scholarly journals Cleavage of the Murine Leukemia Virus Transmembrane Env Protein by Human Immunodeficiency Virus Type 1 Protease: Transdominant Inhibition by Matrix Mutations

1998 ◽  
Vol 72 (12) ◽  
pp. 9621-9627 ◽  
Author(s):  
Rosemary E. Kiernan ◽  
Eric O. Freed
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Wild Type ◽  
Immunodeficiency Virus ◽  
Env Protein ◽  
Hiv 1 ◽  
Murine Leukemia

ABSTRACT We have identified mutations in the human immunodeficiency virus type 1 (HIV-1) matrix protein (MA) which block infectivity of virions pseudotyped with murine leukemia virus (MuLV) envelope (Env) glycoproteins without affecting infectivity conferred by HIV-1 Env or vesicular stomatitis virus G glycoproteins. This inhibition is very potent and displays a strong transdominant effect; infectivity is reduced more than 100-fold when wild-type and mutant molecular clones are cotransfected at a 1:1 ratio. This phenomenon is observed with both ecotropic and amphotropic MuLV Env. The MA mutations do not affect the incorporation of MuLV Env into virions. We demonstrate that in HIV-1 virions pseudotyped with MuLV Env, the HIV-1 protease (PR) efficiently catalyzes the cleavage of the p15(E) transmembrane (TM) protein to p12(E). Immunoprecipitation analysis of pseudotyped virions reveals that the mutant MA blocks this HIV-1 PR-mediated cleavage of MuLV TM. Furthermore, the transdominant inhibition exerted by the mutant MA on wild-type infectivity correlates with the relative level of p15(E) cleavage. Consistent with the hypothesis that abrogation of infectivity imposed by the mutant MA is due to inhibition of p15(E) cleavage, mutant virions are significantly more infectious when pseudotyped with a truncated p12(E) form of MuLV Env. These results indicate that HIV-1 Gag sequences can influence the viral PR-mediated processing of the MuLV TM Env protein p15(E). These findings have implications for the development of HIV-1-based retroviral vectors pseudotyped with MuLV Env, since p15(E) cleavage is essential for activating membrane fusion and virus infectivity.

scholarly journals Evidence that Ecotropic Murine Leukemia Virus Contamination in TZM-bl Cells Does Not Affect the Outcome of Neutralizing Antibody Assays with Human Immunodeficiency Virus Type 1

2009 ◽  
Vol 83 (16) ◽  
pp. 8289-8292 ◽  
Author(s):  
Emily J. Platt ◽  
Miroslawa Bilska ◽  
Susan L. Kozak ◽  
David Kabat ◽  
David C. Montefiori
Keyword(s):  
Human Immunodeficiency Virus ◽  
Cell Line ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Immunodeficiency Virus ◽  
Hiv 1 ◽  
Murine Leukemia

ABSTRACT The TZM-bl cell line that is commonly used to assess neutralizing antibodies against human immunodeficiency virus type 1 (HIV-1) was recently reported to be contaminated with an ecotropic murine leukemia virus (MLV) (Y. Takeuchi, M. O. McClure, and M. Pizzato, J. Virol. 82:12585-12588, 2008), raising questions about the validity of results obtained with this cell line. Here we confirm this observation and show that HIV-1 neutralization assays performed with a variety of serologic reagents in a similar cell line that does not harbor MLV yield results that are equivalent to those obtained in TZM-bl cells. We conclude that MLV contamination has no measurable effect on HIV-1 neutralization when TZM-bl cells are used as targets for infection.

scholarly journals Chimeric Human Immunodeficiency Virus Type 1 Containing Murine Leukemia Virus Matrix Assembles in Murine Cells

2002 ◽  
Vol 76 (1) ◽  
pp. 436-443 ◽  
Author(s):  
Margaret Reed ◽  
Roberto Mariani ◽  
Liana Sheppard ◽  
Katja Pekrun ◽  
Nathaniel R. Landau ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Cyclin T1 ◽  
Immunodeficiency Virus ◽  
Hiv 1 ◽  
Murine Leukemia

ABSTRACT Murine cells do not support efficient assembly and release of human immunodeficiency virus type 1 (HIV-1) virions. HIV-1-infected mouse cells that express transfected human cyclin T1 synthesize abundant Gag precursor polyprotein, but inefficiently assemble and release virions. This assembly defect may result from a failure of the Gag polyprotein precursor to target to the cell membrane. Plasma membrane targeting of the precursor is mediated by the amino-terminal region of polyprotein. To compensate for the assembly block, we substituted the murine leukemia virus matrix coding sequences into an infectious HIV-1 clone. Transfection of murine fibroblasts expressing cyclin T1 with the chimeric proviruses resulted in viruses that were efficiently assembled and released. Chimeric viruses, in which the cytoplasmic tail of the transmembrane subunit, gp41, was truncated to prevent potential interference between the envelope glycoprotein and the heterologous matrix, could infect human and murine cells. They failed to further replicate in the murine cells, but replicated with delayed kinetics in human MT-4 cells. These findings may be useful for establishing a murine model for HIV-1 replication.

scholarly journals The Retroviruses Human Immunodeficiency Virus Type 1 and Moloney Murine Leukemia Virus Adopt Radically Different Strategies To Regulate Promoter-Proximal Polyadenylation

2001 ◽  
Vol 75 (23) ◽  
pp. 11735-11746 ◽  
Author(s):  
Andre Furger ◽  
Joan Monks ◽  
Nick J. Proudfoot
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Moloney Murine Leukemia Virus ◽  
Immunodeficiency Virus ◽  
A Site ◽  
Hiv 1 ◽  
Murine Leukemia

ABSTRACT Maximal gene expression in retroviruses requires that polyadenylation in the 5′ long terminal repeat (LTR) is suppressed. In human immunodeficiency virus type 1 (HIV-1) the promoter-proximal poly(A) site is blocked by interaction of U1 snRNP with the closely positioned major splice donor site (MSD) 200 nucleotides downstream. Here we investigated whether the same mechanism applies to down-regulate 5′ LTR polyadenylation in Moloney murine leukemia virus (MoMLV). Although the same molecular architecture is present in both viruses, the MoMLV poly(A) signal in the 5′ LTR is active whether or not the MSD is mutated. This surprising difference between the two retroviruses is not due to their actual poly(A) signals or MSD sequences, since exchange of either element between the two viral sequences does not alter their ability to regulate 5′ LTR poly(A) site use. Instead we demonstrate that sequence between the cap and AAUAAA is required for MSD-dependent poly(A) regulation in HIV-1, indicating a key role for this part of the LTR in poly(A) site suppression. We also show that the MoMLV poly(A) signal is an intrinsically weak RNA-processing signal. This suggests that in the absence of a poly(A) site suppression mechanism, MoMLV is forced to use a weak poly(A) signal.

scholarly journals Revealing Domain Structure through Linker-Scanning Analysis of the Murine Leukemia Virus (MuLV) RNase H and MuLV and Human Immunodeficiency Virus Type 1 Integrase Proteins

2006 ◽  
Vol 80 (19) ◽  
pp. 9497-9510 ◽  
Author(s):  
Jennifer Puglia ◽  
Tan Wang ◽  
Christine Smith-Snyder ◽  
Marie Cote ◽  
Michael Scher ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Rnase H ◽  
C Terminus ◽  
Immunodeficiency Virus ◽  
Hiv 1 ◽  
Murine Leukemia

ABSTRACT Linker-scanning libraries were generated within the 3′ terminus of the Moloney murine leukemia virus (M-MuLV) pol gene encoding the connection-RNase H domains of reverse transcriptase (RT) as well as the structurally related M-MuLV and human immunodeficiency virus type 1 (HIV-1) integrase (IN) proteins. Mutations within the M-MuLV proviral vectors were Tn7 based and resulted in 15-bp insertions. Mutations within an HIV-1 IN bacterial expression vector were based on Tn5 and resulted in 57-bp insertions. The effects of the insertions were examined in vivo (M-MuLV) and in vitro (HIV-1). A total of 178 individual M-MuLV constructs were analyzed; 40 in-frame insertions within RT connection-RNase H, 108 in-frame insertions within IN, 13 insertions encoding stop codons within RNase H, and 17 insertions encoding stop codons within IN. For HIV-1 IN, 56 mutants were analyzed. In both M-MuLV and HIV-1 IN, regions are identified which functionally tolerate multiple-linker insertions. For MuLV, these correspond to the RT-IN proteolytic junction, the junction between the IN core and C terminus, and the C terminus of IN. For HIV-1 IN, in addition to the junction between the IN core and C terminus and the C terminus of IN, insertions between the N terminus and core domains maintained integration and disintegration activity. Of the 40 in-frame insertions within the M-MuLV RT connection-RNase H domains, only the three C-terminal insertions mapping to the RT-IN proteolytic junction were viable. These results correlate with deletion studies mapping the domain and subdomain boundaries of RT and IN. Importantly, these genetic footprints provide a means to identify nonessential regions within RT and IN for targeted gene therapy applications.

scholarly journals ATPγS Disrupts Human Immunodeficiency Virus Type 1 Virion Core Integrity

2005 ◽  
Vol 79 (9) ◽  
pp. 5557-5567 ◽  
Author(s):  
Cagan Gurer ◽  
Anders Höglund ◽  
Stefan Höglund ◽  
Jeremy Luban
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Reverse Transcription ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Immunodeficiency Virus ◽  
Hiv 1 ◽  
Murine Leukemia ◽  
Virion Core

ABSTRACT Heat shock protein 70 (Hsp70) is incorporated within the membrane of primate lentiviral virions. Here we demonstrate that Hsp70 is also incorporated into oncoretroviral virions and that it remains associated with membrane-stripped human immunodeficiency virus type 1 (HIV-1) virion cores. To determine if Hsp70 promotes virion infectivity, we attempted to generate Hsp70-deficient virions with gag deletion mutations, Hsp70 transdominant mutants, or RNA interference, but these efforts were confounded, largely because they disrupt virion assembly. Given that polypeptide substrates are bound and released by Hsp70 in an ATP-hydrolytic reaction cycle, we supposed that incubation of HIV-1 virions with ATP would perturb Hsp70 interaction with substrates in the virion and thereby decrease infectivity. Treatment with ATP or ADP had no observable effect, but ATPγS and GTPγS, nucleotide triphosphate analogues resistant to Hsp70 hydrolysis, dramatically reduced the infectivity of HIV-1 and murine leukemia virus virions. ATPγS-treated virions were competent for fusion with susceptible target cells, but viral cDNA synthesis was inhibited to an extent that correlated with the magnitude of decrease in infectivity. Intravirion reverse transcription by HIV-1, simian immunodeficiency virus, or murine leukemia virus was also inhibited by ATPγS. The effects of ATPγS on HIV-1 reverse transcription appeared to be indirect, resulting from disruption of virion core morphology that was evident by transmission electron microscopy. Consistent with effects on capsid conformation, ATPγS-treated viruslike particles failed to saturate host antiviral restriction activity. Our observations support a model in which the catalytic activity of virion-associated Hsp70 is required to maintain structural integrity of the virion core.

scholarly journals The LEM Domain Proteins Emerin and LAP2α Are Dispensable for Human Immunodeficiency Virus Type 1 and Murine Leukemia Virus Infections

2008 ◽  
Vol 82 (12) ◽  
pp. 5860-5868 ◽  
Author(s):  
Alok Mulky ◽  
Tatiana V. Cohen ◽  
Serguei V. Kozlov ◽  
Barbara Korbei ◽  
Roland Foisner ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Knockout Mice ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Wild Type ◽  
Immunodeficiency Virus ◽  
Early Replication ◽  
Hiv 1 ◽  
Murine Leukemia

ABSTRACT The human nuclear envelope proteins emerin and lamina-associated polypeptide 2α (LAP2α) have been proposed to aid in the early replication steps of human immunodeficiency virus type 1 (HIV-1) and murine leukemia virus (MLV). However, whether these factors are essential for HIV-1 or MLV infection has been questioned. Prior studies in which conflicting results were obtained were highly dependent on RNA interference-mediated gene silencing. To shed light on these contradictory results, we examined whether HIV-1 or MLV could infect primary cells from mice deficient for emerin, LAP2α, or both emerin and LAP2α. We observed HIV-1 and MLV infectivity in mouse embryonic fibroblasts (MEFs) from emerin knockout, LAP2α knockout, or emerin and LAP2α double knockout mice to be comparable in infectivity to wild-type littermate-derived MEFs, indicating that both emerin and LAP2α were dispensable for HIV-1 and MLV infection of dividing, primary mouse cells. Because emerin has been suggested to be important for infection of human macrophages by HIV-1, we also examined HIV-1 transduction of macrophages from wild-type mice or knockout mice, but again we did not observe a difference in susceptibility. These findings prompted us to reexamine the role of human emerin in supporting HIV-1 and MLV infection. Notably, both viruses efficiently infected human cells expressing high levels of dominant-negative emerin. We thus conclude that emerin and LAP2α are not required for the early replication of HIV-1 and MLV in mouse or human cells.

scholarly journals Characterization of the murine leukemia virus protease and its comparison with the human immunodeficiency virus type 1 protease

2006 ◽  
Vol 87 (5) ◽  
pp. 1321-1330 ◽  
Author(s):  
Anita Fehér ◽  
Péter Boross ◽  
Tamás Sperka ◽  
Gabriella Miklóssy ◽  
János Kádas ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Activity Measurements ◽  
Immunodeficiency Virus ◽  
Hiv 1 ◽  
Murine Leukemia

The protease (PR) of Murine leukemia virus (MLV) was expressed in Escherichia coli, purified to homogeneity and characterized by using various assay methods, including HPLC-based, photometric and fluorometric activity measurements. The specificity of the bacterially expressed PR was similar to that of virion-extracted PR. Compared with human immunodeficiency virus type 1 (HIV-1) PR, the pH optimum of the MLV enzyme was higher. The specificity of the MLV PR was further compared with that of HIV-1 PR by using various oligopeptides representing naturally occurring cleavage sites in MLV and HIV-1, as well as by using bacterially expressed proteins having part of the MLV Gag. Inhibitors designed against HIV-1 PR were also active on MLV PR, although all of the tested ones were substantially less potent on this enzyme than on HIV-1 PR. Nevertheless, amprenavir, the most potent inhibitor against MLV PR, was also able to block Gag processing in MLV-infected cells. These results indicate that, in spite of the similar function in the life cycle of virus infection, the two PRs are only distantly related in their specificity.

scholarly journals Association of Murine Leukemia Virus Pol with Virions, Independent of Gag-Pol Expression

1999 ◽  
Vol 73 (11) ◽  
pp. 9632-9637 ◽  
Author(s):  
Gary L. Buchschacher ◽  
Lei Yu ◽  
Fukashi Murai ◽  
Theodore Friedmann ◽  
Atsushi Miyanohara
Keyword(s):  
Human Immunodeficiency Virus ◽  
Fusion Protein ◽  
Reverse Transcriptase ◽  
Human Immunodeficiency Virus Type ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Wild Type ◽  
Immunodeficiency Virus ◽  
Murine Leukemia

ABSTRACT During the replication cycle of murine leukemia virus (MLV), Pol is normally synthesized as part of a Gag-Pol fusion protein. In this study, the ability of free MLV Pol to be incorporated into virions was examined. When MLV Gag and MLV Pol were coexpressed from separate plasmids in cells, reverse transcriptase (RT) activity associated with Gag core particles at a slightly lower level than did RT activity generated from wild-type Gag-Pol expression. Particles produced in this manner were somewhat less infectious than those produced with wild-type Gag-Pol. A smaller amount of MLV Pol also associated with heterologous human immunodeficiency virus type 1 Gag cores.

scholarly journals Human Immunodeficiency Virus Type 1 Genetic Recombination Is More Frequent Than That of Moloney Murine Leukemia Virus despite Similar Template Switching Rates

2003 ◽  
Vol 77 (8) ◽  
pp. 4577-4587 ◽  
Author(s):  
Adewunmi Onafuwa ◽  
Wenfeng An ◽  
Nicole D. Robson ◽  
Alice Telesnitsky
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Template Switching ◽  
Immunodeficiency Virus ◽  
Hiv 1 ◽  
Murine Leukemia

ABSTRACT Retroviral recombinants result from template switching between copackaged viral genomes. Here, marker reassortment between coexpressed vectors was measured during single replication cycles, and human immunodeficiency virus type 1 (HIV-1) recombination was observed six- to sevenfold more frequently than murine leukemia virus (MLV) recombination. Template switching was also assayed by using transduction-type vectors in which donor and acceptor template regions were joined covalently. In this situation, where RNA copackaging could not vary, MLV and HIV-1 template switching rates were indistinguishable. These findings argue that MLV's lower intermolecular recombination frequency does not reflect enzymological differences. Instead, these data suggest that recombination rates differ because coexpressed MLV RNAs are less accessible to the recombination machinery than are coexpressed HIV RNAs. This hypothesis provides a plausible explanation for why most gammaretrovirus recombinants, although relatively rare, display evidence of multiple nonselected crossovers. By implying that recombinogenic template switching occurs roughly four times on average during the synthesis of every MLV or HIV-1 DNA, these results suggest that virtually all products of retroviral replication are biochemical recombinants.

scholarly journals Ubiquitin Is Covalently Attached to the p6GagProteins of Human Immunodeficiency Virus Type 1 and Simian Immunodeficiency Virus and to the p12Gag Protein of Moloney Murine Leukemia Virus

1998 ◽  
Vol 72 (4) ◽  
pp. 2962-2968 ◽  
Author(s):  
David E. Ott ◽  
Lori V. Coren ◽  
Terry D. Copeland ◽  
Bradley P. Kane ◽  
Donald G. Johnson ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Simian Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Moloney Murine Leukemia Virus ◽  
Immunodeficiency Virus ◽  
Hiv 1 ◽  
Murine Leukemia

ABSTRACT Host proteins are incorporated into retroviral virions during assembly and budding. We have examined three retroviruses, human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency virus (SIV), and Moloney murine leukemia virus (Mo-MuLV), for the presence of ubiquitin inside each of these virions. After a protease treatment to remove exterior viral as well as contaminating cellular proteins, the proteins remaining inside the virion were analyzed. The results presented here show that all three virions incorporate ubiquitin molecules at approximately 10% of the level of Gag found in virions. In addition to free ubiquitin, covalent ubiquitin-Gag complexes were detected, isolated, and characterized from all three viruses. Our immunoblot and protein sequencing results on treated virions showed that approximately 2% of either HIV-1 or SIV p6Gag was covalently attached to a single ubiquitin molecule inside the respective virions and that approximately 2 to 5% of the p12Gag in Mo-MuLV virions was monoubiquitinated. These results show that ubiquitination of Gag is conserved among these retroviruses and occurs in the p6Gag portion of the Gag polyprotein, a region that is likely to be involved in assembly and budding.

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