboratory mice. Transduction of human hematopoietic stem cells with GALV retroviral vectors is more efficient than with amphotropic vectors. In this study, a Moloney murine leukemia virus-gibbon ape leukemia virus (MoMLV-GALV) vector was constructed by replacing the natural env gene of the full-length Moloney MLV genome with the GALV env gene. To monitor viral transmission by green fluorescent protein (GFP) expression, internal ribosomal entry site-enhanced GFP (IRES-EGFP) was positioned between the GALV env gene and the 3' untranslated region (3' UTR) to obtain pMoMLV-GALV-EGFP. The MoMLV-GALV-EGFP vector was able to replicate with high titer in TE671 human rhabdomyosarcoma cells and U-87 human glioma cells. To evaluate the potential of the MoMLV-GALV vector as a therapeutic agent, the gene for the fusogenic envelope G glycoprotein of vesicular stomatitis virus (VSV-G) was incorporated into the vector. Infection with the resulting MoMLV-GALV-VSV-G vector resulted in lysis of the U-87 cells due to syncytium formation. Syncytium formation was also observed in the transfected human prostate cancer cell line LNCaP after extended cultivation of cells. In addition, we deleted the GALV env gene from the MoMLV-GALV-VSV-G vector to improve viral genome stability. This MoMLV-VSV-G vector is also replication competent and induces syncytium formation in 293T, HT1080, TE671 and U-87 cells. These results suggest that replication of the MoMLV-GALV-VSV-G vector or MoMLV-VSV-G vector may directly lead to cytotoxicity. Therefore, the vectors developed in this study are potentially useful tools for cancer gene therapy.

Disrupting MLV integrase:BET protein interaction biases integration into quiescent chromatin and delays but does not eliminate tumor activation in a MYC/Runx2 mouse model.

Murine leukemia virus (MLV) integrase (IN) lacking the C-terminal tail peptide (TP) loses its interaction with the host bromodomain and extraterminal (BET) proteins and displays decreased integration at promoter/enhancers and transcriptional start sites/CpG islands. MLV lacking the IN TP via an altered open reading frame was used to infect tumorigenesis mouse model (MYC/Runx2) animals to observe integration patterns and phenotypic effects, but viral passage resulted in the restoration of the IN TP through small deletions. Mice subsequently infected with an MLV IN lacking the TP coding sequence (TP-) showed an improved median survival by 15 days compared to wild type (WT) MLV infection. Recombination with polytropic endogenous retrovirus (ERV), Pmv20, was identified in seven mice displaying both fast and slow tumorigenesis, highlighting the strong selection within the mouse to maintain the full-length IN protein. Mapping the genomic locations of MLV in tumors from an infected mouse with no observed recombination with ERVs, TP-16, showed fewer integrations at TSS and CpG islands, compared to integrations observed in WT tumors. However, this mouse succumbed to the tumor in relatively rapid fashion (34 days). Analysis of the top copy number integrants in the TP-16 tumor revealed their proximity to known MLV common insertion site genes while maintaining the MLV IN TP- genotype. Furthermore, integration mapping in K562 cells revealed an insertion preference of MLV IN TP- within chromatin profile states associated with weakly transcribed heterochromatin with fewer integrations at histone marks associated with BET proteins (H3K4me1/2/3, and H3K27Ac). While MLV IN TP- showed a decreased overall rate of tumorigenesis compared to WT virus in the MYC/Runx2 model, MLV integration still occurred at regions associated with oncogenic driver genes independently from the influence of BET proteins, either stochastically or through trans-complementation by functional endogenous Gag-Pol protein.

HIV-1 Matrix Trimerization-Impaired Mutants Are Rescued by Matrix Substitutions That Enhance Envelope Glycoprotein Incorporation.

The matrix (MA) domain of HIV-1 Gag plays key roles in virus assembly by targeting the Gag precursor to the plasma membrane and directing the incorporation of the viral envelope (Env) glycoprotein into virions. The latter function appears to be in part dependent on trimerization of the MA domain of Gag during assembly, as disruption of the MA trimer interface impairs Env incorporation. Conversely, many MA mutations that impair Env incorporation can be rescued by compensatory mutations in the trimer interface. In this study, we sought to investigate further the biological significance of MA trimerization by isolating and characterizing compensatory mutations that rescue MA trimer interface mutants with severely impaired Env incorporation. By serially propagating MA trimerization-defective mutants in T cell lines, we identified a number of changes in MA, both within and distant from the trimer interface. The compensatory mutations located within or near the trimer interface restored Env incorporation and particle infectivity and permitted replication in culture. The structure of the MA lattice was interrogated by measuring the cleavage of the murine leukemia virus (MLV) transmembrane Env protein by the viral protease in MLV Env-pseudotyped HIV-1 particles bearing the MA mutations and by performing crystallographic studies of in vitro-assembled MA lattices. These results demonstrate that rescue is associated with structural alterations in MA organization and rescue of MA domain trimer formation. Our data highlight the significance of the trimer interface of the MA domain of Gag as a critical site of protein-protein interaction during HIV-1 assembly and establish the functional importance of trimeric MA for Env incorporation.IMPORTANCE The immature Gag lattice is a critical structural feature of assembling HIV-1 particles, which is primarily important for virion formation and release. While Gag forms a hexameric lattice, driven primarily by the capsid domain, the MA domain additionally trimerizes where three Gag hexamers meet. MA mutants that are defective for trimerization are deficient for Env incorporation and replication, suggesting a requirement for trimerization of the MA domain of Gag in Env incorporation. This study used a gain-of-function, forced viral evolution approach to rescue HIV-1 mutants that are defective for MA trimerization. Compensatory mutations that rescue virus replication do so by restoring Env incorporation and MA trimer formation. This study supports the importance of MA domain trimerization in HIV-1 replication and the potential of the trimer interface as a therapeutic target.