scholarly journals Retroviral Restriction Factors Fv1 and TRIM5α Act Independently and Can Compete for Incoming Virus before Reverse Transcription

2006 ◽  
Vol 80 (5) ◽  
pp. 2100-2105 ◽  
Author(s):  
Luca D. Passerini ◽  
Zuzana Keckesova ◽  
Greg J. Towers
Keyword(s):  
Life Cycle ◽  
Reverse Transcription ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Viral Life Cycle ◽  
Human Cells ◽  
Restriction Factors ◽  
Retroviral Infection ◽  
The Relationship ◽  
Murine Leukemia

ABSTRACT The restriction factors Fv1 and TRIM5α provide dominant blocks to retroviral infection, targeting incoming capsids at a postentry, preintegration step. They both restrict N-tropic murine leukemia virus with similar specificity yet act at different points in the viral life cycle. TRIM5α-restricted virus is usually unable to reverse transcribe, whereas Fv1-restricted virus reverse transcribes normally. Here we investigate the relationship between these two restriction factors by expressing Fv1 alleles in human cells. We demonstrate that Fv1 is able to compete with TRIM5α for virus before reverse transcription. In human cells expressing Fv1b, N-tropic restricted virus becomes less infectious but reverse transcribes more efficiently, indicating competition between the two antiviral molecules and protection of the virus from TRIM5α by Fv1. Our findings suggest that, like TRIM5α, Fv1 interacts with virus before reverse transcription, but the consequences of this interaction are not realized until a later stage of the life cycle. We also demonstrate that Fv1 is functionally independent of TRIM5α when expressed in human cells.

scholarly journals Characterizationof Murine Leukemia Virus Restriction inMammals

2003 ◽  
Vol 77 (24) ◽  
pp. 13403-13406 ◽  
Author(s):  
Caroline Besnier ◽  
Laura Ylinen ◽  
Benjamin Strange ◽  
Adrian Lister ◽  
Yasuhiro Takeuchi ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Human Cells ◽  
Restriction Factor ◽  
Human Host ◽  
Murine Leukemia ◽  
In Cells

ABSTRACT Restriction of murine leukemia virus (MLV) was examined in cells from a range of mammals. All nonmurine restrictions were saturable blocks to N-tropic MLV infection, and several were prior to reverse transcription. We demonstrate restriction in cells from bat and show that if we express the murine restriction factor Fv1 in human cells, then Fv1, not the human host, defines the stage at which infection is blocked.

scholarly journals High-Throughput, Library-Based Selection of a Murine Leukemia Virus Variant To Infect Nondividing Cells

2006 ◽  
Vol 80 (18) ◽  
pp. 8981-8988 ◽  
Author(s):  
Julie H. Yu ◽  
David V. Schaffer
Keyword(s):  
Life Cycle ◽  
High Throughput ◽  
Murine Leukemia Virus ◽  
High Titer ◽  
Leukemia Virus ◽  
Retroviral Vectors ◽  
Viral Life Cycle ◽  
Nuclear Localization Signals ◽  
Murine Leukemia ◽  
Selection Of

ABSTRACT Gammaretroviruses, such as murine leukemia virus (MLV), are functionally distinguished from lentiviruses, such as human immunodeficiency virus, by their inability to infect nondividing cells. Attempts to engineer this property into MLV have been hindered by an incomplete understanding of early events in the viral life cycle. We utilized a transposon-based method to generate saturated peptide insertion libraries of MLV gag-pol variants with nuclear localization signals randomly incorporated throughout these overlapping genes. High-throughput selection of the libraries via iterative retroviral infection of nondividing cells led to the identification of a novel variant that successfully transduced growth-arrested cells. Vector packaging by cotransfection of the gag-pol.NLS variant with wild-type gag-pol produced high-titer virions capable of infecting neurons in vitro and in vivo. The capacity of mutant virions to transduce nondividing cells could help to elucidate incompletely understood mechanisms of the viral life cycle and greatly broaden the gene therapy applications of retroviral vectors. Furthermore, the ability to engineer key intracellular viral infection steps has potential implications for the understanding, design, and control of other postentry events. Finally, this method of library generation and selection for a desired phenotype directly in a mammalian system can be readily expanded to address other challenges in protein engineering.

scholarly journals Isolation of Cell Lines That Show Novel, Murine Leukemia Virus-Specific Blocks to Early Steps of Retroviral Replication

2005 ◽  
Vol 79 (20) ◽  
pp. 12969-12978 ◽  
Author(s):  
James W. Bruce ◽  
Kenneth A. Bradley ◽  
Paul Ahlquist ◽  
John A. T. Young
Keyword(s):  
Cell Lines ◽  
Reverse Transcription ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Mutant Cell ◽  
Chinese Hamster ◽  
High Volume ◽  
Early Stages ◽  
Retroviral Replication ◽  
Murine Leukemia

ABSTRACT In order to identify cellular proteins required for early stages of retroviral replication, a high volume screening with mammalian somatic cells was performed. Ten pools of chemically mutagenized Chinese hamster ovary (CHO-K1) cells were challenged with a murine leukemia virus (MLV) vector pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-G), and cells that failed to be transduced were enriched by cell sorting. Each pool yielded a clonally derived cell line with a 5-fold or greater resistance to virus infection, and five cell lines exhibited a >50-fold resistance. These five cell lines were efficiently infected by a human immunodeficiency virus vector pseudotyped with VSV-G. When engineered to express the TVA receptor for subgroup A avian sarcoma and leukosis virus (ASLV-A), the five cell lines were resistant to infection with a MLV vector pseudotyped with the ASLV-A envelope protein but were fully susceptible to infection with an ASLV-A vector. Thus, the defect in these cells resides after virus-cell membrane fusion and, unlike those in other mutant cell lines that have been described, is specific for the MLV core. To identify the specific stages of MLV infection that are impaired in the resistant cell lines, real-time quantitative PCR analyses were employed and two phenotypic groups were identified. Viral infection of three cell lines was restricted before reverse transcription; in the other two cell lines, it was blocked after reverse transcription, nuclear localization, and two-long terminal repeat circle formation but before integration. These data provide genetic evidence that at least two distinct intracellular gene products are required specifically for MLV infection. These cell lines are important tools for the biochemical and genetic analysis of early stages in retrovirus infection.

scholarly journals Mutational Analysis of the N-Terminal Domain of Moloney Murine Leukemia Virus Capsid Protein

2007 ◽  
Vol 81 (22) ◽  
pp. 12337-12347 ◽  
Author(s):  
Marcy R. Auerbach ◽  
Kristy R. Brown ◽  
Ila R. Singh
Keyword(s):  
Murine Leukemia Virus ◽  
Mutational Analysis ◽  
Leukemia Virus ◽  
Host Protein ◽  
Moloney Murine Leukemia Virus ◽  
Structural Defects ◽  
Retroviral Infection ◽  
Functional Region ◽  
Terminal Domain ◽  
Murine Leukemia

ABSTRACT Retroviral capsid (CA) proteins contain a structurally conserved N-terminal domain (NTD) consisting of a β-hairpin and six to seven α-helices. To examine the role of this domain in Moloney murine leukemia virus (MoMLV) replication, we analyzed 18 insertional mutations in this region. All mutants were noninfectious. Based on the results of this analysis and our previous studies on additional mutations in this domain, we were able to divide the NTD of MoMLV CA into three functional regions. The first functional region included the region near the N terminus that forms the β-hairpin and was shown to control normal maturation of virions. The second region included the helix 4/5 loop and was essential for the formation of spherical cores. The third region encompassed most of the NTD except for the above loop. Mutants of this region assembled imperfect cores, as seen by detailed electron microscopy analyses, yet the resulting particles were efficiently released from cells. The mutants were defective at a stage immediately following entry of the core into cells. Despite possessing functional reverse transcriptase machinery, these mutant virions did not initiate reverse transcription in cells. This block could be due to structural defects in the assembling core or failure of an essential host protein to interact with the mutant CA protein, both of which may prevent correct disassembly upon entry of the virus into cells. Future studies are needed to understand the mechanism of these blocks and to target these regions pharmacologically to inhibit retroviral infection at additional stages.

scholarly journals Role of Murine Leukemia Virus Reverse Transcriptase Deoxyribonucleoside Triphosphate-Binding Site in Retroviral Replication and In Vivo Fidelity

2000 ◽  
Vol 74 (22) ◽  
pp. 10349-10358 ◽  
Author(s):  
Elias K. Halvas ◽  
Evguenia S. Svarovskaia ◽  
Vinay K. Pathak
Keyword(s):  
Amino Acids ◽  
Viral Replication ◽  
Reverse Transcriptase ◽  
Binding Site ◽  
Reverse Transcription ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Deoxyribonucleoside Triphosphate ◽  
Murine Leukemia

ABSTRACT Retroviral populations exhibit a high evolutionary potential, giving rise to extensive genetic variation. Error-prone DNA synthesis catalyzed by reverse transcriptase (RT) generates variation in retroviral populations. Structural features within RTs are likely to contribute to the high rate of errors that occur during reverse transcription. We sought to determine whether amino acids within murine leukemia virus (MLV) RT that contact the deoxyribonucleoside triphosphate (dNTP) substrate are important for in vivo fidelity of reverse transcription. We utilized the previously described ANGIE P encapsidating cell line, which expresses the amphotropic MLV envelope and a retroviral vector (pGA-1). pGA-1 expresses the bacterial β-galactosidase gene (lacZ), which serves as a reporter of mutations. Extensive mutagenesis was performed on residues likely to interact with the dNTP substrate, and the effects of these mutations on the fidelity of reverse transcription were determined. As expected, most substitution mutations of amino acids that directly interact with the dNTP substrate significantly reduced viral titers (>10,000-fold), indicating that these residues played a critical role in catalysis and viral replication. However, the D153A and A154S substitutions, which are predicted to affect the interactions with the triphosphate, resulted in statistically significant increases in the mutation rate. In addition, the conservative substitution F155W, which may affect interactions with the base and the ribose, increased the mutation rate 2.8-fold. Substitutions of residues in the vicinity of the dNTP-binding site also resulted in statistically significant decreases in fidelity (1.3- to 2.4-fold). These results suggest that mutations of residues that contact the substrate dNTP can affect viral replication as well as alter the fidelity of reverse transcription.

Functional analysis of reverse transcription by a frameshift pol mutant of murine leukemia virus

Virology ◽  
1985 ◽  
Vol 146 (1) ◽  
pp. 146-152 ◽  
Author(s):  
Lesley I. Messer ◽  
Kathleen M. Currey ◽  
Blanche J. O'Neill ◽  
Jacob V. Maizel ◽  
Judith G. Levin ◽  
...  
Keyword(s):  
Functional Analysis ◽  
Reverse Transcription ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Murine Leukemia
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