Capsid is an important determinant for functional complementation of murine leukemia virus and spleen necrosis virus Gag proteins

ABSTRACT Murine leukemia virus (MLV)-based vector RNA can be packaged and propagated by the proteins of spleen necrosis virus (SNV). We recently demonstrated that MLV proteins cannot support the replication of an SNV-based vector; RNA analysis revealed that MLV proteins cannot efficiently package SNV-based vector RNA. The domain in Gag responsible for the specificity of RNA packaging was identified using chimericgag-pol expression constructs. A competitive packaging system was established by generating a cell line that expresses one viral vector RNA containing the MLV packaging signal (Ψ) and another viral vector RNA containing the SNV packaging signal (E). The chimericgag-pol expression constructs were introduced into the cells, and vector titers as well as the efficiency of RNA packaging were examined. Our data confirm that Gag is solely responsible for the selection of viral RNAs. Furthermore, the nucleocapsid (NC) domain in the SNV Gag is responsible for its ability to interact with both SNV E and MLV Ψ. Replacement of the SNV NC with the MLV NC generated a chimeric Gag that could not package SNV RNA but retained its ability to package MLV RNA. A construct expressing SNV gag-MLVpol supported the replication of both MLV and SNV vectors, indicating that the gag and pol gene products from two different viruses can functionally cooperate to perform one cycle of retroviral replication. Viral titer data indicated that SNVcis-acting elements are not ideal substrates for MLVpol gene products since infectious viruses were generated at a lower efficiency. These results indicate that the nonreciprocal recognition between SNV and MLV extends beyond the Gag-RNA interaction and also includes interactions between Pol and othercis-acting elements.

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Nonreciprocal Pseudotyping: Murine Leukemia Virus Proteins Cannot Efficiently Package Spleen Necrosis Virus-Based Vector RNA

Journal of Virology ◽

10.1128/jvi.72.7.5408-5413.1998 ◽

1998 ◽

Vol 72 (7) ◽

pp. 5408-5413 ◽

Cited By ~ 17

Author(s):

Jeanine L. Certo ◽

Betsy F. Shook ◽

Philip D. Yin ◽

John T. Snider ◽

Wei-Shau Hu

Keyword(s):

Murine Leukemia Virus ◽

Leukemia Virus ◽

Rna Structures ◽

Packaging Signal ◽

Necrosis Virus ◽

Hairpin Rna ◽

Spleen Necrosis Virus ◽

Retroviral Replication ◽

Spleen Necrosis ◽

Murine Leukemia

ABSTRACT It has been documented that spleen necrosis virus (SNV) can package murine leukemia virus (MLV) RNA efficiently and propagate MLV vectors to the same titers as it propagates SNV-based vectors. Although the SNV packaging signal (E) and MLV packaging signal (Ψ) have little sequence homology, similar double-hairpin RNA structures were predicted and supported by experimental evidence. To test whether SNV RNA can be packaged by MLV proteins, we modified an SNV vector to be expressed in an MLV-based murine helper cell line. Surprisingly, we found that MLV proteins could not support the replication of SNV vectors. The decrease in titer was approximately 2,000- to 20,000-fold in one round of retroviral replication. RNA analysis revealed that SNV RNA was not efficiently packaged by MLV proteins. RNA hybridization of the cellular and viral RNAs indicated that SNV RNA was packaged at least 25-fold less efficiently than MLV RNA, which was the sensitivity limit of the hybridization assay. The contrast between the MLV and SNV packaging specificity is striking. SNV proteins can recognize both SNV E and MLV Ψ, but MLV can recognize only MLV Ψ. This is the first demonstration of two retroviruses with nonreciprocal packaging specificities.

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Cooperative Effect of Gag Proteins p12 and Capsid during Early Events of Murine Leukemia Virus Replication

Journal of Virology ◽

10.1128/jvi.79.7.4159-4169.2005 ◽

2005 ◽

Vol 79 (7) ◽

pp. 4159-4169 ◽

Cited By ~ 19

Author(s):

Sook-Kyung Lee ◽

Kunio Nagashima ◽

Wei-Shau Hu

Keyword(s):

Virus Replication ◽

Murine Leukemia Virus ◽

Leukemia Virus ◽

Virus Production ◽

Cooperative Effect ◽

Gag Proteins ◽

Rous Sarcoma ◽

Reverse Transcription Step ◽

Spleen Necrosis ◽

Murine Leukemia

ABSTRACT The Gag polyprotein of murine leukemia virus (MLV) is processed into matrix (MA), p12, capsid (CA), and nucleocapsid (NC) proteins. p12 affects early events of virus replication and contains a PPPY motif important for virus release. To probe the functions of p12 in the early steps of MLV replication, we tested whether p12 can be replaced by spleen necrosis virus (SNV) p18, human immunodeficiency virus type 1 p6, or Rous sarcoma virus p2b. Analyses revealed that all chimeras generated virions at levels similar to that of MLV gag-pol; however, none of them could support MLV vector replication, and all of them exhibited severely reduced DNA synthesis upon virus infection. Because a previously reported SNV gag-MLV pol chimera, but not the MLV hybrid with SNV p18, can support replication of an MLV vector, we hypothesized that other Gag proteins act cooperatively with p12 during the early phase of virus replication. To test this hypothesis, we generated three more MLV-based chimeras containing SNV CA, p18-CA, or p18-CA-NC. We found that the MLV chimera containing SNV p18-CA or p18-CA-NC could support MLV vector replication, but the chimera containing SNV CA could not. Furthermore, viruses derived from the MLV chimera with SNV CA could synthesize viral DNA upon infection but were blocked at a post-reverse-transcription step and generated very little two long terminal repeat circle DNA, thereby producing a phenotype similar to that of the provirus formation-defective p12 mutants. Taken together, our data indicate that when p12/p18 or CA was from different viruses, despite abundant virus production and proper Gag processing, the resulting viruses were not infectious. However, when p12/p18 and CA were from the same virus, even though they were from SNV and not MLV, the resulting viruses were infectious. Therefore, these results suggest a cooperative effect of p12 and CA during the early events of MLV replication.

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Mutant murine leukemia virus Gag proteins lacking proline at the N-terminus of the capsid domain block infectivity in virions containing wild-type Gag

Virology ◽

10.1016/j.virol.2005.12.012 ◽

2006 ◽

Vol 347 (2) ◽

pp. 364-371 ◽

Cited By ~ 25

Author(s):

S.J. Rulli ◽

D. Muriaux ◽

K. Nagashima ◽

J. Mirro ◽

M. Oshima ◽

...

Keyword(s):

Murine Leukemia Virus ◽

Leukemia Virus ◽

Wild Type ◽

Gag Proteins ◽

N Terminus ◽

Domain Block ◽

Murine Leukemia

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Targeting of Murine Leukemia Virus Gag to the Plasma Membrane Is Mediated by PI(4,5)P2/PS and a Polybasic Region in the Matrix

Journal of Virology ◽

10.1128/jvi.01134-09 ◽

2009 ◽

Vol 84 (1) ◽

pp. 503-515 ◽

Cited By ~ 58

Author(s):

E. Hamard-Peron ◽

F. Juillard ◽

J. S. Saad ◽

C. Roy ◽

P. Roingeard ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Plasma Membrane ◽

Murine Leukemia Virus ◽

Leukemia Virus ◽

Gag Proteins ◽

Immunodeficiency Virus ◽

The Matrix ◽

Polybasic Region ◽

Murine Leukemia

ABSTRACT Membrane targeting of the human immunodeficiency virus Gag proteins is dependent on phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2] located in the plasma membrane. In order to determine if evolutionarily distant retroviral Gag proteins are targeted by a similar mechanism, we generated mutants of the matrix (MA) domain of murine leukemia virus (MuLV) Gag, examined their binding to membrane models in vitro, and analyzed their phenotypes in cell culture. In vitro, we showed that MA bound all the phosphatidylinositol phosphates with significant affinity but displayed a strong specificity for PI(4,5)P2 only if enhanced by phosphatidylserine. Mutations in the polybasic region in MA dramatically reduced this affinity. In cells, virus production was strongly impaired by PI(4,5)P2 depletion under conditions of 5ptaseIV overexpression, and mutations in the MA polybasic region altered Gag localization, membrane binding, and virion production. Our results suggest that the N-terminal polybasic cluster of MA is essential for Gag targeting to the plasma membrane. The binding of the MA domain to PI(4,5)P2 appears to be a conserved feature among retroviruses despite the fact that the MuLV-MA domain is structurally different from that of human immunodeficiency virus types 1 and 2 and lacks a readily identifiable PI(4,5)P2 binding cleft.

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Transport and assembly of gag proteins into Moloney murine leukemia virus.

Journal of Virology ◽

10.1128/jvi.64.11.5306-5316.1990 ◽

1990 ◽

Vol 64 (11) ◽

pp. 5306-5316 ◽

Cited By ~ 37

Author(s):

M Hansen ◽

L Jelinek ◽

S Whiting ◽

E Barklis

Keyword(s):

Murine Leukemia Virus ◽

Leukemia Virus ◽

Moloney Murine Leukemia Virus ◽

Gag Proteins ◽

Murine Leukemia

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Late Domain-Independent Rescue of a Release-Deficient Moloney Murine Leukemia Virus by the Ubiquitin Ligase Itch

Journal of Virology ◽

10.1128/jvi.01319-09 ◽

2009 ◽

Vol 84 (2) ◽

pp. 704-715 ◽

Cited By ~ 23

Author(s):

Joshua A. Jadwin ◽

Victoria Rudd ◽

Paola Sette ◽

Swathi Challa ◽

Fadila Bouamr

Keyword(s):

Ubiquitin Ligase ◽

Murine Leukemia Virus ◽

Leukemia Virus ◽

Dominant Negative ◽

Moloney Murine Leukemia Virus ◽

Independent Manner ◽

Gag Proteins ◽

Aaa Atpase ◽

Domain Independent ◽

Murine Leukemia

ABSTRACT Moloney murine leukemia virus (MoMLV) Gag utilizes its late (L) domain motif PPPY to bind members of the Nedd4-like ubiquitin ligase family. These interactions recruit components of the cell's budding machinery that are critical for virus release. MoMLV Gag contains two additional L domains, PSAP and LYPAL, that are believed to drive residual MoMLV release via interactions with cellular proteins Tsg101 and Alix, respectively. We found that overexpression of Tsg101 or Alix failed to rescue the release of PPPY-deficient MoMLV via these other L domains. However, low-level expression of the ubiquitin ligase Itch potently rescued the release and infectivity of MoMLV lacking PPPY function. In contrast, other ubiquitin ligases such as WWP1, Nedd4.1, Nedd4.2, and Nedd4.2s did not rescue this release-deficient virus. Efficient rescue required the ubiquitin ligase activity of Itch and an intact C2 domain but not presence of the endophilin-binding site. Additionally, we found Itch to immunoprecipitate with MoMLV Gag lacking the PPPY motif and to be incorporated into rescued MoMLV particles. The PSAP and LYPAL motifs were dispensable for Itch-mediated virus rescue, and their absence did not affect the incorporation of Itch into the rescued particles. Itch-mediated rescue of release-defective MoMLV was sensitive to inhibition by dominant-negative versions of ESCRT-III components and the VPS4 AAA ATPase, indicating that Itch-mediated correction of MoMLV release defects requires the integrity of the host vacuolar sorting protein pathway. RNA interference knockdown of Itch suppressed the residual release of the MoMLV lacking the PPPY motif. Interestingly, Itch stimulation of the PPPY-deficient MoMLV release was accompanied by the enhancement of Gag ubiquitination and the appearance of new ubiquitinated Gag proteins in virions. Together, these results suggest that Itch can facilitate MoMLV release in an L domain-independent manner via a mechanism that requires the host budding machinery and involves Gag ubiquitination.

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Lower mutation rate of bovine leukemia virus relative to that of spleen necrosis virus.

Journal of Virology ◽

10.1128/jvi.68.1.494-499.1994 ◽

1994 ◽

Vol 68 (1) ◽

pp. 494-499 ◽

Cited By ~ 55

Author(s):

L M Mansky ◽

H M Temin

Keyword(s):

Mutation Rate ◽

Bovine Leukemia Virus ◽

Leukemia Virus ◽

Necrosis Virus ◽

Spleen Necrosis Virus ◽

Spleen Necrosis

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Binding of Murine Leukemia Virus Gag Polyproteins to KIF4, a Microtubule-Based Motor Protein

Journal of Virology ◽

10.1128/jvi.72.8.6898-6901.1998 ◽

1998 ◽

Vol 72 (8) ◽

pp. 6898-6901 ◽

Cited By ~ 47

Author(s):

Wankee Kim ◽

Yao Tang ◽

Yasushi Okada ◽

Ted A. Torrey ◽

Sisir K. Chattopadhyay ◽

...

Keyword(s):

Murine Leukemia Virus ◽

Mammalian Cells ◽

Leukemia Virus ◽

Motor Protein ◽

Cellular Protein ◽

Gag Proteins ◽

C Terminus ◽

Murine Leukemia

ABSTRACT A cDNA clone encoding a cellular protein that interacts with murine leukemia virus (MuLV) Gag proteins was isolated from a T-cell lymphoma library. The sequence of the clone is identical to the C terminus of a cellular protein, KIF4, a microtubule-associated motor protein that belongs to the kinesin superfamily. KIF4-MuLV Gag associations have been detected in vitro and in vivo in mammalian cells. We suggest that KIF4 could be involved in Gag polyprotein translocation from the cytoplasm to the cell membrane.

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