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. Author manuscript; available in PMC: 2009 Jul 7.
Published in final edited form as: Virology. 2006 Dec 6;360(2):388–397. doi: 10.1016/j.virol.2006.10.038

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

Sook-Kyung Lee 1, Vitaly Boyko 1, Wei-Shau Hu 1,*
PMCID: PMC2706498  NIHMSID: NIHMS21075  PMID: 17156810

Abstract

In this report, we examined the abilities and requirements of heterologous Gag proteins to functionally complement each other to support viral replication. Two distantly related gammaretroviruses, murine leukemia virus (MLV) and spleen necrosis virus (SNV), were used as a model system because SNV proteins can support MLV vector replication. Using chimeric or mutant Gag proteins that could not efficiently support MLV vector replication, we determined that a homologous capsid (CA) domain was necessary for the functional complementation of MLV and SNV Gag proteins. Findings from the bimolecular fluorescence complementation assay revealed that MLV and SNV Gag proteins were capable of colocalizing and interacting in cells. Taken together, our results indicated that MLV and SNV Gag proteins can interact in cells; however, a homologous CA domain is needed for functional complementation of MLV and SNV Gag proteins to complete virus replication. This requirement of homologous Gag most likely occurs at a postassembly step(s) of the viral replication.

Keywords: Retroviruses, Capsid, Infectivity, Coassembly, Complementation, Phenotypic mixing

Introduction

All retroviruses carry at least three genes – gag, pol, and env – encoding proteins essential for viral replication. These genes were first translated as polyproteins and then processed into mature proteins. The gag gene, which encodes the structural proteins, is first translated to a Gag polyprotein and then cleaved by the viral protease into smaller mature proteins during or soon after virus assembly. Although Gag polyproteins from different retroviruses may have only limited sequence homology, they share conserved domains that are organized similarly. Gag polyproteins from all of the retroviruses contain matrix (MA), capsid (CA), and nucleocapsid (NC) domains, arranged in the same order from the N to C terminus. In addition to these three domains, Gag polyproteins from different viruses often have other domains, located either between the three conserved domains or at the C terminus of the polyproteins. For example, murine leukemia virus (MLV) and spleen necrosis virus (SNV) have an additional p12 and p18 domain between MA and CA, respectively. Human immunodeficiency virus type 1 (HIV-1) has an additional p2 domain between CA and NC, and two additional domains (p1 and p6) downstream from NC.

During virus assembly, Gag is targeted to the assembly sites, coordinates the incorporation of other viral proteins and viral RNA, and interacts with the host cell machinery for proper release of the viral particle (Freed, 2002; Gottlinger, 2001; Swanstrom, 1997). Therefore, Gag plays a central role in virus assembly. Even in the absence of all other viral components, Gag or a portion of Gag can form virus-like particles in both cultured cells and in vitro reactions (Campbell and Rein, 1999; Campbell and Vogt, 1997; Gheysen et al., 1989; Gottlinger, 2001; Morikawa, Goto, and Sano, 1999; Wills and Craven, 1991).

In addition to assembly defects, mutations in gag can cause defects in virion maturations, reverse transcription, and integration (Cairns and Craven, 2001; Forshey et al., 2002; Fu et al., 2006; Oshima et al., 2004; Tang et al., 2001; von Schwedler et al., 2003). The NC proteins have well-known functions in the processes of reverse transcription and integration, because mutations in NC can cause defects in these processes (Buckman, Bosche, and Gorelick, 2003; Gorelick et al., 1993; Gorelick et al., 1999; Thomas et al., 2006). Mutations in the MLV p12, Rous sarcoma virus (RSV) CA, and HIV-1 CA can cause defects in generating reverse transcription products (Cairns and Craven, 2001; Forshey et al., 2002; Tang et al., 2001; von Schwedler et al., 2003; Yuan, Li, and Goff, 1999). Additionally, mutations in MLV p12 can cause defects in step(s) leading to integration (Yuan et al., 2002; Yuan, Li, and Goff, 1999). In this phenotype, reverse transcription products were generated and preintegration complexes isolated from the infected cells were capable of the integration reaction in vitro; however, the viruses appeared to have defects in integration (Yuan et al., 2002). Therefore, these studies indicated that Gag products play important roles in the early events in the virus replication cycle.

During the past decade, it was demonstrated that functional Gag chimeras could be generated with domains from distantly related viruses, indicating that a Gag domain could be functionally replaced by a sequence-divergent counterpart from another retrovirus. For example, various functional and nonfunctional Gag chimeras have been generated between MLV and HIV-1 (Berkowitz et al., 1995; Chen et al., 2001; Deminie and Emerman, 1993; Yamashita and Emerman, 2004; Zhang and Barklis, 1995), RSV and MLV (Dupraz and Spahr, 1992), RSV and HIV-1 (Bennett, Nelle, and Wills, 1993; Parent et al., 1995), and SNV and MLV (Certo et al., 1999; Fu and Hu, 2003; Lee, Nagashima, and Hu, 2005). One of the most dramatic examples of a functional chimera is RSV Gag in which the p2b domain was replaced by the HIV-1 p6 domain (Parent et al., 1995). RSV p2b and HIV-1 p6 do not share significant sequence homology and are located in different positions in Gag; it was later demonstrated that the PPPY motif in p2b and the PTAP motif in p6 interact with different cellular proteins in the same endosomal sorting pathway to enable proper virion release (Demirov et al., 2002; Garrus et al., 2001; Kikonyogo et al., 2001; Pornillos, Garrus, and Sundquist, 2002). In different studies, the definition of the functional chimeras varied; some of these chimeras have been shown to generate virions or to package viral RNA, whereas others have been shown to support one or limited rounds of viral replication.

The fact that functional Gag chimeras can be generated indicated that the assembly pathways of different viruses might share many similarities, which raises the questions of whether Gag polyproteins from different retroviruses can coassemble into the same virus and if coassembly occurs, whether viruses containing Gag proteins from two different viruses can be infectious. When proteins from two viruses are present in the same virions, it is unclear whether heterologous Gag proteins can functionally complement or interfere with each other. Coassembly of heterologous Gag proteins was investigated using RSV and MLV Gag (Bennett and Wills, 1999; Craven and Parent, 1996). It was found that the unmodified RSV and MLV Gag did not coassemble; however, these two Gag polyproteins could coassemble when both Gag polyproteins contained the membrane-targeting signal from the Src oncoprotein (Bennett and Wills, 1999). This study demonstrated that there are barriers to coassembly of heterologous Gag polyproteins; in this particular pair, the step of membrane targeting is the barrier. In another study, it was suggested that MLV and HIV-1 Gag polyproteins do not coassemble because MLV Gag did not crosslink to HIV-1 Gag (Hansen and Barklis, 1995). Using imaging analyses, it was recently reported that a homologous CA domain is required for coassembly of HIV-1 and RSV Gag proteins (Ako-Adjei, Johnson, and Vogt, 2005). It was observed that although HIV-1 Gag and RSV Gag do not colocalize and coassemble, if one of the Gag polyprotein was modified to contain the CA domain of the other Gag, these two proteins could colocalize and coassemble. Because gag-expression constructs were used in the aforementioned MLV/RSV and HIV-1/RSV systems, the potential infectivity of these mixed virions could not be determined. Therefore, it is not known whether these mixed particles will encounter other blocks in viral replication.

In this study, we examined whether Gag polyproteins from two distantly related retroviruses can functionally complement each other to complete virus replication. In order for complementation to occur, heterologous Gag proteins need to be coassembled and functionally assist each other. We selected MLV, a murine virus, and SNV, an avian virus, for the following reasons. First, the Gag polyproteins of these two viruses contain only limited (36%) amino acid sequence identity. Second, despite the limited homology, SNV proteins can support MLV vector replication (Certo et al., 1998; Embretson and Temin, 1987); this feature allows us to examine the infectivity of the coassembled virions. Third, functional Gag chimeras can be generated between MLV and SNV (Certo et al., 1999; Fu and Hu, 2003; Lee, Nagashima, and Hu, 2005); this attribute allows us to further pursue the barrier to functional complementation if MLV and SNV Gag polyproteins fail to do so. Using this system, we found that coexpression of mutant MLV and SNV Gag polyproteins did not produce infectious virions. However, Gag chimeras containing homologous CA domains were able to coassemble and produce infectious virions. Therefore, the CA domain plays a critical role in the functional complementation of MLV and SNV Gag polyproteins.

Results

Strategy and system used to study domain(s) in Gag important for functional complementation of heterologous Gag proteins

Both MLV and SNV proteins can package and support the replication of MLV vectors. Using this feature, we examined whether MLV vector replication can be supported by coexpression of various MLV or SNV gag-pol mutants or chimeras. Individually, these mutants or chimeric gag-pol expression constructs cannot efficiently support MLV vector replication; a significant increase in the MLV vector titer upon coexpression of two gag-pol constructs would indicate coassembly and functional complementation of two gag-pol products. To take this approach, we constructed multiple chimeras and also used previously described mutants or chimeras. We selected gag-pol mutants or chimeras based on the following criteria. First, these chimeras or mutants should express Gag at a level detectable by Western analyses. Additionally, these chimeras or mutants should have a deficiency in supporting MLV vector replication that would yield a minimal 1000-fold decrease in vector titer compared with that of the MLV gag-pol expression construct.

We generated various constructs based on the previously described pWZH30 or pRD136 (Martinez and Dornburg, 1995; Zhang et al., 2002), which express MLV or SNV Gag/Gag-Pol, respectively. These expression constructs contain large deletions of the packaging signals and their RNAs are not efficiently packaged by viral proteins. The general structures of these constructs are shown in Fig. 1A. For the chimeras, one or more Gag domains were replaced by the counterpart from the other virus. The names of the constructs reflect the composition of the sequences: the first letter after p indicates whether the construct is based on MLV (M) or SNV (S), and the letter following the slash indicates the heterologous domain(s) from the other virus. For example, pM/Sp18 is an MLV-based construct with the SNV p18 domain replacing the MLV p12 domain. In addition to the chimeras, two Gag mutants were also used: pWZH35 and pSΔPY. pWZH35 is a previously described MLV gag-pol expression construct with a CCHC-to-SSHS mutation in NC (Zhang et al., 2002). This mutant displays robust virion production but cannot support virus replication effectively because of a defect in viral RNA encapsidation into virions. pSΔPY is an SNV-based gag-pol expression construct with a deletion of the PPPPY sequence in the p18 domain of Gag (Fig. 1A).

Fig. 1.

Fig. 1

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Expression constructs used to examine functional complementation of heterologous Gag proteins. (A) Structures of the gag-pol expression constructs. MLV- and SNV-derived sequences are shown as white and gray boxes, respectively. Construct pWZH35 contains a mutation in NC (shown as N*), in which the conserved CCHC motif was changed to SSHC. Construct pSΔPY contains a deletion mutation in which the PPPPY motif (amino acids 40 to 44) in the p18 domain was removed. (B) MLV vector titers generated by various gag-pol expression constructs in one round of virus replication. Viral titers are shown as CFU/ml (mean ± standard deviation). Results shown are summarized from three independent sets of experiments.

To examine their phenotypes, the mutant gag-pol expression constructs were transiently transfected into SR2-293T cells along with pSV-a-MLV-env, which expresses amphotropic MLV Env. SR2-293T cells are a pool of 293T cells containing proviruses from the MLV vector SR2 (Cheslock et al., 2000). SR2 does not express viral proteins but encodes the hygromycin phosphotransferase gene (hygro), which confers resistance to hygromycin, and the green fluorescence protein gene (gfp). Viruses were harvested from the transfected cells and used to infect target D17 cells; the resulting SR2 titers were determined by the number of hygromycin-resistant colonies. The SR2 viral titers generated by any of the mutant constructs were at least 1000-fold lower than that of pWZH30 (Fig. 1B). As a negative control, SR2-293T cells were transfected with only pSV-a-MLV-env (shown as Mock). Western analyses were performed to examine the expression of viral proteins in the transfected cells and cell-free supernatants. Antibodies against MLV MA, MLV CA, and SNV CA were used individually or in combination to detect viral proteins in cell or virus lysates. For all of the constructs shown in Fig. 1A, Gag expression was detected in transfected cell lysates (data not shown).

The presence of homologous MA, p12/p18, or NC is not sufficient to mediate functional MLV and SNV Gag/Gag-Pol complementation

We first tested whether an MLV NC mutant (pWZH35) and an SNV late-domain mutant (pSΔPY) could complement one another and rescue viral titers (shown as pair 1 in Fig. 2A). We cotransfected these two mutant gag-pol expression constructs along with an env expression construct into SR2-293T cells and determined the SR2 titers generated by the resulting viruses. The resulting viruses generated SR2 titers of approximately 102 CFU/ml, which were similar to the sum of the SR2 titers generated by the two vectors independently and far lower than the wild-type viral titers (pWZH30, 105 CFU/ml). We also tested the complementation of a pair of chimeric gag-encoding vectors, pM/Sp18 and pS/Mp12 (Fig. 2A, pair 2), that do not contain any homologous Gag domains; the viral titers generated by this pair of vectors were also much lower than the wild-type viral titers. These results indicate that without a homologous domain(s), coexpression of mutant or chimeric MLV and SNV Gag/Gag-Pol does not rescue the viral titers to the levels of the wild-type viruses.

Fig. 2.

Fig. 2

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Complementation of various gag-pol expression constructs as indicated by MLV vector titers in one round of vector replication. (A) A homologous CA domain is required for successful complementation that generates high vector titers. (B) A homologous CA domain is not sufficient for successful complementation. Results shown are summarized from three independent sets of experiments.

To test whether it is possible to achieve viral titers at levels comparable to those of the wild-type viruses via complementation, we cotransfected two constructs that share three common Gag domains: MA, CA, and NC (Fig. 2A, pair 3). The resulting viruses indeed have titers comparable to those of wild-type viruses, indicating that element(s) important for functional coassembly and complementation resides in these three domains. Additionally, these results indicate that homologous p18 or p12 is not required for coassembly of heterologous Gag proteins and functional complementation of viral proteins for vector replication.

It was previously suggested that MLV and RSV Gag proteins cannot coassemble because these two proteins are targeted to different regions of the plasma membrane (Bennett and Wills, 1999). Thus, it is possible that MLV and SNV Gag proteins are targeted to different parts of the plasma membrane, thereby affecting efficient coassembly and complementation. Because MA plays a critical role in directing Gag membrane targeting, we examined whether MLV and SNV Gag chimeras with a homologous MA domain could mediate efficient complementation by cotransfecting pM/Sp18 and pM/SCANC (Fig. 2A, pair 4). The viral titers generated by pair 4 were comparable to those produced by pairs 1 and 2, indicating that homologous MA alone is not sufficient to mediate efficient complementation. We then tested the effects of homologous MA and NC (Fig. 2A, pair 5), or homologous p18 and NC (Fig. 2A, pair 6), and found that neither pair produced viral titers comparable to those of wild-type viruses. Taken together, these results indicated that the presence of homologous MA, p18, and/or NC was not sufficient for functional coassembly and complementation of MLV and SNV Gag proteins to achieve wild-type viral titers, and suggested that CA plays an important role in these interactions.

A homologous CA domain is critical for functional MLV and SNV Gag/Gag-Pol complementation

To test the role of the CA domain in functional complementation of the MLV and SNV Gag proteins, we cotransfected constructs that contained homologous MA and CA (Fig. 2A, pair 7) or homologous CA and NC (Fig. 2A, pair 8). Viruses derived from pair 7 or pair 8 had viral titers comparable to those of wild-type viruses, indicating successful complementation. Because pairs 7 and 8 both have a homologous CA domain, we questioned whether a homologous CA domain alone is sufficient for mediating functional complementation. To answer this question, we cotransfected constructs that shared only an MLV CA domain (Fig. 2A, pair 9) or an SNV CA domain (Fig. 2A, pair 10), and observed that viruses generated from either pair had viral titers similar to those of wild-type viruses. To further confirm the role of the CA domain in functional complementation, we cotransfected vectors containing homologous MA, p12, and NC domains but heterologous CA domains (Fig. 2A, pair 11); the viral titers from viruses generated by pair 11 were similar to those of pairs 1 and 2, in which the two constructs did not contain homologous domains. Taken together, these results indicate that a homologous CA domain is necessary for efficient coassembly and complementation of MLV and SNV polyproteins.

Coassembly and functional complementation of viral proteins from different expression constructs resulted in the rescue of the viral titers

As shown in Fig. 2A, cotransfection of two gag-pol expression vectors could significantly rescue the MLV vector titers. Most likely, the rescued viral titers were a consequence of the functional complementation of the coassembled viral proteins. However, to rule out the remote possibility that protein complementation occurred between coinfected viruses and not from viruses with coassembled proteins, we performed the following experiments. We selected the two constructs used in pair 3 in Fig. 2A and generated viruses derived from pM/Sp18 or pWZH35 individually, used these two viruses for coinfection, and measured the viral titers. This coinfection resulted in viral titers of 102 CFU/ml (Fig. 3), which are similar to titers generated by each construct alone and very different from the viral titers resulting from coexpression of the two constructs that allow coassembly to occur (Fig. 2A, pair 3). Similar results were obtained (Fig. 3) using other pairs of constructs including those used in pairs 7 and 9 in Fig. 2A. Taken together, these results indicate that the rescue of the viral titers resulted from complementation between coassembled proteins, not from complementation between coinfected viruses.

Fig. 3.

Fig. 3

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MLV vector titers from coinfection of two virus stocks that were each derived from a singly transfected gag-pol expression construct. Results shown are summarized from three independent sets of experiments.

Homologous CA is necessary but not sufficient for functional complementation of MLV and SNV Gag proteins – homologous p12/p18 and CA are required for virus replication

Although we demonstrated that a homologous CA domain is necessary for functional complementation of MLV and SNV Gag polyproteins, it was unclear whether homologous CA is sufficient to mediate such complementation. In order to rescue the viral titers, functional complementation of the heterologous Gag proteins requires coassembly of the heterologous proteins, proper processing of the coassembled proteins, and compatible mature proteins to support the early events of virus replication. Previously, using MLV/SNV chimeras, we demonstrated that homologous p12 and CA are required for efficient MLV replication, probably because these two proteins act cooperatively to complete the early steps of viral infection (Lee, Nagashima, and Hu, 2005). Therefore, we hypothesized that sharing the homologous CA between MLV and SNV Gag proteins is necessary but not sufficient for coassembly and functional complementation because other factors, such as the presence of homologous p12/p18 and CA, are also required. To test our hypothesis, we cotransfected two gag-pol expression constructs that both contain MLV p12 and SNV CA. We found that the viruses generated from such cotransfection did not restore MLV vector titers (Fig. 2B, pM/SCA and pS/Mp12); we also observed similar results from cotransfection of two gag-pol expression constructs that both contained SNV p18 and MLV CA (Fig. 2B, pM/Sp18 and pS/MCANC). These data are in sharp contrast to those obtained from cotransfection of vector pairs 3 and 7–10 in Fig. 2A, which recovered wild-type level viral titers. The important determinant is that in the aforementioned pairs in Fig. 2A, one of the vectors contained p12/p18 and CA from the same virus, whereas in the two pairs in Fig. 2B, both vectors contained p12/p18 from one virus and CA from another virus. These findings confirm our previous observation that homologous p12/p18 and CA are needed for efficient MLV replication, and demonstrate that homologous CA is necessary but not sufficient for functional coassembly and complementation of MLV and SNV Gag proteins.

Interactions and coassembly of MLV and SNV Gag proteins

The work described above indicated that CA plays a very important role in the functional complementation of MLV and SNV Gag proteins. Recent work on the coassembly of HIV-1 and RSV Gag indicated that homologous CA is required for the colocalization and coassembly of the heterologous Gag proteins. In our system, it was unclear whether homologous CA is required for the coassembly of the MLV and SNV Gag polyproteins, or is needed at a later stage during the replication of the coassembled virions.

To examine whether MLV and SNV Gag proteins colocalize and interact in cells, we performed the bimolecular fluorescence complementation (BiFC) assay on these proteins (Hu and Kerppola, 2003). In this assay, a fluorescent protein gene, such as the venus yellow fluorescence protein gene (yfp) (Nagai et al., 2002), was split into N-terminal (ny) and C-terminal (cy) portions and fused to proteins of interest. Neither the NY nor the CY portion of the YFP can confer fluorescence by itself. If the proteins of interest interact with each other and bring the NY and CY portions together, then fluorescence can be detected. If the two proteins of interest do not interact with each other, fluorescence is not detected.

We fused MLV gag to ny and cy to generate m-gag-ny and m-gag-cy, respectively (Fig. 4). Neither construct alone could generate YFP fluorescence signals (data not shown). However, when m-gag-ny and m-gag-cy were coexpressed, MLV Gag-Gag interaction allowed the complementation of NY and CY, thereby generating the YFP fluorescence signal (Fig. 5A, left panel). Similarly, coexpression of SNV gag fused to ny or cy (s-gag-ny and s-gag-cy) also generated YFP fluorescence signals (Fig. 5A, right panel). To test whether the YFP fluorescence signals were generated by specific protein-protein interactions, we used BiFC to examine the interactions between SNV Gag and a cell surface protein, human CD8 α unit (CD8) (Moody et al., 2001). In these experiments, a plasmid expressing red fluorescence protein gene (rfp) (Campbell et al., 2002) was added and used as a transfection control. We generated cd8-ny and cd8-cy constructs (Fig. 4). Human CD8 α unit is known to dimerize; upon coexpression of cd8-ny and cd8-cy, we observed YFP fluorescence signals (Fig. 5B, upper left panel). However, when cd8-ny and s-gag-cy were coexpressed, YFP fluorescence signals were not detected, although the RFP fluorescence signal was detected, indicating successful transfection (Fig. 5B, lower panels). Coexpression of cd8-cy and s-gag-ny, or coexpression of cd8-ny and m-gag-cy also failed to produce detectable YFP fluorescence signals (data not shown). These results indicated that BiFC can easily detect interactions between homologous Gag proteins, and the fluorescence signals detected were based on protein-protein interactions and not from nonspecific signals due to coexpression of ny and cy fusion proteins.

Fig. 4.

Fig. 4

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Structures of constructs used in the BiFC assay to examine Gag-Gag interactions in cells. MLV and SNV Gag sequences are shown as white and gray boxes, respectively. Human cd8 is shown as a hatched box whereas the hinge region and yfp sequences are shown as stippled boxes.

Fig. 5.

Fig. 5

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Analyses of Gag-Gag interaction by the BiFC assay. (A) Interactions of homologous Gag. (B) Interactions between CD8 and CD8, and SNV Gag and CD8. These samples were cotransfected with a plasmid that express rfp, which served as a transfection control. (C) Interactions between heterologous Gag proteins.

We then examined coexpression of two pairs of constructs containing chimeric or mutant gag genes. We generated constructs m-NC*-ny, m-Sp18-cy, and m-SCA-cy (Fig. 4), which contained the gag sequences of pWZH35, pM/Sp18, and pM/SCA, respectively. Coexpression of pWZH35 and pM/Sp18 resulted in successful complementation (Fig 2A, pair 3); as expected, we detected YFP fluorescence signals when m-NC*-ny and m-Sp18-cy were coexpressed (Fig. 5C, top left panel). Coexpression of pWZH35 and pM/SCA did not result in successful complementation (Fig. 2A, pair 11). However, abundant YFP fluorescence signals were detected when m-NC*-ny and m-SCA-cy were coexpressed (Fig. 5C, top right panel), indicating that these Gag proteins can interact despite their different CA domains. To further determine whether a homologous CA domain or any homologous domain was required for interactions between these heterologous Gag proteins, we coexpressed m-gag-ny and s-gag-cy that contained MLV and SNV Gag, respectively, and observed abundant YFP fluorescent signals (Fig. 5C, bottom panel). These data indicated that MLV and SNV Gag proteins can colocalize and interact in cells. Therefore, the requirement for homologous CA domain is likely to occur at a postassembly step(s).

Discussion

In this study, we investigated the requirements for functional complementation of Gag proteins from MLV and SNV, two genetically distinct gammaretroviruses. We used mutant or chimeric Gag proteins that could not efficiently support MLV vector replication, and tested whether two Gag proteins can functionally complement each other by the restoration of vector titers. We found that MLV and SNV mutant Gag proteins did not complement each other, whereas coexpression of chimeric Gag polyproteins containing the same CA domain could restore MLV vector titers. We then explored whether the homologous CA domain is required for the colocalization of heterologous Gag proteins, and found that SNV and MLV Gag proteins colocalize and interact in cells. From these results, we conclude that homologous CA domains are required for functional complementation of MLV and SNV Gag polyproteins and the requirement for homologous CA most likely occurs at a postassembly step(s).

A homologous N-terminal MA domain was needed for the coassembly of MLV and RSV Gag proteins, suggesting that these Gag proteins were targeted to different regions of the plasma membrane (Bennett and Wills, 1999). Previous studies indicated that membrane targeting is a prerequisite for Gag multimerization in both MLV and SNV (Schultz and Rein, 1989; Weaver and Panganiban, 1990). Our BiFC data demonstrated colocalization and interaction between MLV and SNV Gag proteins (Fig. 5C). In addition, our complementation results demonstrated that Gag with SNV MA and Gag with MLV MA could coassemble and generate infectious viruses (Fig. 2A, pairs 8–10). Taken together, these two lines of results indicate that MLV and SNV Gag proteins are targeted to the same locations of the plasma membrane during virus assembly.

In contrast to the coassembly of RSV and HIV-1 Gag proteins, a homologous CA domain is not needed for MLV and SNV Gag proteins to colocalize and interact with each other. Our observation also revealed that Gag proteins with and without the charged assembly helix (CAH) motif could interact. We previously described the CAH motif in MLV CA as a critical factor for proper assembly of the virus particle (Cheslock et al., 2003). This predicted helical motif is located at the C terminus of MLV CA. Deletions or other mutations that disturb the phases of the helices cause MLV assembly defects that can be identified phenotypically by drastic reduction of particle production and the formation of mutant virions with aberrant sizes and RNA content (Cheslock et al., 2003). The CAH motif is conserved among most gammaretroviruses, but not in SNV (Cheslock et al., 2003). Therefore, although CAH is strictly required for proper MLV virus assembly, interactions can occur between Gag with CAH and Gag lacking the CAH motif.

CA plays multiple roles during retrovirus replication. As a domain, CA plays an important role in virus assembly; as a mature protein, CA forms the virion core, participates in the uncoating process, and possibly plays a role in reverse transcription and the transport of the replication complex (Alin and Goff, 1996; Cairns and Craven, 2001; Lee, Nagashima, and Hu, 2005; Swanstrom, 1997). Among the MLV and SNV Gag domains, CA has the highest identity (42%) in amino acid sequence; the identities of MA, p12/p18, and NC are 31%, 23%, and 33%, respectively. There are certain similar features in the MLV and SNV CA sequences. Besides the major homology domains located near the central part of CA, there are patches of similar sequences elsewhere in CA; for example, the amino acid sequence in a part of the conserved α-helical structures, such as α3, is identical in MLV and SNV (Fu et al., 2006). Similar to HIV-1 CA, the mature MLV CA refolds after proteolytic cleavage and generates the β1-β2 N-terminal hairpin loop, which is critical for CA-CA interaction; this refolding is thought to involve the formation of a salt bridge between Pro 1 and Asp 54 in MLV CA (Mortuza et al., 2004). Interestingly, SNV CA also has Pro 1 and an equivalent Asp 60, which is embedded in the conserved α3 in a manner identical to MLV Asp 54. This sequence conservation suggests that a similar hairpin loop is also formed in mature SNV CA. Despite these conserved features between MLV and SNV CA, it is unclear whether these two mature CA proteins can properly interact with each other to form the virus core. Further studies are required to delineate the precise motif(s) or structure(s) that lead to the requirement for the same CA.

To our knowledge, there are three other studies examining elements needed for coassembly of heterologous Gag proteins. The previous studies using MLV and RSV, and RSV and HIV-1 used viruses from different genera. In both cases, a homologous domain is needed for coassembly of the viral proteins. We have performed this study and a separate study examining the interactions of Gag proteins from viruses of the same genus. In both studies, we found that the heterologous Gag proteins can colocalize and interact. In our study of the interactions of HIV-1 and HIV-2 Gag proteins, we found that these Gag proteins can coassemble and some mutants can functionally complement each other (Boyko et al., 2006). In the current study, we found that although MLV and SNV Gag proteins can interact, a homologous CA domain is needed for functional complementation of the two Gag proteins. Taken together, these studies indicate the different levels of interactions between heterologous Gag proteins and different barriers presented in the coassembly and functional complementation of the heterologous Gag proteins. One of the implications of coassembly of viral proteins is a potential to alter the property of the phenotypically mixed particles and provide a possible gateway for viruses to adapt to a new environment and to evolve. Therefore, our work not only has revealed elements important for coassembly and complementation of heterologous Gag, but also has important implications in virus evolution.

Materials and methods

Plasmid construction

Standard cloning procedures were used to construct all plasmids (Sambrook, 1989). The previously described plasmid pWZH30 (Zhang et al., 2002) was derived from pLGPS (Miller and Buttimore, 1986) and expresses MLV gag-pol from a truncated MLV 5′ long terminal repeat (LTR) but lacks the packaging signal and the 3′ LTR. Plasmid pRD136 has also been described (Martinez and Dornburg, 1995) and expresses SNV gag-pol.

All the mutant or chimeric gag-pol expression constructs were generated by overlapping PCR and cloning of the PCR fragments. To ensure that inadvertent mutations were not introduced into these chimeras or mutants, the sequences of all regions subjected to PCR were confirmed by DNA sequencing. Plasmids pM/Sp18, pM/SCA, and pM/SCANC have been previously described (Lee, Nagashima, and Hu, 2005). To generate pS/Mp12, pS/Mp12CA, pS/Mp12CANC, pS/MCANC, and pSΔPY, two PCR products were generated and later combined using overlapping PCR. DNA fragments containing a hybrid or mutant gag generated by overlapping PCR was digested with StuI and BlnI, and inserted into StuI-BlnI digested pRD136. Plasmids pM/SNC, pS/MNC, and pWZH35 have been described previously (Certo et al., 1999; Zhang et al., 2002).

All constructs used for the BiFC assay were derived from two expression vectors, HIV-2 gag-ny and HIV-2 gag-cy (Boyko et al., 2006). These constructs are HIV-1 based and contain HIV-1 LTRs, 5′ untranslated region, and truncations in pol, env, vif, vpr, and vpu; they also express HIV-1 tat, rev, and nef. Additionally, these two constructs express HIV-2 gag fused to either the ny portion or the cy portion of YFP. The ny portion contains amino acids 1–172 and the cy portion contains amino acids 173–238 of YFP; a hinge region is located between the gag and yfp sequences. DNA fragments containing various gag genes were amplified by PCR, digested with PauI and XmaI, and cloned into the PauI-XmaI-digested HIV-2 gag-ny or HIV-2 gag-cy constructs to replaced the HIV-2 gag in these constructs. The resulting constructs express various gag genes fused in-frame to ny or cy portions of yfp. Previously described cd8-ny and cd8-cy have the same structures, except that gag was replaced by the human CD8 α unit gene (cd8).

Cell culture, DNA transfection, and virus infection

D17 cells are dog osteosarcoma cells permissive to amphotropic MLV infection (Riggs, McAllister, and Lennette, 1974); 293T cells are human embryonic kidney cells (DuBridge et al., 1987). SR2-293T cells are a pool of 293T cells containing SR2 proviruses derived from the MLV-based vector pSR2 (Cheslock et al., 2000), which expresses the hygromycin phosphotransferase B gene (hygro) (Gritz and Davies, 1983) and the green fluorescent protein gene (gfp) (Chalfie et al., 1994). Cells were maintained at 37°C with 5% CO2 in Dulbecco’s modified Eagle’s medium supplemented with 6% calf serum (D17) or 10% fetal calf serum (293T) in the presence of penicillin (50 U/ml) and streptomycin (50 μg/ml). Transfection was performed using the calcium phosphate precipitation method (Sambrook, 1989) (MBS transfection kit, Stratagene) with a mixture of DNA containing the gag-pol expression construct and pSV-a-MLV-env at a 5:1 (w/w) ratio, respectively. Plasmid pSV-a-MLV-env expresses amphotropic MLV env (Landau, Page, and Littman, 1991). To examine whether two Gag proteins can functionally coassemble, SR2-293T cells were cotransfected with the same amount of each gag-pol expression construct.

Viruses were harvested 48 h posttransfection and filtered through a 0.45-μm-pore-size membrane (Millipore) to remove cell debris. Serial 10-fold dilutions of the virus samples were used to infect D17 cells in the presence of Polybrene (50 μg/ml final concentration). Infected cells were selected with hygromycin (120 μg/ml final concentration), and viral titers were determined by scoring the number of hygromycin-resistant colonies. For coinfection, the same volume of each virus supernatant was mixed, diluted serially, and used to infect D17 cells in the presence of Polybrene (50 μg/ml final concentration).

Western analyses and reverse transcriptase (RT) assays

Viruses were concentrated by ultracentrifugation at 25,000 rpm for 90 min at 4°C using a TH-641 rotor (Sorvall); pelleted viruses were then resuspended in phosphate-buffered saline for Western analyses and RT assays. Viral proteins in virions or in transfected cells were examined by Western analyses using polyclonal rabbit anti-MLV-CA, anti-MLV-MA (a kind gift from the AIDS Vaccine Program, SAIC-Frederick), or anti-SNV-CA (a kind gift from Nancy Rice, NCI-Frederick) antibodies. RT assays were performed using standard procedures (Cheslock et al., 2000; Temin and Mizutani, 1970).

Confocal microscopy

Plasmid DNAs were transfected into HeLa cells or 293T cells, and images were collected on a Zeiss 510 META confocal microscope with Plan-Neofluar 40 X/N.A. 1.3 Oil DIC objective, 514 nm or 543 nm laser excitation lines and the emission window of 527–559 nm for the venus YFP signals and 602–655 nm for RFP signals. The images were processed using Zeiss LSM 5 Image Browser and Adobe Photoshop 7.0.1. software.

Acknowledgments

We thank Dr. Vinay K. Pathak for discussions and intellectual input in this work. We also thank Anne Arthur for her expert editorial help, and Drs. Eric Freed, Alan Rein, and Vinay Pathak for critical reading of the manuscript. This research was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.

Footnotes

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