Differential Effects of C-Terminal Molecular Tagged Integrase on Replication Competent Moloney Murine Leukemia Virus

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Methods have been developed for the comprehensive analysis of a gene by construction of a saturating or near-saturating library of mutants (5,78,83). This approach has defined domain boundaries, provided functional maps, and given insights into previously predicted unstructured loops (4,5,50,71,73,78,83). In this report, this method of insertional functional mapping is applied to three catalytically related domains: the Moloney murine leukemia virus (M-MuLV) RNase H domain of the reverse transcriptase (RT), and the M-MuLV and human immunodeficiency virus type 1 (HIV-1) integrase (IN) proteins. Inclusion of the HIV-1 IN protein assisted comparison and model building, since structural information is available (7,18,26,34,35,37,87). In the retroviral life cycle, the RNase H activity is required for viral replication during the conversion of the viral RNA (vRNA) into double-stranded DNA through the RNA-DNA intermediate. The IN protein is required for the insertion of the double-stranded DNA into the host chromosome, establishing the integrated provirus.The replication and integration of retroviral particles are two distinct yet interrelated processes. Replicative complexes and preintegrative complexes have been purified and characterized from infected cells (6, 9, 17, 28-30, 39, 52, 53, 55, 56, 66, 67). Within viral species as well as between viral species, the composition of replicative complexes differs from that of preintegrative complexes. Interactions between RT and IN are also reported (40,69,89,90,96), and multiple mutations of IN are known to alter viral replication (27,(58)(59)(60). Despite extensive efforts, the assembly of these complexes is not well understood. These studies have been assisted by structural studies. A structure of the M-MuLV RT has recently been reported (21), as have structures of related retroviral IN subdomains (8,14,18,19,26,35,43,87,94). However, to date, neither a structure of a complete retroviral IN protein nor one of a subdomain in complex with DNA has been obtained.The ability of retroviral particles to stably integrate into the host genome is a great benefit for gene delivery, but the potential for insertional mutagenesis cannot be overlooked (15,22,38,63). Schemes to target integration into alternative positions within the host chromosome to avoid this issue frequently involve generation of fusion proteins with novel targeting domains (10,48,84). The linker insertion genetic footprint provides a means to identify nonessential regions

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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

Puglia¹,

Wang

Smith-Snyder³

et al. 2006

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“…The SV40 NLS sequence was inserted within this nonessential region, resulting in the truncation of the terminal 23 amino acids of IN (Fig. 1A) (65). The IN-SV40 NLS protein was generated by PCR amplification of the template plasmid pNCA-C-XN-SU8 harboring the NLS fusion (65).…”

Section: Expression Of In and In-sv40 Nls Proteinsmentioning

confidence: 99%

“…IN proteins tagged with either six-His or hemagglutinin (HA) epitope tags were shown to be genetically stable during replication. In contrast, insertion of the SV40 large T-antigen NLS (SV40 NLS) was not stable, resulting in the selection and propagation of viruses in which the basic NLS sequence was disrupted (65). This study examines the stability of alternative NLS sequences within Mo-MuLV IN and the influence the NLS sequences have on the nuclear transport properties of Mo-MuLV.…”

mentioning

confidence: 99%

Inserting a Nuclear Targeting Signal into a Replication-Competent Moloney Murine Leukemia Virus Affects Viral Export and Is Not Sufficient for Cell Cycle-Independent Infection

Seamon¹,

Jones

Miller³

et al. 2002

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Following entry into the cytoplasm of the host cell, reverse transcription of the retroviral RNA genome into doublestranded viral DNA occurs within large nucleoprotein complexes derived from the core of the infecting virion, termed preintegration complexes (PICs). These complexes must then gain access to host cell chromosomes within the nucleus to achieve stable integration. Human immunodeficiency virus type 1 (HIV-1) can infect certain types of nondividing cells (5,22,46,74). Although the ability of lentiviruses to infect nondividing cells has been exploited in the development of vectors for human gene therapy, their application is limited by safety concerns. In contrast, nuclear accumulation of Moloney murine leukemia virus (Mo-MuLV) viral DNA increases only after infected cells pass through mitosis (61). This property limits the range of cells and target tissues that can use murinebased retroviral vectors for gene transfer or gene therapy.Many studies put forth the model that HIV-1 PICs are actively imported into the nucleus. In support of this model, nuclear localization signals (NLSs) have been reported on a number of viral proteins that comprise the HIV-1 PIC. The role or roles that these sequences play in viral replication, however, remain unclear. In addition, a cis-acting viral DNA structure generated during lentivirus-specific reverse transcription, termed the central DNA flap, may also be involved in the nuclear import of the HIV-1 genome (77). To date, no NLSs have been identified in MuLV PIC components. This suggests that it is the disassembly of the nuclear envelope during mitosis that renders the chromosomes accessible to the MuLV PIC.Interestingly, the avian sarcoma virus (ASV), which has a novel NLS sequence (44, 45), presents an intermediate phenotype in which mitosis-independent replication can occur, but at a lower efficiency than HIV-1 (31).Previous analysis indicated various molecular tags placed at the C terminus of the Mo-MuLV integrase (IN) protein could be incorporated into viral particles when expressed within the context of a replication-competent virus. IN proteins tagged with either six-His or hemagglutinin (HA) epitope tags were shown to be genetically stable during replication. In contrast, insertion of the SV40 large T-antigen NLS (SV40 NLS) was not stable, resulting in the selection and propagation of viruses in which the basic NLS sequence was disrupted (65). This study examines the stability of alternative NLS sequences within Mo-MuLV IN and the influence the NLS sequences have on the nuclear transport properties of Mo-MuLV. A similar approach to alter nuclear transport properties was reported to be successful for spleen necrosis virus (SNV), in which the efficiency of infecting nondividing cells was altered by incorporating an HIV-1 matrix (MA) protein NLS sequence into the SNV MA protein (56).Expression of IN and IN-SV40 NLS proteins. The effects of inserting the SV40 NLS into M-MuLV IN were first examined in an IN expression system in the absence of additional viral protein...

“…However, insertion of the HIV-1 MA NLS into the SNV MA sequence confers enhanced viral replication in stationary cells. Similarly, NLSs derived from simian virus 40 T antigen or nucleoplasmin inserted into the murine leukemia virus IN protein disrupted virus infectivity, while NLSs from HIV-1 MA, RSV IN, or the M9 sequence of hnRNP were compatible with virus replication (34,35). These results provide strong support for the idea that the route and regulation of nuclear entry for viral proteins is a critical determinant of virus infectivity.…”

Section: Discussionmentioning

confidence: 54%

Insertion of a Classical Nuclear Import Signal into the Matrix Domain of the Rous Sarcoma Virus Gag Protein Interferes with Virus Replication

Garbitt¹,

Bone

Parent

2004

The Rous sarcoma virus Gag protein undergoes transient nuclear trafficking during virus assembly. Nuclear import is mediated by a nuclear targeting sequence within the MA domain. To gain insight into the role of nuclear transport in replication, we investigated whether addition of a "classical " nuclear localization signal (NLS) in Gag would affect virus assembly or infectivity. A bipartite NLS derived from nucleoplasmin was inserted into a region of the MA domain of Gag that is dispensable for budding and infectivity. Gag proteins bearing the nucleoplasmin NLS insertion displayed an assembly defect. Mutant virus particles (RC.V8.NLS) were not infectious, although they were indistinguishable from wild-type virions in Gag, Gag-Pol, Env, and genomic RNA incorporation and Gag protein processing. Unexpectedly, postinfection viral DNA synthesis was also normal, as similar amounts of two-long-terminal-repeat junction molecules were detected for RC.V8.NLS and wild type, suggesting that the replication block occurred after nuclear entry of proviral DNA. Phenotypically revertant viruses arose after continued passage in culture, and sequence analysis revealed that the nucleoplasmin NLS coding sequence was deleted from the gag gene. To determine whether the nuclear targeting activity of the nucleoplasmin sequence was responsible for the infectivity defect, two critical basic amino acids in the NLS were altered. This virus (RC.V8.KR/AA) had restored infectivity, and the MA.KR/AA protein showed reduced nuclear localization, comparable to the wild-type MA protein. These data demonstrate that addition of a second NLS, which might direct MA and/or Gag into the nucleus by an alternate import pathway, is not compatible with productive virus infection.The retroviral Gag polyprotein is sufficient to direct virus assembly, as demonstrated by its ability to form virus-like particles in the absence of all other viral gene products (reviewed in reference 39). Previously, it was thought that Gag proteins of type C retroviruses were synthesized on cytosolic ribosomes and then targeted directly to the plasma membrane, where budding occurs. However, we discovered that the Rous sarcoma virus (RSV) Gag polyprotein traffics through the nucleus (32). Nuclear import is mediated by a nuclear localization signal (NLS) within the N-terminal matrix (MA) domain. A CRM1-dependent nuclear export signal (NES) within the p10 domain of Gag mediates nuclear egress, after which Gag travels to the plasma membrane for particle release. Treatment of cells with leptomycin B (LMB), a specific inhibitor of CRM1, results in the accumulation of Gag proteins in the nucleus (32).Nuclear import of macromolecules, including all proteins greater than 50 kDa, occurs by signal-mediated active transport across the nuclear membrane through the nuclear pore complex. The majority of proteins bearing specific NLS sequences are imported through an interaction with soluble transport factors belonging to the importin superfamily (reviewed in reference 9). The "classical" NLSs in...