Gv1, a Zinc Finger Gene Controlling Endogenous MLV Expression.

The genomes of inbred mice harbor around 50 endogenous murine leukemia virus (MLV) loci, although the specific complement varies greatly between strains. The Gv1 locus is known to control the transcription of endogenous MLVs and to be the dominant determinant of cell-surface presentation of MLV envelope, the GIX antigen. Here, we identify a single Krüppel-associated box zinc finger protein (ZFP) gene, Zfp998, as Gv1 and show it to be necessary and sufficient to determine the GIX+ phenotype. By long-read sequencing of bacterial artificial chromosome clones from 129 mice, the prototypic GIX+ strain, we reveal the source of sufficiency and deficiency as splice-acceptor variations and highlight the varying origins of the chromosomal region encompassing Gv1. Zfp998 becomes the second identified ZFP gene responsible for epigenetic suppression of endogenous MLVs in mice and further highlights the prominent role of this gene family in control of endogenous retroviruses.

Continuous low pH viral inactivation: Operation and scaling strategy informs viral clearance study.

A continuous viral inactivation (CVI) tubular reactor was designed for low pH viral inactivation within a continuous downstream system across multiple scales of operation. The reactors were designed to provide a minimum residence time of >60 min. The efficacy of this tubular reactor was tested with xenotropic murine leukemia virus (X-MuLV) through pulse injection experiments. It was determined that the minimum residence time of the small-scale reactor design, when operated at the target process flow rate, occurred between 63 and 67 min. Inactivation kinetics were compared between continuous operation and standard batch practices using three monoclonal antibodies. The quantification of the virus log reduction values (LRV) was similar between the two modes of operation and most of the acid-treated samples had virus concentrations below the limit of detection. However, residual infectivity was still present in the endpoint batch samples of two experiments while the continuous samples always remained below the limit of detection. This provides the foundation for leveraging a standard batch-based model to quantify the LRV for a CVI unit operation.

HIV-1 Nef promotes infection by excluding SERINC5 from virion incorporation.

HIV-1 Nef, a protein important for the development of AIDS, has well-characterized effects on host membrane trafficking and receptor downregulation. By an unidentified mechanism, Nef increases the intrinsic infectivity of HIV-1 virions in a host-cell-dependent manner. Here we identify the host transmembrane protein SERINC5, and to a lesser extent SERINC3, as a potent inhibitor of HIV-1 particle infectivity that is counteracted by Nef. SERINC5 localizes to the plasma membrane, where it is efficiently incorporated into budding HIV-1 virions and impairs subsequent virion penetration of susceptible target cells. Nef redirects SERINC5 to a Rab7-positive endosomal compartment and thereby excludes it from HIV-1 particles. The ability to counteract SERINC5 was conserved in Nef encoded by diverse primate immunodeficiency viruses, as well as in the structurally unrelated glycosylated Gag from murine leukaemia virus. These examples of functional conservation and convergent evolution emphasize the fundamental importance of SERINC5 as a potent anti-retroviral factor.

Truly continuous low pH viral inactivation for biopharmaceutical process integration.

Continuous virus inactivation (VI) has received little attention in the efforts to realize fully continuous biomanufacturing in the future. Implementation of continuous VI must assure a specific minimum incubation time, typically 60 min. To guarantee the minimum incubation time, we implemented a packed bed continuous viral inactivation reactor (CVIR) with narrow residence time distribution (RTD) for low pH incubation. We show that the RTD does not broaden significantly over a wide range of linear flow velocities-which highlights the flexibility and robustness of the design. Prolonged exposure to acidic pH has no impact on bed stability, assuring constant RTD throughout long term operation. The suitability of the packed bed CVIR for low pH inactivation is shown with two industry-standard model viruses, that is xenotropic murine leukemia virus and pseudorabies virus. Controls at neutral pH showed no system-induced VI. At low pH, significant VI is observed, even after only 15 min. Based on the low pH inactivation kinetics, the continuous process is equivalent to traditional batch operation. This study establishes a concept for continuous low pH inactivation and, together with previous reports, highlights the versatility of the packed bed reactor for continuous VI, regardless of the inactivation method.

NXF1 and CRM1 nuclear export pathways orchestrate nuclear export, translation and packaging of murine leukaemia retrovirus unspliced RNA.

Cellular mRNAs are exported from the nucleus as fully spliced RNAs. Proofreading mechanisms eliminate unprocessed and irregular pre-mRNAs to control the quality of gene expression. Retroviruses need to export partially spliced and unspliced full-length RNAs to the cytoplasm where they serve as templates for protein synthesis and/or as encapsidated RNA in progeny viruses. Genetically complex retroviruses such as HIV-1 use Rev-equivalent proteins to export intron-retaining RNA from the nucleus using the cellular CRM1-driven nuclear export machinery. By contrast, genetically simpler retroviruses such as murine leukaemia virus (MLV) recruit the NXF1 RNA export machinery. In this study, we reveal for the first time that MLV hijacks both NXF1 and CRM1-dependent pathways to achieve optimal replication capacity. The CRM1-pathway marks the MLV full-length RNA (FL RNA) for packaging, while NXF1-driven nuclear export is coupled to translation. Thus, the cytoplasmic function of the viral RNA is determined early in the nucleus. Depending on the nature of ribonucleoprotein complex formed on FL RNA cargo in the nucleus, the FL RNA will be addressed to the translation machinery sites or to the virus-assembly sites at the plasma membrane.

Equine Myxovirus Resistance Protein 2 Restricts Lentiviral Replication by Blocking Nuclear Uptake of Capsid Protein.

Human myxovirus resistance protein 2 (huMxB) has been shown to be a determinant type I interferon (IFN)-induced host factor involved in the inhibition of human immunodeficiency virus type 1 (HIV-1) as well as many other primate lentiviruses. This blocking occurs after the reverse transcription of viral RNA and ahead of integration into the host DNA, which is closely connected to the ability of the protein to bind the viral capsid. To date, Mx2s derived from nonprimate animals have shown no capacity for HIV-1 suppression. In this study, we examined the restrictive effect of equine Mx2 (eqMx2) on both equine infectious anemia virus (EIAV) and HIV-1 and investigated possible mechanisms for its specific function. We demonstrated that IFN-α/ß upregulates the expression of eqMx2 in equine monocyte-derived macrophages (eMDMs). The overexpression of eqMx2 significantly suppresses the replication of EIAV, HIV-1, and simian immunodeficiency viruses (SIVs) but not that of murine leukemia virus (MLV). The knockdown of eqMx2 transcription weakens the inhibition of EIAV replication by type I interferon. Interestingly, data from immunofluorescence assays suggest that the subcellular localization of eqMx2 changes following virus infection, from being dispersed in the cytoplasm to being accumulated at the nuclear envelope. Furthermore, eqMx2 blocks the nuclear uptake of the proviral genome by binding to the viral capsid. The N-terminally truncated mutant of eqMx2 lost the ability to bind the viral capsid as well as the restriction effect for lentiviruses. These results improve our understanding of the Mx2 protein in nonprimate animals.IMPORTANCE Previous research has shown that the antiviral ability of Mx2s is confined to primates, particularly humans. EIAV has been shown to be insensitive to restriction by human MxB. Here, we describe the function of equine Mx2. This protein plays an important role in the suppression of EIAV, HIV-1, and SIVs. The antiviral activity of eqMx2 depends on its subcellular location as well as its capsid binding capacity. Our results showed that following viral infection, eqMx2 changes its original cytoplasmic location and accumulates at the nuclear envelope, where it binds to the viral capsid and blocks the nuclear entry of reverse-transcribed proviral DNAs. In contrast, huMxB does not bind to the EIAV capsid and shows no EIAV restriction effect. These studies expand our understanding of the function of the equine Mx2 protein.

Virus study for continuous low pH viral inactivation inside a coiled flow inverter.

Continuous processing for the production of monoclonal antibodies (mAb) gains more and more importance. Several solutions exist for all the necessary production steps, leading to the possibility to build fully continuous processes. Low pH viral inactivation is a part of the standard platform process for mAb production. Consequently, Klutz et al. introduced the coiled flow inverter (CFI) as a tool for continuous low pH viral inactivation. Besides theoretical calculations of viral reduction, no viral clearance study has been presented so far. In addition, the validation of continuous viral clearance is often neglected in the already existing studies for continuous processing. This study shows in detail the development and execution of a virus study for continuous low pH viral inactivation inside a CFI. The concept presented is also valid for adaptation to other continuous viral clearance steps. The development of this concept includes the technical rationale for an experimental setup, a valid spiking procedure, and finally a sampling method. The experimental results shown represent a viral study using xenotropic murine leukemia virus as a model virus. Two different protein A (ProtA) chromatography setups with varying pH levels were tested. In addition, one of these setups was tested against a batch experiment utilizing the same process material. The results show that sufficient low pH viral inactivation (decadic logarithm reduction value >4) was achieved in all experiments. Complete viral inactivation took place within the first 14.5 min for both continuous studies and the batch study, hence showing similar results. This study therefore represents a successful virus study concept and experiment for a continuous viral inactivation step. Moreover, it was shown that the transfer from batch results to the continuous process is possible. This is accomplished by the narrow residence time distribution of the CFI, showing how close the setup approaches the ideal plug flow and with that batch operation.

Use of a new RNA next generation sequencing approach for the specific detection of virus infection in cells.

The utilization of the current combination of in vitro, in vivo and PCR assays for the identification of adventitious viruses in production cells has a limited range of detection. While Next Generation Sequencing (NGS) has a broader breadth of detection, it is unable to differentiate sequences from replicating viruses versus background inert sequences. In order to improve NGS specificity, we have designed a new NGS approach which targets subsets of viral RNAs only synthesized during cell infection. In order to evaluate the performance of this approach for detecting low levels of adventitious viruses, we selected two difficult virus/cell systems. This included B95-8 cells persistently infected by Human herpesvirus 4 (HHV-4) and serially diluted into HHV-4 negative Ramos cells and Madin-Darby bovine kidney cells with an early infection produced via a low dose of Bovine viral diarrhea virus. We demonstrated that the sensitivity of our RNA NGS approach was equivalent to targeted PCR with an increased specificity for the detection of viral infection. We were also able to identify a previously undetected Murine Leukemia Virus contaminant in Ramos cells. Based on these results, we conclude that this new RNA NGS approach is suitable for conducting viral safety evaluations of cells.

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.

Resurrection of endogenous retroviruses in antibody-deficient mice.

The mammalian host has developed a long-standing symbiotic relationship with a considerable number of microbial species. These include the microbiota on environmental surfaces, such as the respiratory and gastrointestinal tracts, and also endogenous retroviruses (ERVs), comprising a substantial fraction of the mammalian genome. The long-term consequences for the host of interactions with these microbial species can range from mutualism to parasitism and are not always completely understood. The potential effect of one microbial symbiont on another is even less clear. Here we study the control of ERVs in the commonly used C57BL/6 (B6) mouse strain, which lacks endogenous murine leukaemia viruses (MLVs) able to replicate in murine cells. We demonstrate the spontaneous emergence of fully infectious ecotropic MLV in B6 mice with a range of distinct immune deficiencies affecting antibody production. These recombinant retroviruses establish infection of immunodeficient mouse colonies, and ultimately result in retrovirus-induced lymphomas. Notably, ERV activation in immunodeficient mice is prevented in husbandry conditions associated with reduced or absent intestinal microbiota. Our results shed light onto a previously unappreciated role for immunity in the control of ERVs and provide a potential mechanistic link between immune activation by microbial triggers and a range of pathologies associated with ERVs, including cancer.

Native structure of a retroviral envelope protein and its conformational change upon interaction with the target cell.

Enveloped viruses enter their host cells by membrane fusion. The process of attachment and fusion in retroviruses is mediated by a single viral envelope glycoprotein (Env). Conformational changes of Env in the course of fusion are a focus of intense studies. Here we provide further insight into the changes occurring in retroviral Env during its initial interaction with the cell, employing murine leukemia virus (MLV) as model system. We first determined the structure of both natively membrane anchored MLV Env and MLV Env tagged with YFP in the proline rich region (PRR) by electron cryo tomography (cET) and sub-volume averaging. At a resolution of ∼20Å, native MLV Env presents as a hollow trimer (height ∼85Å, diameter ∼120Å) composed of step-shaped protomers. The major difference to the YFP-tagged protein was in regions outside of the central trimer. Next, we focused on elucidating the changes in MLV Env upon interaction with a host cell. Virus interaction with the plasma membrane occurred over a large surface and Env clustering on the binding site was observed. Sub-volume averaging did yield a low-resolution structure of Env interacting with the cell, which had lost its threefold symmetry and was elongated by ∼35Å in comparison to the unbound protein. This indicates a major rearrangement of Env upon host cell binding. At the site of virus interaction, the otherwise clearly defined bilayer structure of the host cell plasma membrane was much less evident, indicative of integral membrane protein accumulation and/or a change in membrane lipid composition.

Dynein Regulators Are Important for Ecotropic Murine Leukemia Virus Infection.

UNLABELLED: During the early steps of infection, retroviruses must direct the movement of the viral genome into the nucleus to complete their replication cycle. This process is mediated by cellular proteins that interact first with the reverse transcription complex and later with the preintegration complex (PIC), allowing it to reach and enter the nucleus. For simple retroviruses, such as murine leukemia virus (MLV), the identities of the cellular proteins involved in trafficking of the PIC in infection are unknown. To identify cellular proteins that interact with the MLV PIC, we developed a replication-competent MLV in which the integrase protein was tagged with a FLAG epitope. Using a combination of immunoprecipitation and mass spectrometry, we established that the microtubule motor dynein regulator DCTN2/p50/dynamitin interacts with the MLV preintegration complex early in infection, suggesting a direct interaction between the incoming viral particles and the dynein complex regulators. Further experiments showed that RNA interference (RNAi)-mediated silencing of either DCTN2/p50/dynamitin or another dynein regulator, NudEL, profoundly reduced the efficiency of infection by ecotropic, but not amphotropic, MLV reporters. We propose that the cytoplasmic dynein regulators are a critical component of the host machinery needed for infection by the retroviruses entering the cell via the ecotropic envelope pathway. IMPORTANCE: Retroviruses must access the chromatin of host cells to integrate the viral DNA, but before this crucial event, they must reach the nucleus. The movement through the cytoplasm-a crowded environment where diffusion is slow-is thought to utilize retrograde transport along the microtubule network by the dynein complex. Different viruses use different components of this multisubunit complex. We found that the preintegration complex of murine leukemia virus (MLV) interacts with the dynein complex and that regulators of this complex are essential for infection. Our study provides the first insight into the requirements for retrograde transport of the MLV preintegration complex.

From cytopenia to leukemia: the role of Gfi1 and Gfi1b in blood formation.

The DNA-binding zinc finger transcription factors Gfi1 and Gfi1b were discovered more than 20 years ago and are recognized today as major regulators of both early hematopoiesis and hematopoietic stem cells. Both proteins function as transcriptional repressors by recruiting histone-modifying enzymes to promoters and enhancers of target genes. The establishment of Gfi1 and Gfi1b reporter mice made it possible to visualize their cell type-specific expression and to understand their function in hematopoietic lineages. We now know that Gfi1 is primarily important in myeloid and lymphoid differentiation, whereas Gfi1b is crucial for the generation of red blood cells and platelets. Several rare hematologic diseases are associated with acquired or inheritable mutations in the GFI1 and GFI1B genes. Certain patients with severe congenital neutropenia carry mutations in the GFI1 gene that lead to the disruption of the C-terminal zinc finger domains. Other mutations have been found in the GFI1B gene in families with inherited bleeding disorders. In addition, the Gfi1 locus is frequently found to be a proviral integration site in retrovirus-induced lymphomagenesis, and new, emerging data suggest a role of Gfi1 in human leukemia and lymphoma, underlining the role of both factors not only in normal hematopoiesis, but also in a wide spectrum of human blood diseases.

An equilibrium-dependent retroviral mRNA switch regulates translational recoding.

Most retroviruses require translational recoding of a viral messenger RNA stop codon to maintain a precise ratio of structural (Gag) and enzymatic (Pol) proteins during virus assembly. Pol is expressed exclusively as a Gag-Pol fusion either by ribosomal frameshifting or by read-through of the gag stop codon. Both of these mechanisms occur infrequently and only affect 5-10% of translating ribosomes, allowing the virus to maintain the critical Gag to Gag-Pol ratio. Although it is understood that the frequency of the recoding event is regulated by cis RNA motifs, no mechanistic explanation is currently available for how the critical protein ratio is maintained. Here we present the NMR structure of the murine leukaemia virus recoding signal and show that a protonation-dependent switch occurs to induce the active conformation. The equilibrium is such that at physiological pH the active, read-through permissive conformation is populated at approximately 6%: a level that correlates with in vivo protein quantities. The RNA functions by a highly sensitive, chemo-mechanical coupling tuned to ensure an optimal read-through frequency. Similar observations for a frameshifting signal indicate that this novel equilibrium-based mechanism may have a general role in translational recoding.

Genome-wide Profiling Reveals Remarkable Parallels Between Insertion Site Selection Properties of the MLV Retrovirus and the piggyBac Transposon in Primary Human CD4(+) T Cells.

The inherent risks associated with vector insertion in gene therapy need to be carefully assessed. We analyzed the genome-wide distributions of Sleeping Beauty (SB) and piggyBac (PB) transposon insertions as well as MLV retrovirus and HIV lentivirus insertions in human CD4(+) T cells with respect to a panel of 40 chromatin states. The distribution of SB transposon insertions displayed the least deviation from random, while the PB transposon and the MLV retrovirus showed unexpected parallels across all chromatin states. Both MLV and PB insertions are enriched at transcriptional start sites (TSSs) and co-localize with BRD4-associated sites. We demonstrate physical interaction between the PB transposase and bromodomain and extraterminal domain proteins (including BRD4), suggesting convergent evolution of a tethering mechanism that directs integrating genetic elements into TSSs. We detect unequal biases across the four systems with respect to targeting genes whose deregulation has been previously linked to serious adverse events in gene therapy clinical trials. The SB transposon has the highest theoretical chance of targeting a safe harbor locus in the human genome. The data underscore the significance of vector choice to reduce the mutagenic load on cells in clinical applications.

Expression levels of Fv1: effects on retroviral restriction specificities.

BACKGROUND: The mouse protein Fv1 is a factor that can confer resistance to retroviral infection. The two major Fv1 alleles from laboratory mice, Fv1 (n) and Fv1 (b) , restrict infection by different murine leukaemia viruses (MLVs). Fv1(n) restricts B-tropic MLV, but not N-tropic MLV or NB-tropic MLV. In cells expressing Fv1(b) at natural levels, only N-MLV is restricted, however restriction of NB-MLV and partial restriction of B-MLV were observed when recombinant Fv1(b) was expressed from an MLV promoter in Fv1 null Mus dunni tail fibroblast cells. To investigate the relationship between expression level and restriction specificity we have developed new retroviral delivery vectors which allow inducible expression of Fv1, and yet allow sufficient production of fluorescent reporter proteins for analysis in our FACS-based restriction assay. RESULTS: We demonstrated that at concentrations close to the endogenous expression level, Fv1(b) specifically restricts only N-MLV, but restriction of NB-MLV, and to a lesser extent B-MLV, could be gained by increasing the protein level of Fv1(b). By contrast, we found that even when Fv1(n) is expressed at very high levels, no significant inhibition of N-MLV or NB-MLV could be observed. Study of Fv1 mutants using this assay led to the identification of determinants for N/B tropism at an expression level close to that of endogenous Fv1(n) and Fv1(b). We also compared the recently described restriction activities of wild mice Fv1 proteins directed against non-MLV retroviruses when expressed at different levels. Fv1 from M. spretus restricted N-MLV, B-MLV and equine infectious anaemia virus equally even at low concentrations, while Fv1 from M. macedonicus showed even stronger restriction against equine infectious anaemia virus than to N-MLV. Restriction of feline foamy virus by Fv1 of M. caroli occurred at levels equivalent to MLV restriction. CONCLUSIONS: Our data indicate that for some but not all Fv1 proteins, gain of restriction activities could be achieved by increasing the expression level of Fv1. However such a concentration dependent effect is not seen with most Fv1s and cannot explain the recently reported activities against non-MLVs. It will be interesting to examine whether overexpression of other capsid binding restriction factors such as TRIM5α or Mx2 result in novel restriction specificities.

Macropinocytosis is the entry mechanism of amphotropic murine leukemia virus.

UNLABELLED: The entry mechanism of murine amphotropic retrovirus (A-MLV) has not been unambiguously determined. We show here that A-MLV is internalized not by caveolae or other pinocytic mechanisms but by macropinocytosis. Thus, A-MLV infection of mouse embryonic fibroblasts deficient for caveolin or dynamin, and NIH 3T3 cells knocked down for caveolin expression, was unaffected. Conversely, A-MLV infection of NIH 3T3 and HeLa cells was sensitive to amiloride analogues and actin-depolymerizing drugs that interfere with macropinocytosis. Further manipulation of the actin cytoskeleton through conditional expression of dominant positive or negative mutants of Rac1, PAK1, and RhoG, to increase or decrease macropinocytosis, similarly correlated with an augmented or inhibited infection with A-MLV, respectively. The same experimental perturbations affected the infection of viruses that use clathrin-coated-pit endocytosis or other pathways for entry only mildly or not at all. These data agree with immunofluorescence studies and cryo-immunogold labeling for electron microscopy, which demonstrate the presence of A-MLV in protrusion-rich areas of the cell surface and in cortical fluid phase (dextran)-filled macropinosomes, which also account for up to a half of the cellular uptake of the cell surface-binding lectin concanavalin A. We conclude that A-MLV use macropinocytosis as the predominant entry portal into cells. IMPORTANCE: Binding and entry of virus particles into mammalian cells are the first steps of infection. Understanding how pathogens and toxins exploit or divert endocytosis pathways has advanced our understanding of membrane trafficking pathways, which benefits development of new therapeutic schemes and methods of drug delivery. We show here that amphotropic murine leukemia virus (A-MLV) pseudotyped with the amphotropic envelope protein (which expands the host range to many mammalian cells) gains entry into host cells by macropinocytosis. Macropinosomes form as large, fluid-filled vacuoles (up to 10 µm) following the collapse of cell surface protrusions and membrane scission. We used drugs or the introduction of mutant proteins that affect the actin cytoskeleton and cell surface dynamics to show that macropinocytosis and A-MLV infection are correlated, and we provide both light- and electron-microscopic evidence to show the localization of A-MLV in macropinosomes. Finally, we specifically exclude some other potential entry portals, including caveolae, previously suggested to internalize A-MLV.

N-linked glycosylation protects gammaretroviruses against deamination by APOBEC3 proteins.

UNLABELLED: Retroviruses are pathogens with rapid infection cycles that can be a source of disease, genome instability, and tumor development in their hosts. Host intrinsic restriction factors, such as APOBEC3 (A3) proteins, are constitutively expressed and dedicated to interfering with the replication cycle of retroviruses. To survive, propagate, and persist, retroviruses must counteract these restriction factors, often by way of virus genome-encoded accessory proteins. Glycosylated Gag, also called glycosylated Pr80 Gag (gPr80), is a gammaretrovirus genome-encoded protein that inhibits the antiretroviral activity of mouse A3 (mA3). Here we show that gPr80 exerts two distinct inhibitory effects on mA3: one that antagonizes deamination-independent restriction and another one that inhibits its deaminase activity. More specifically, we find that the number of N-glycosylated residues in gPr80 inversely correlates with the sensitivity of a gammaretrovirus to deamination by mouse A3 and also, surprisingly, by human A3G. Finally, our work highlights that retroviruses which have successfully integrated into the mouse germ line generally express a gPr80 with fewer glycosylated sites than exogenous retroviruses. This observation supports the suggestion that modulation of A3 deamination intensity could be a desirable attribute for retroviruses to increase genetic diversification and avoid immune detection. Overall, we present here the first description of how gammaretroviruses employ posttranslational modification to antagonize and modulate the activity of a host genome-encoded retroviral restriction factor. IMPORTANCE: APOBEC3 proteins are host factors that have a major role in protecting humans and other mammals against retroviruses. These enzymes hinder their replication and intensely mutate their DNA, thereby inactivating viral progeny and the spread of infection. Here we describe a newly recognized way in which some retroviruses protect themselves against the mutator activity of APOBEC3 proteins. We show that gammaretroviruses expressing an accessory protein called glycosylated Gag, or gPr80, use the host's posttranslational machinery and, more specifically, N-linked glycosylation as a way to modulate their sensitivity to mutations by APOBEC3 proteins. By carefully controlling the amount of mutations caused by APOBEC3 proteins, gammaretroviruses can find a balance that helps them evolve and persist.

HIV-1 Is Restricted prior to Integration of Viral DNA in Primary Cord-Derived Human CD34+ Cells.

Certain cells have the ability to block retroviral infection at specific stages of the viral cycle by the activities of well-characterized factors and transcriptional silencing machinery. Infection of murine stem cells (MSCs) by the murine leukemia viruses (MLVs) is profoundly blocked postintegration by transcriptional silencing. Here, we show that a dominant point of restriction of HIV-1 in human CD34+ cells is prior to integration of viral DNA and that HIV-1 restriction by human CD34+ cells is fundamentally different from MLV restriction by mouse cells.

An interleukin-1 beta-encoding retrovirus exhibits enhanced replication in vivo.

UNLABELLED: Interleukin-1 beta (IL-1ß) is an inflammatory cytokine that is secreted in response to inflammasome activation by innate microbe-sensing pathways. Although some retroviruses can trigger IL-1ß secretion through the DNA-sensing molecule IFI16, the effect of IL-1ß on the course of infection is unknown. To test whether IL-1ß secretion affects retroviral replication in vivo, I constructed a novel murine leukemia virus strain (FMLV-IL-1ß) that encodes the mature form of IL-1ß. This virus replicated with kinetics similar to that of wild-type virus in tissue culture but caused a dramatically more aggressive infection of both C57BL/6 and BALB/c mice. By 7 days postinfection (PI), mice infected with FMLV-IL-1ß exhibited splenomegaly and viral loads 300-fold higher than those in mice infected with wild-type FMLV. Furthermore, the enlarged spleens of FMLV-IL-1ß-infected mice correlated with a large expansion of Gr-1(+) CD11b(+) myeloid-derived suppressor cells, as well as elevated levels of immune activation. Although FMLV-IL-1ß infection was controlled by C57BL/6 mice by 14 days p.i., FMLV-IL-1ß was able to establish a significant persistent infection and immune activation in BALB/c mice. These results demonstrate that IL-1ß secretion is a powerful positive regulator of retroviral infection and that FMLV-IL-1ß represents a new model of proinflammatory retroviral infection. IMPORTANCE: Interleukin-1 beta (IL-1ß) is an inflammatory cytokine released in response to activation of innate pathogen-sensing pathways during microbial infection. To examine the potential impact of IL-1ß on retroviral replication in vivo, I constructed a novel mouse retrovirus strain (FMLV-IL-1ß) that encodes IL-1ß and promotes abundant IL-1ß secretion from infected cells. This virus replicates with normal kinetics in cultured cells but displays a dramatically enhanced ability to replicate in mice and caused persistent infection and immune activation in the BALB/c strain of mice. These results establish IL-1ß as a positive regulator of retroviral replication and suggest that targeting this pathway may have therapeutic benefits in infections with proinflammatory retroviruses. This virus can also be used to further study the impact of inflammatory pathways on retroviral infection.