Murine Leukemia Virus Infection Research Articles

The Furin Protease Dependence and Antiviral GBP2 Sensitivity of Murine Leukemia Virus Infection Are Determined by the Amino Acid Sequence at the Envelope Glycoprotein Cleavage Site.

Host restriction factor GBP2 suppresses the replication of the ecotropic Moloney murine leukemia virus (E-MLV) by inhibiting furin protease, which cleaves the viral envelope glycoprotein (Env) into surface (SU) and transmembrane (TM) subunits. We analyzed the impacts of GBP2 on the infection efficiency mediated by MLV Envs of different strains of ecotropic Moloney, polytropic Friend, amphotropic, and xenotropic MLV-related (XMRV) viruses. Interestingly, the Envs of ecotropic Moloney and polytropic Friend MLV were sensitive to the antiviral activity of GBP2, while XMRV and amphotropic Envs showed resistance. Consistent with the sensitivity to GBP2, the amino acid sequences of the sensitive Envs at the SU-TM cleavage site were similar, as were the sequences of the resistant Envs. SU-TM cleavage of the GBP2-sensitive Env protein was inhibited by furin silencing, whereas that of GBP2-resistant Env was not. The substitution of the ecotropic Moloney cleavage site sequence with that of XMRV conferred resistance to both GBP2 and furin silencing. Reciprocally, the substitution of the XMRV cleavage site sequence with that of the ecotropic sequence conferred sensitivity to GBP2 and furin silencing. According to the SU-TM cleavage site sequence, there were sensitive and resistant variants among ecotropic, polytropic, and xenotropic MLVs. This study found that the dependence of MLV Env proteins on furin cleavage and GBP2-mediated restriction is determined by the amino acid sequences at the SU-TM cleavage site.

IFI207, a young and fast-evolving protein, controls retroviral replication via the STING pathway.

Mammalian AIM-2-like receptor (ALR) proteins bind nucleic acids and initiate production of type I interferons or inflammasome assembly, thereby contributing to host innate immunity. In mice, the Alr locus is highly polymorphic at the sequence and copy number level, and we show here that it is one of the most dynamic regions of the genome. One rapidly evolving gene within this region, Ifi207, was introduced to the Mus genome by gene conversion or an unequal recombination event a few million years ago. Ifi207 has a large, distinctive repeat region that differs in sequence and length among Mus species and even closely related inbred Mus musculus strains. We show that IFI207 controls murine leukemia virus (MLV) infection in vivo and that it plays a role in the STING-mediated response to cGAMP, dsDNA, DMXXA, and MLV. IFI207 binds to STING, and inclusion of its repeat region appears to stabilize STING protein. The Alr locus and Ifi207 provide a clear example of the evolutionary innovation of gene function, possibly as a result of host-pathogen co-evolution.IMPORTANCEThe Red Queen hypothesis predicts that the arms race between pathogens and the host may accelerate evolution of both sides, and therefore causes higher diversity in virulence factors and immune-related proteins, respectively . The Alr gene family in mice has undergone rapid evolution in the last few million years and includes the creation of two novel members, MndaL and Ifi207. Ifi207, in particular, became highly divergent, with significant genetic changes between highly related inbred mice. IFI207 protein acts in the STING pathway and contributes to anti-retroviral resistance via a novel mechanism. The data show that under the pressure of host-pathogen coevolution in a dynamic locus, gene conversion and recombination between gene family members creates new genes with novel and essential functions that play diverse roles in biological processes.

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.

Dynein Light-Chain Dynlrb2 Is Essential for Murine Leukemia Virus Traffic and Nuclear Entry.

Murine leukemia virus (MLV) requires the infected cell to divide to access the nucleus to integrate into the host genome. It has been determined that MLV uses the microtubule and actin network to reach the nucleus at the early stages of infection. Several studies have shown that viruses use the dynein motor protein associated with microtubules for their displacement. We have previously reported that dynein light-chain roadblock type 2 (Dynlrb2) knockdown significantly decreases MLV infection compared to nonsilenced cells, suggesting a functional association between this dynein light chain and MLV preintegration complex (PIC). In this study, we aimed to determine if the dynein complex Dynlrb2 subunit plays an essential role in the retrograde transport of MLV. For this, an MLV mutant containing the green fluorescent protein (GFP) fused to the viral protein p12 was used to assay the PIC localization and speed in cells in which the expression of Dynlrb2 was modulated. We found a significant decrease in the arrival of MLV PIC to the nucleus and a reduced net speed of MLV PICs when Dynlrb2 was knocked down. In contrast, an increase in nuclear localization was observed when Dynlrb2 was overexpressed. Our results suggest that Dynlrb2 plays an essential role in MLV retrograde transport. IMPORTANCE Different viruses use different components of cytoplasmic dynein complex to traffic to their replication site. We have found that murine leukemia virus (MLV) depends on dynein light-chain Dynlrb2 for infection, retrograde traffic, and nuclear entry. Our study provides new information regarding the molecular requirements for retrograde transport of MLV preintegration complex and demonstrates the essential role of Dynlrb2 in MLV infection.

SERINC5 Potently Restricts Retrovirus Infection In Vivo.

The serine incorporator (SERINC) proteins are multipass transmembrane proteins that affect sphingolipid and phosphatidylserine synthesis. Human SERINC5 and SERINC3 were recently shown to possess antiretroviral activity for a number of retroviruses, including human immunodeficiency virus (HIV), murine leukemia virus (MLV), and equine infectious anemia virus (EIAV). In the case of MLV, the glycosylated Gag (glyco-Gag) protein was shown to counteract SERINC5-mediated restriction in in vitro experiments and the viral envelope was found to determine virion sensitivity or resistance to SERINC5. However, nothing is known about the in vivo function of SERINC5. Antiretroviral function of a host factor in vitro is not always associated with antiretroviral function in vivo Using SERINC5-/- mice that we had generated, we showed that mouse SERINC5 (mSERINC5) restriction of MLV infection in vivo is influenced not only by glyco-Gag but also by the retroviral envelope. Finally, we also examined the in vivo function of the other SERINC gene with known antiretroviral functions, SERINC3. By using SERINC3-/- mice, we found that the murine homologue, mSERINC3, had no antiretroviral role either in vivo or in vitro To our knowledge, this report provides the first data showing that SERINC5 restricts retrovirus infection in vivo and that restriction of retrovirus infectivity in vivo is dependent on the presence of both glyco-Gag and the viral envelope.IMPORTANCE This study examined for the first time the in vivo function of the serine incorporator (SERINC) proteins during retrovirus infection. SERINC3 and SERINC5 (SERINC3/5) restrict a number of retroviruses, including human immunodeficiency virus 1 (HIV-1) and murine leukemia virus (MLV), by blocking their entry into cells. Nevertheless, HIV-1 and MLV encode factors, Nef and glycosylated Gag, respectively, that counteract SERINC3/5 in vitro We recently developed SERINC3 and SERINC5 knockout mice to examine the in vivo function of these genes. We found that SERINC5 restriction is dependent on the absence of glycosylated Gag and the expression of a specific viral envelope glycoprotein. On the other hand, SERINC3 had no antiviral function. Our findings have implications for the development of therapeutics that target SERINC5 during retrovirus infection.

Cathepsin B Protease Facilitates Chikungunya Virus Envelope Protein-Mediated Infection via Endocytosis or Macropinocytosis.

Chikungunya virus (CHIKV) is an enveloped virus that enters host cells and transits within the endosomes before starting its replication cycle, the precise mechanism of which is yet to be elucidated. Endocytosis and endosome acidification inhibitors inhibit infection by CHIKV, murine leukemia virus (MLV), or SARS-coronavirus, indicating that these viral entries into host cells occur through endosomes and require endosome acidification. Although endosomal cathepsin B protease is necessary for MLV, Ebola virus, and SARS-CoV infections, its role in CHIKV infection is unknown. Our results revealed that endocytosis inhibitors attenuated CHIKV-pseudotyped MLV vector infection in 293T cells but not in TE671 cells. In contrast, macropinocytosis inhibitors attenuated CHIKV-pseudotyped MLV vector infection in TE671 cells but not in 293T cells, suggesting that CHIKV host cell entry occurs via endocytosis or macropinocytosis, depending on the cell lines used. Cathepsin B inhibitor and knockdown by an shRNA suppressed CHIKV-pseudotyped MLV vector infection both in 293T and TE671 cells. These results show that cathepsin B facilitates CHIKV infection regardless of the entry pathway.

Intact Viral Particle Counts Measured by Flow Virometry Provide Insight into the Infectivity and Genome Packaging Efficiency of Moloney Murine Leukemia Virus

Murine leukemia viruses (MLVs) have long been used as a research model to further our understanding of retroviruses. These simple gammaretroviruses have been studied extensively in various facets of science for nearly half a century, yet we have surprisingly little quantitative information about some of the basic features of these viral particles. These include parameters such as the genome packaging efficiency and the number of particles required for a productive infection. The reason for this knowledge gap relies primarily on the technical challenge of accurately measuring intact viral particles from infected cell supernatants. Virus-infected cells are well known to release soluble viral proteins, defective viruses, and extracellular vesicles (EVs) harboring viral proteins that may mimic viruses, all of which can skew virus titer quantifications. Flow virometry, also known as nanoscale flow cytometry or simply small-particle flow cytometry, is an emerging analytical method enabling high-throughput single-virus phenotypic characterizations. By utilizing the viral envelope glycoprotein (Env) and monodisperse light scattering characteristics as discerning parameters of intact virus particles, here, we analyzed the basic properties of Moloney MLV (M-MLV). We show that <24% of the total p30 capsid protein measured in infected cell supernatants is associated with intact viruses. We calculate that about one in five M-MLV particles contains a viral RNA genome pair and that individual intact particle infectivity is about 0.4%. These findings provide new insights into the characteristics of an extensively studied prototypical retrovirus while highlighting the benefits of flow virometry for the field of virology.IMPORTANCE Gammaretroviruses, or, more specifically, murine leukemia viruses (MLVs), have been a longstanding model for studying retroviruses. Although being extensively analyzed and dissected for decades, several facets of MLV biology are still poorly understood. One of the primary challenges has been enumerating total intact virus particles in a sample. While several analytical methods can precisely measure virus protein amounts, MLVs are known to induce the secretion of soluble and vesicle-associated viral proteins that can skew these measurements. With recent technological advances in flow cytometry, it is now possible to analyze viruses down to 90 nm in diameter with an approach called flow virometry. The technique has the added benefit of being able to discriminate viruses from extracellular vesicles and free viral proteins in order to confidently provide an intact viral particle count. Here, we used flow virometry to provide new insights into the basic characteristics of Moloney MLV.

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.

DDX41 Recognizes RNA/DNA Retroviral Reverse Transcripts and Is Critical for In Vivo Control of Murine Leukemia Virus Infection.

ABSTRACTHost recognition of viral nucleic acids generated during infection leads to the activation of innate immune responses essential for early control of virus. Retrovirus reverse transcription creates numerous potential ligands for cytosolic host sensors that recognize foreign nucleic acids, including single-stranded RNA (ssRNA), RNA/DNA hybrids, and double-stranded DNA (dsDNA). We and others recently showed that the sensors cyclic GMP-AMP synthase (cGAS), DEAD-box helicase 41 (DDX41), and members of the Aim2-like receptor (ALR) family participate in the recognition of retroviral reverse transcripts. However, why multiple sensors might be required and their relative importance in in vivo control of retroviral infection are not known. Here, we show that DDX41 primarily senses the DNA/RNA hybrid generated at the first step of reverse transcription, while cGAS recognizes dsDNA generated at the next step. We also show that both DDX41 and cGAS are needed for the antiretroviral innate immune response to murine leukemia virus (MLV) and HIV in primary mouse macrophages and dendritic cells (DCs). Using mice with cell type-specific knockout of the Ddx41 gene, we show that DDX41 sensing in DCs but not macrophages was critical for controlling in vivo MLV infection. This suggests that DCs are essential in vivo targets for infection, as well as for initiating the antiviral response. Our work demonstrates that the innate immune response to retrovirus infection depends on multiple host nucleic acid sensors that recognize different reverse transcription intermediates.

The Effects of Curcuma longa L., Purple Sweet Potato, and Mixtures of the Two on Immunomodulation in C57BL/6J Mice Infected with LP-BM5 Murine Leukemia Retrovirus.

The immune response is stimulated to protect the body from external antigens and is controlled by several types of immune cells. In the present study, the immunomodulatory effects of Curcuma longa L., purple sweet potato, and mixtures of the two (CPM) were investigated in C57BL/6 mice infected with LP-BM5 murine leukemia virus (MuLV). Mice were divided into seven groups as follows: normal control, infected control (LP-BM5 MuLV infection), positive control (LP-BM5 MuLV infection+dietary supplement of red ginseng 300 mg/kg body weight), the original powder of C. longa L. (C; LP-BM5 MuLV infection+dietary supplement of C 189 mg/kg body weight), the original powder of purple sweet potato (P; LP-BM5 MuLV infection+dietary supplement of P 1811 mg/kg body weight), CPM Low (CPL; LP-BM5 MuLV infection+CPM 2 g/kg body weight), and CPM High (CPH; LP-BM5 MuLV infection+CPM 5 g/kg body weight). Dietary supplementation lasted for 12 weeks. Dietary supplementation of CPM inhibited LP-BM5 MuLV-induced lymphadenopathy and splenomegaly and inhibited reduction of messenger RNA (mRNA) expression of major histocompatibility complex (MHC) I and II. Moreover, CPM reduced the decrease in T- and B cell proliferation, reduced the population of CD4(+)/CD8(+) T cells, and remedied the unbalanced production of T helper-1 (Th1)/T helper-2 (Th2) cytokines in LP-BM5 MuLV-infected mice. In addition, CPM inhibited reduction of phagocytosis in peritoneal macrophages and decreased serum levels of immunoglobulin A (IgA), immunoglobulin E (IgE), and immunoglobulin G (IgG). These results suggest that CPM had a positive effect on immunomodulation in C57BL/6 mice induced by LP-BM5 leukemia retrovirus infection.

Functional Evidence of the Involvement of the Dynein Light Chain DYNLRB2 in Murine Leukemia Virus Infection.

How murine leukemia virus (MLV) travels from the cell membrane to the nucleus and the mechanism for nuclear entry of MLV DNA in dividing cells still remain unclear. It seems likely that the MLV preintegration complex (PIC) interacts with cellular proteins to perform these tasks. We recently published that the microtubule motor cytoplasmic dynein complex and its regulator proteins interact with the MLV PIC at early times of infection, suggesting a functional interaction between the incoming viral particles, the dynein complex, and dynein regulators. To better understand the role of the dynein complex in MLV infection, we performed short hairpin RNA (shRNA) screening of the dynein light chains on MLV infection. We found that silencing of a specific light chain of the cytoplasmic dynein complex, DYNLRB2, reduced the efficiency of infection by MLV reporter viruses without affecting HIV-1 infection. Furthermore, the overexpression of DYNLRB2 increased infection by MLV. We conclude that the DYNLRB2 light chain of the cytoplasmic dynein complex is an important and specific piece of the host machinery needed for MLV infection.IMPORTANCE Retroviruses must reach the chromatin of their host to integrate their viral DNA, but first they must get into the nucleus. The cytoplasm is a crowded environment in which simple diffusion is slow, and thus viruses utilize retrograde transport along the microtubule network, mediated by the dynein complex. Different viruses use different components of this multisubunit complex. We have found that murine leukemia virus (MLV) associates functionally and specifically with the dynein light chain DYNLRB2, which is required for infection. Our study provides more insight into the molecular requirements for retrograde transport of the MLV preintegration complex and demonstrates, for the first time, a role for DYNLRB2 in viral infection.

Latent murine leukemia virus infection characterized by the release of non-infectious virions

Clonal cell lines derived from cultures infected with a polytropic MuLV release vastly different levels of infectious virions ranging from undetectable to very high. Low producing clones release an overwhelming proportion of non-infectious virions containing retroviral RNA but deficient in the Env protein. Non-infectious virion production is not due to an inability of the cells to support infectious MuLV production or to an inherent replicative defectiveness of the proviruses. Reinfection of the lowest producing lines with the polytropic or an ecotropic MuLV results in enormous increases in the specific infectivity of the released virions. This indicates a reversible state of retroviral latency characterized by the release of non-infectious virions that is likely the result of insufficient levels of Env protein required for infectivity. The latency state described here may have important roles in in vivo retroviral infections including alterations of the immune response and the production of defective interfering particles.

Endogenous retroviruses mobilized during friend murine leukemia virus infection

We have demonstrated in a mouse model that infection with a retrovirus can lead not only to the generation of recombinants between exogenous and endogenous gammaretrovirus, but also to the mobilization of endogenous proviruses by pseudotyping entire polytropic proviral transcripts and facilitating their infectious spread to new cells. However, the frequency of this occurrence, the kinetics, and the identity of mobilized endogenous proviruses was unclear. Here we find that these mobilized transcripts are detected after only one day of infection. They predominate over recombinant polytropic viruses early in infection, persist throughout the course of disease and are comprised of multiple different polytropic proviruses. Other endogenous retroviral elements such as intracisternal A particles (IAPs) were not detected. The integration of the endogenous transcripts into new cells could result in loss of transcriptional control and elevated expression which may facilitate pathogenesis, perhaps by contributing to the generation of polytropic recombinant viruses.

Effects of Standardized Eriobotrya japonica Extract in LP-BM5 Murine Leukemia Viruses-Induced Murine Immunodeficiency Syndrome

ABSTRACTFolk medicine has long employed leaves from Eriobotrya japonica Lindl. (Rosaceae) (LEJ) as relieving many diseases including chronic bronchitis and high fever. In this study, we investigated the immunomodulatory effects of leaves from LEJ water extracts (LEJE) in LP-BM5 murine leukemia viruses (MuLV)-induced immune-deficient animal model. Dietary supplementation of LEJE (100, 300, 500 mg/kg) began on the day of LP-BM5 MuLV infection and continued for 12 weeks. Dietary supplementation of LEJE inhibited LP-BM5 MuLV-induced splenomegaly and lymphadenopathy. Moreover, LEJE attenuated reductions of T- and B-cell proliferation and Th1/Th2 cytokine imbalance in LP-BM5. We found that dietary supplements of LEJE suppressed the hypergammaglobulinemia by ameliorating LP-BM5 MuLV infection-induced B-cell dysfunction and production of pro-inflammatory cytokines. We suggest that Eriobotrya japonica may have beneficial immunomodulatory effects, improving the balance of Th1/Th2 cytokines and anti-inflammatory effects.

Ecotropic Murine Leukemia Virus Infection of Glial Progenitors Interferes with Oligodendrocyte Differentiation: Implications for Neurovirulence.

Certain murine leukemia viruses (MLVs) are capable of inducing fatal progressive spongiform motor neuron disease in mice that is largely mediated by viral Env glycoprotein expression within central nervous system (CNS) glia. While the etiologic mechanisms and the glial subtypes involved remain unresolved, infection of NG2 glia was recently observed to correlate spatially and temporally with altered neuronal physiology and spongiogenesis. Since one role of NG2 cells is to serve as oligodendrocyte (OL) progenitor cells (OPCs), we examined here whether their infection by neurovirulent (FrCasE) or nonneurovirulent (Fr57E) ecotropic MLVs influenced their viability and/or differentiation. Here, we demonstrate that OPCs, but not OLs, are major CNS targets of both FrCasE and Fr57E. We also show that MLV infection of neural progenitor cells (NPCs) in culture did not affect survival, proliferation, or OPC progenitor marker expression but suppressed certain glial differentiation markers. Assessment of glial differentiation in vivo using transplanted transgenic NPCs showed that, while MLVs did not affect cellular engraftment or survival, they did inhibit OL differentiation, irrespective of MLV neurovirulence. In addition, in chimeric brains, where FrCasE-infected NPC transplants caused neurodegeneration, the transplanted NPCs proliferated. These results suggest that MLV infection is not directly cytotoxic to OPCs but rather acts to interfere with OL differentiation. Since both FrCasE and Fr57E viruses restrict OL differentiation but only FrCasE induces overt neurodegeneration, restriction of OL maturation alone cannot account for neuropathogenesis. Instead neurodegeneration may involve a two-hit scenario where interference with OPC differentiation combined with glial Env-induced neuronal hyperexcitability precipitates disease. A variety of human and animal retroviruses are capable of causing central nervous system (CNS) neurodegeneration manifested as motor and cognitive deficits. These retroviruses infect a variety of CNS cell types; however, the specific role each cell type plays in neuropathogenesis remains to be established. The NG2 glia, whose CNS functions are only now emerging, are a newly appreciated viral target in murine leukemia virus (MLV)-induced neurodegeneration. Since one role of NG2 glia is that of oligodendrocyte progenitor cells (OPCs), we investigated here whether their infection by the neurovirulent MLV FrCasE contributed to neurodegeneration by affecting OPC viability and/or development. Our results show that both neurovirulent and nonneurovirulent MLVs interfere with oligodendrocyte differentiation. Thus, NG2 glial infection could contribute to neurodegeneration by preventing myelin formation and/or repair and by suspending OPCs in a state of persistent susceptibility to excitotoxic insult mediated by neurovirulent virus effects on other glial subtypes.

Dynamics and restriction of murine leukemia virus cores in mitotic and interphase cells

BackgroundMurine leukemia viruses (MLVs) naturally infect unsynchronized T and B lymphocytes, thus, the incoming virus encounters both interphase and mitotic cells. While it is well accepted that MLV requires cell division to complete its replication cycle, it is not known if ab initio infection of mitotic cells can result in productive infection. This question is highly relevant since the milieu of mitotic cells is markedly different from this of interphase cells; e.g. lacking radial microtubule network and intact nuclear envelope. To follow MLV infection in mitotic and interphase cells in real-time, we employed our recently developed infectious MLV particles with labeled cores, cellular models expressing fluorescence markers of different intracellular compartments and protocols for reversible mitotic arrest of MLV-susceptible cells.ResultsMulti-wavelength live cell imaging was employed to simultaneously visualize GFP-labeled MLV cores, DiD-labeled viral or cellular membranes, and fluorescently-labeled microtubules or chromosomes. Cells were imaged either at interphase or upon mitotic arrest with microtubule poisons. Analysis of virus localization and trajectories revealed entry by endocytosis at interphase and mitosis, and correlation between viral mobility parameters and presence or absence of polymerized interphase microtubules. The success of infection of viruses that entered cells in mitosis was evidenced by their ability to reverse transcribe, their targeting to condensed chromosomes in the absence of radial microtubule network, and gene expression upon exit from mitosis. Comparison of infection by N, B or NB -tropic viruses in interphase and mitotic human cells revealed reduced restriction of the N-tropic virus, for infection initiated in mitosis.ConclusionsThe milieu of the mitotic cells supports all necessary requirements for early stages of MLV infection. Such milieu is suboptimal for restriction of N-tropic viruses, most likely by TRIM5α.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-015-0220-2) contains supplementary material, which is available to authorized users.

Phosphorylation of mouse SAMHD1 regulates its restriction of human immunodeficiency virus type 1 infection, but not murine leukemia virus infection

Human SAMHD1 (hSAMHD1) restricts HIV-1 infection in non-dividing cells by depleting intracellular dNTPs to limit viral reverse transcription. Phosphorylation of hSAMHD1 at threonine (T) 592 by cyclin-dependent kinase (CDK) 1 and CDK2 negatively regulates HIV-1 restriction. Mouse SAMHD1 (mSAMHD1) restricts HIV-1 infection in non-dividing cells, but whether its phosphorylation regulates retroviral restriction is unknown. Here we identified six phospho-sites of mSAMHD1, including T634 that is homologous to T592 of hSAMHD1 and phosphorylated by CDK1 and CDK2. We found that wild-type (WT) mSAMHD1 and a phospho-ablative mutant, but not a phospho-mimetic mutant, restricted HIV-1 infection in differentiated U937 cells. Murine leukemia virus (MLV) infection of dividing NIH3T3 cells was modestly restricted by mSAMHD1 WT and phospho-mutants, but not by a dNTPase-defective mutant. Our results suggest that phosphorylation of mSAMHD1 at T634 by CDK1/2 negatively regulates its HIV-1 restriction in differentiated cells, but does not affect its MLV restriction in dividing cells.

Mouse Siglec-1 Mediates trans-Infection of Surface-bound Murine Leukemia Virus in a Sialic Acid N-Acyl Side Chain-dependent Manner

Siglec-1 (sialoadhesin, CD169) is a surface receptor on human cells that mediates trans-enhancement of HIV-1 infection through recognition of sialic acid moieties in virus membrane gangliosides. Here, we demonstrate that mouse Siglec-1, expressed on the surface of primary macrophages in an interferon-α-responsive manner, captures murine leukemia virus (MLV) particles and mediates their transfer to proliferating lymphocytes. The MLV infection of primary B-cells was markedly more efficient than that of primary T-cells. The major structural protein of MLV particles, Gag, frequently co-localized with Siglec-1, and trans-infection, primarily of surface-bound MLV particles, efficiently occurred. To explore the role of sialic acid for MLV trans-infection at a submolecular level, we analyzed the potential of six sialic acid precursor analogs to modulate the sialylated ganglioside-dependent interaction of MLV particles with Siglec-1. Biosynthetically engineered sialic acids were detected in both the glycolipid and glycoprotein fractions of MLV producer cells. MLV released from cells carrying N-acyl-modified sialic acids displayed strikingly different capacities for Siglec-1-mediated capture and trans-infection; N-butanoyl, N-isobutanoyl, N-glycolyl, or N-pentanoyl side chain modifications resulted in up to 92 and 80% reduction of virus particle capture and trans-infection, respectively, whereas N-propanoyl or N-cyclopropylcarbamyl side chains had no effect. In agreement with these functional analyses, molecular modeling indicated reduced binding affinities for non-functional N-acyl modifications. Thus, Siglec-1 is a key receptor for macrophage/lymphocyte trans-infection of surface-bound virions, and the N-acyl side chain of sialic acid is a critical determinant for the Siglec-1/MLV interaction.

Immunomodulatory and Antioxidant Effects of Purple Sweet Potato Extract in LP-BM5 Murine Leukemia Virus-Induced Murine Acquired Immune Deficiency Syndrome.

The immunomodulatory effects of a dietary supplement of purple sweet potato extract (PSPE) in LP-BM5 murine leukemia virus (MuLV)-induced immune-deficient mice were investigated. Mice were divided into six groups: normal control, infected control (LP-BM5 MuLV infection), positive control (LP-BM5 MuLV infection+dietary supplement of red ginseng 300 mg/kg), purple sweet potato water extract (PSPWE) (LP-BM5 MuLV infection+dietary supplement of PSPE 300 mg/kg), PSP10EE (LP-BM5 MuLV infection+dietary supplement of 10% ethanol PSPE 300 mg/kg), and PSP80EE (LP-BM5 MuLV infection+dietary supplement of 80% ethanol PSPE 300 mg/kg). Dietary supplementation began on the day of LP-BM5 MuLV infection and continued for 12 weeks. Dietary supplementation of PSPE inhibited LP-BM5 MuLV-induced splenomegaly and lymphadenopathy and attenuated the suppression of T- and B-cell proliferation and T helper 1/T helper 2 cytokine imbalance in LP-BM5 MuLV-infected mice. Dietary supplement of PSPE increased the activity of the antioxidant enzymes, superoxide dismutase and glutathione peroxidase. The data suggest that PSPE may ameliorate immune dysfunction due to LP-BM5 MuLV infection by modulating antioxidant defense systems.

High-throughput RNA sequencing-based virome analysis of 50 lymphoma cell lines from the Cancer Cell Line Encyclopedia project.

Using high-throughput RNA sequencing data from 50 common lymphoma cell culture models from the Cancer Cell Line Encyclopedia project, we performed an unbiased global interrogation for the presence of a panel of 740 viruses and strains known to infect human and other mammalian cells. This led to the findings of previously identified infections by Epstein-Barr virus (EBV), Kaposi's sarcoma herpesvirus (KSHV), and human T-lymphotropic virus type 1 (HTLV-1). In addition, we also found a previously unreported infection of one cell line (DEL) with a murine leukemia virus. High expression of murine leukemia virus (MuLV) transcripts was observed in DEL cells, and we identified four transcriptionally active integration sites, one being in the TNFRSF6B gene. We also found low levels of MuLV reads in a number of other cell lines and provided evidence suggesting cross-contamination during sequencing. Analysis of HTLV-1 integrations in two cell lines, HuT 102 and MJ, identified 14 and 66 transcriptionally active integration sites with potentially activating integrations in immune regulatory genes, including interleukin-15 (IL-15), IL-6ST, STAT5B, HIVEP1, and IL-9R. Although KSHV and EBV do not typically integrate into the genome, we investigated a previously identified integration of EBV into the BACH2 locus in Raji cells. This analysis identified a BACH2 disruption mechanism involving splice donor sequestration. Through viral gene expression analysis, we detected expression of stable intronic RNAs from the EBV BamHI W repeats that may be part of long transcripts spanning the repeat region. We also observed transcripts at the EBV vIL-10 locus exclusively in the Hodgkin's lymphoma cell line, Hs 611.T, the expression of which were uncoupled from other lytic genes. Assessment of the KSHV viral transcriptome in BCP-1 cells showed expression of the viral immune regulators, K2/vIL-6, K4/vIL-8-like vCCL1, and K5/E2-ubiquitin ligase 1 that was significantly higher than expression of the latency-associated nuclear antigen. Together, this investigation sheds light into the virus composition across these lymphoma model systems and provides insights into common viral mechanistic principles. Viruses cause cancer in humans. In lymphomas the Epstein-Barr virus (EBV), Kaposi's sarcoma herpesvirus (KSHV) and human T-lymphotropic virus type 1 are major contributors to oncogenesis. We assessed virus-host interactions using a high throughput sequencing method that facilitates the discovery of new virus-host associations and the investigation into how the viruses alter their host environment. We found a previously unknown murine leukemia virus infection in one cell line. We identified cellular genes, including cytokine regulators, that are disrupted by virus integration, and we determined mechanisms through which virus integration causes deregulation of cellular gene expression. Investigation into the KSHV transcriptome in the BCP-1 cell line revealed high-level expression of immune signaling genes. EBV transcriptome analysis showed expression of vIL-10 transcripts in a Hodgkin's lymphoma that was uncoupled from lytic genes. These findings illustrate unique mechanisms of viral gene regulation and to the importance of virus-mediated host immune signaling in lymphomas.