Cell, Vol. 63, 77-66,

October

5, 1990, Copyright

0 1990 by Cell Press

A Single Amino Acid Substitution within the Matrix Protein of a Type D Retrovirus Converts Its Morphogenesis to That of a Type C Retrovirus Sung S. Rhee and Eric Hunter Department of Microbiology University of Alabama at Birmingham Birmingham, Alabama 35294

Summary Two different morphogenic processes of retroviral capsid assembly have been observed: the capsid is either assembled at the plasma membrane during the budding process (type C), or preassembled within the cytoplasm (types B and D). We describe here a gag mutant of Mason-Pfizer monkey virus, a type D retrovirus, in which a tryptophan substituted for an arginine in the matrix protein results in efficient assembly of capsids at the plasma membrane through a morphogenie process similar to that of type C retroviruses. We conclude that a type D retrovirus Gag polyprotein contains an additional, dominant signal that pfevents immediate transport of precursors from the site of biosynthesis to the plasma membrane. Instead, they are directed to and retained at a cytoplasmic site where a concentration sufficient for self-assembly into capsids occurs. Thus, capsid assembly processes for different retroviruses appear to differ only in the intracellular site to which capsid precursors are directed. Introduction Regardless of whether they are enveloped or not, most viruses assemble a protein shell (or capsid) in which they package their genetic information prior to release from an infected cell. Although in a few viruses X-ray crystallographic studies (Burnett, 1985; Hogle et al., 1985; Luo et al., 1987; Roberts et al., 1986) and molecular genetic studies (Arnold et al., 1987; Chow et al., 1987; Gelderblom et al., 1987; Marc et al., 1989) have successfully elucidated the structure of capsids and the processes of capsid assembly, it remains unclear how the individually synthesized components are brought together into this noncovalently bonded, complex structure. This lack of understanding is even greater for the retrovirus family: viruses containing an RNA genome and an RNA-dependent DNA polymerase (Fenner, 1975). The mature retrovirus contains an icosahedral capsid that is composed of an envelope-associated outer shell and an inner ribonucleoprotein core (Marx et al., 1988; Nermut et al., 1972). These are in turn surrounded by a viral glycoproteincontaining lipid bilayer that is acquired during budding from the plasma membrane of the infected cell (Rifkin and Compans, 197l). The capsid protein subunits are synthesized as cytoplasmic polyproteins from genomic-sized mRNA molecules on free polysomes, and are assembled into an immature capsid either within the cytoplasm or at the plasma membrane, depending on the retrovirus. The polyproteins in the immature capsid

are then proteolytically cleaved to the structural proteins of the mature capsid during or shortly after virus budding. Early electron microscopic studies (Bernhard, 1958, 1960; Fine and Schochetman, 1978) revealed two morphologically distinct pathways for capsid assembly in different types of retroviruses. In type C morphogenic viruses, including type C oncoviruses (e.g., murine leukemia virus and Rous sarcoma virus) and lentiviruses (e.g., human immunodeficiency virus), the capsid is assembled from individually transported capsid precursor polyproteins (Gag polyproteins) at the cytoplasmic side of the plasma membrane. This assembly event occurs simultaneously with the process of virion budding from the membrane. In contrast, in type B and D viruses (e.g., mouse mammary tumor virus and Mason-Pfizer monkey virus [M-PMV], respectively) an immature capsid is preassembled within the cytoplasm prior to transport to the cell membrane. In the latter viruses, therefore, only the preassembled capsid appears to possess the necessary signals for intracytoplasmic transport to the plasma membrane. Indeed, our previous studies of M-PMV showed that myristylation of the Gag polyproteins plays a crucial role in conferring transport competency on preassembled type D capsids (Rhee and Hunter, 1987). In its absence, assembled capsids accumulated within the cytoplasm of infected cells; none were observed near the plasma membrane, and no virions were released. It has been shown for both types of retroviruses that in the absence of envelope glycoproteins capsids are still assembled and released from the cells in a manner equivalent to that seen with wild-type virus (Kawai and Hanafusa, 1973; Linial et al., 1980; Rhee et al., 1990). This indicates that the newly synthesized capsid polyproteins contain intrinsic information that specifies the site of capsid selfassembly and directs intracellular transport to the plasma membrane. Nevertheless, neither the detailed mechanisms of the assembly process nor the signals for intracellular transport are understood for either morphogenic group. In this paper we report on the identification of a signal in the Gag polyprotein of M-PMV, which is responsible for intracytoplasmic capsid assembly in type D retroviruses. A mutant (T2111R55W) of M-PMV that has two amino acid substitutions in the matrix (MA) protein at positions 21 (threonine to isoleucine) and 55 (arginine to tryptophan) shows an altered morphogenesis. Cells transfected with the mutant genome synthesized normal levels of Gag polyproteins that were processed somewhat more rapidly than in wild-type infected cells. However, in contrast to cells infected with wild-type M-PMV, no preassembled intracytoplasmic capsids could be detected, despite the fact that equivalent amounts of mutant virions were released into the culture medium. The released virions were, however, noninfectious. Electron microscopic studies of cells transfected with mutant M-PMV genomic DNA showed that a majority of the Gag polyproteins assembled into capsids at the plasma membrane through a morphogenic

CA I

I

oD2CM16

I

012

1

027

NC I

014

loll 1’

I

Figure 1. Schematio tants of M-PMV

Representation

of Mu-

The arrangement of the structural proteins within the gag gene-encoded polyprotein is Ihmdmamm~pw-~ wild-type schematicallv . oresented with the amino acid . sequences of wild-type p10 (MA) protein using T*,,,R55W 1. I:: _>l ~.:~p*)C~~i~~~&~~~ ,’ ,a’% a%&,;*; n.?(e-‘ . ij es single-letter amino acid codes. Mutant 72111 R55W has two amino acid substitutions, at poR55W 1 */ sitions 21 fthreonine to isoleucine) and 55 farginine to tryptophan) within the ‘MA protein. Mutant R55W contains a single amino acid substitution of tryptophan for the arginine residue at position 55. The amino acid sequences of MA protein were deduced from the nucleic acid sequences (Sonigo et al., 1986). The structural gag proteins, ~10, ~27, and ~14, were designated MA (matrix protein), CA (capsid protein), and NC (nucleocapsid protein), respectively (Bradac and Hunter, 1986b). /

“r_

process similar to that of type C retroviruses. Thus the two amino acid substitutions in the MA protein of mutant T2111R55W convert its morphogenesis from that of a type D retrovirus to one of a type C retrovirus. Oligonucleotidedirected mutagenesis yielded mutant R55W, which contained only the tryptophan for arginine substitution and demonstrated that this substitution alone in the MA protein is sufficient to modulate the site of capsid formation in type D retroviruses. We propose here a model for intracytoplasmic capsid assembly in which a signal within the newly synthesized Gag polyproteins of type D viruses appears to act dominantly to target molecules to a region within the cytoplasm where they can self-assemble into immature intracytoplasmic capsids. Without this dominant targetinglretention signal, the Gag polyproteins of M-PMV mutants and presumably type C viruses are individually transferred to a site at the plasma membrane where, concurrently with budding, they self-assemble into a membrane-associated capsid. Results T21l/R55W, a Mutant of M-PMV with ‘ILvo Amino Acid Substltutlons within the MA Protein Mutant l21llR55W is one of a series of mutants of M-PMV that was generated by the site-specific introduction of random point mutations within the MA protein coding domain through sodium bisulfite mutagenesis as described in Experimental Procedures. In this mutant, two amino acid8 within the MA protein, a threonine residue at position 21 and an arginine at position 55, were substituted by isoleutine and tryptophan, respectively (Figure 1). The mutated gag gene sequence was cloned into an infectious proviral genome (pSHRM15) (Rhee et al., 1990) in order to determine the effect of these amino acid substitutions on viral protein biosynthesis, virus assembly, and infectivity. The virus vector (pSHRM15) contains an SV40 origin of replication that allows transient, high level virus protein expression in COS cells. Mutant Gag Polyproteins Are Processed Rapidly into Maturr, Capsid Proteins In M-PMV-infected cells, three gag-related precursor polyproteins (Pr180, Pr95, and Pr7sBag) and one env precursor

glycoprotein (Pr88B”v) are produced (Bradac and Hunter, 1984, 1988a). Pr180 and Pr95 are the precursors of the RNA-dependent DNA polymerase (reverse transcriptase, RT), and the viral protease, respectively, and are presumably synthesized via a frameshifting mechanism as a gag-pro-@fusion protein of 180 kd and agag-pro fusion protein of 95 kd (Bradac and Hunter, 1984; Sonigo et al., 1988). Pr78s8s is the major gag precursor polyprotein (Gag polyprotein) and functions as the predominant protein subunit of the intracytoplasmically assembled capsid. The Gag polyproteins of immature capsids are subsequently cleaved to yield the six nonglycosylated structural protein8 (~10, ~~24-18, ~12, ~27, ~14, and p4) of the mature capsid during or shortly after virus budding (Bradac and Hunter, 1984,1988b; Henderson et al., 1985). Theenv precursor, Pr88em, is cleaved by a host-derived protease into two glycosylated proteins (gp70 and gp22) in the late Golgi complex (Bradac and Hunter, 1988a; Hunter, 1988). The cell-associated form of the M-PMV transmembrane (TM) protein, which we term here gp22, is further proCeSSSd following virus release, pre8UNiably by the virusencoded protease, to gp20 (Rhee, Brody, and Hunter, unpublished data) in a manner analogous to that seen in murine leukemia virus (Karshin et al., 1977). To determine whether mutant Gag polyproteins were synthesized and processed into mature structural proteins in a wild-type manner, 48 hr after transfection COS cells were pulse-labeled with [3H]leucine for 20 min and chased for 2, 4, and 12 hr (Figure 2A). In cells expressing either wild-type or mutant genomes, similar levels of viral precursor8 were synthesized and processed into mature proteins (Figure 2A). In the pulselabeled cells (lane l), the three gag-related precursors (Pr7Bee9, Pr95, Pr180) and the env precursor (Pr889 could be identified. In mutant infected cells, an additional band of 27 kd, corresponding to the major capsid protein ~27, was detected (Figure 2A lane 1). In wild-type infected cells p27 appeared only after a 2 hr chase (lane 2). This result indicates that the processing of mutant Gag polyproteins into mature capsid proteins is taking place within the 20 min pulse-labeling period. In both sets of cells, decreasing amounts of cell-associated p27 were detected following 4 hr and 12 hr chases (Figure 2A, lanes 3 and 4), consistent with release of capsid proteins from cells (see below). These results indicate that the genetic

Morphogenesis 79

Mutant

of Mason-Pfizer

A

Monkey

Virus

B virion-associated

cell-associated wild-type 1234

wild-type

TZll/R55W

TZll/R55W

-7-i-T 123

1234 &- Pm 4 gp70 +

p27 +

Figure 2. lmmunoprecipitation Proteins

4 p27 + 4 gp22 + QP20 +

of Cell-

and

Virion-Associated

Viral

COS cells were transfected with either wild-type pSHRM15 or mutant pT2111R55W DNAs and pulse-labeled with 13H]leucine for 20 min. After a 2, 4, and 12 hr chase, virus-specific intracellular (cell-associated) (A) and extracellular (virion-associated) (6) proteins were immunoprecipitated with anti-M-PMV antiserum as described in Experimental Procedures. (A) In wild-type cells, the gag-related precursor polyproteins (Pr160, PI%, and Pr7w and the env precursor (Pr66y are pulse-labeled (lane I), and their processed products (pm, gp70, and gp22) appear after a 2 hr chase (lane 2). In contrast, in mutant cells bands of p27 as well as precursor polyproteins can be detected after the 20 min pulse-labeling (lane 1). Amounts of both precursors and mature proteins decrease following 4 hr (lane 3) and 12 hr (lane 4) chases in both sets of cells. (B) Extracellular virions were pelleted from the culture fluids of pulselabeled cells after 2 hr (lane l), 4 hr (lane 2), and 12 hr (lane 3) chases, then immunoprecipitated as above.

changes in mutant T2111R55W have no effect on Gag polyprotein biosynthesis, but do result in more rapid precursor processing. Mutant T2111R55W Genome-lkansfected Cells Release Vlrlons Into the Culture Medium, but They Are Noninfectious The results described above showed that mutant Gag polyproteins are processed into mature proteins, and indicated that virus particles were released from the mutant infected cells since the processing of retroviral capsid precursors takes place at a late stage of virus budding from the plasma membrane. However, we could not rule out the possibility of abnormal budding of mutant viruses into intracellular vesicles, which might account for the rapid processing of mutant Gag polyproteins. This was investigated by analysis of sedimented extracellular virions, together with electron microscopic studies of mutant infected cells. An initial analysisof virus particles released into the culture supernatant was made in the pulse-chase experiment described above. Radiolabeled extracellular virions were pelleted from the culture medium of pulse-labeled cells after 2, 4, and 12 hr chase periods, and viral proteins in the pellet were immunoprecipitated and analyzed by SDSPAGE (Figure 28). Both wild-type and mutant DNA-transfected COS cells released viruses into the culture media. In wild-type transfected cells virion-associated p27 as well as two glycoproteins, gp70 and gp20, increased during the

2 hr and 4 hr chases (lanes 1 and 2), and remained stable after a 12 hr chase (lane 3). This indicates that a majority of the pulse-labeled viral proteins are assembled into virions within 4 hr. This is consistent with our previous studies in which we have observed a half-time of Gag polyprotein processing of 2-3 hr (Rhee and Hunter, 1990). In contrast, in mutant genome-transfected cells, a majority of pulse-labeled Gag polyproteins were released as virions during the 2 hr chase period (Figure 28, lane 1) and no significant increase in p27 could be seen following 4 hr and 12 hr chases (lanes 2 and 3). Taken together with the observation of rapid processing of Gag polyproteins in mutant transfected cells, mutant virus particles thus appear to be formed and released with more rapid kinetics than that of wild-type M-PMV It should be noted that much less gp70 and gp20 is found associated with mutant virions (compare the ratios of glycoprotein gp70 to capsid protein p27 in mutant virions with those in wild-type virions), and the kinetics of incorporation of glycoproteins into virions is much slower than that observed with wild-type virus. These results suggest that mutant Gag polyproteins are rapidly assembled into a virion particle and released from the cells, but that the viral glycoproteins are recognized and incorporated into the particles much less efficiently during budding. It was of interest to determine whether the mutant viruses released from T2111R55W genome-transfected cells were infectious. COS cells were transfected with either wild-type or mutant DNAs, and 46 hr after transfection viruses released into the culture media were harvested for 24 hr. An equal number of virus particles, measured by levels of both RT activity and p27 capsid proteins (Figure 3, inset), were used to infect monolayers of HeLa cells, which are susceptible to infection by M-PMV. The spread of infectious virus through the culture was monitored by RT assays of the material pelleted from culture fluids at various days postinfection. Wild-type virus induced the release of RT-containing containing virions 4 days after infection, and a rapid increase in enzyme activity was observed 6 days postinfection. In contrast, no RT activity was detected in the culture fluids of mutant virus-infected cells. This demonstrates that while #r-containing mutant virions are assembled and released from genome-transfected cells, they are noninfectious. Mutant Gag Polyproteins Are Not Preassembled into an lntracytoplasmic Capsid Since mutant viruses containing normally processed capsid proteins are released from T21I/R55W-infected cells, the mutant Gag polyproteins appear to assume a conformation that allowed the intermolecular interactions necessary for capsid self-assembly and precursor cleavage. Moreover, these stages of mutant virus assembly and maturation were shown to occur faster than those of wildtype M-PMV. To examine this further, we initially determined whether mutant Gag polyproteins were assembled into intracytoplasmic capsids with normal kinetics. We have shown that in M-PMV-infected HeLa cells half of the newly synthesized Gag polyproteins are incorporated into

Cdl 60

Two HeLa cell lines, expressing either the mutant genome (line T2111R55W) or wild-type genome (line MPMV15), were pulse-labeled for 20 min and chased for the times indicated (Figure 4A). Cells were lysed in Triton X-100 lysis buffer, and the radiolabeled Gag polyproteins were fractionated by sedimentation into two forms: free polyproteins remained in the soluble fraction (S) and those incorporated into capsids were recovered in the pellet fraction (P). As observed previously (Rhee and Hunter, 1990) newly synthesized Gag polyproteins in wild-type infected cells are found predominantly in the soluble fraction after the pulse-label (Figure 4A, lanes 1 and 6). Approximately 25%~ of the molecules appeared to have been incorporated into capsids during the 20 min pulse; the fraction increased following chases (lanes 2-5 and 7-10). Therefore in M-PMV-infected cells Gag polyproteins are assembled with a half-time of 45 min into intracytoplasmic immature capsids, which are stable and pelletable under mild detergent conditions. Mature capsids appear to be disrupted under these conditions since p27 is found in the soluble fraction after a 3.0 hr chase (Figure 4A, lane 4). This is consistent with earlier observations that the mature capsid of retroviruses is a relatively fragile structure that is easily disrupted in nonionic detergent (Stromberg et al., 1974). In the mutant genome-transfected cells, no Gag polyproteins were recovered in the pelleted fraction during the pulse-labeling period (Figure 4A, lanes 1 and 2) nor after chase periods as long as 4.5 hr (lanes 3-6). Interestingly, processing of the mutant polyproteins was still observed and a band of p27 could be seen in the soluble fractions after a 1.5 hr (Figure 4A, lane 3) and 4.5 hr chase (lane 5). These results thus suggest that in mutant transfected cells, virus particles are released without the formation of stable, pelletable immature capsids within the cytoplasm. The possibility that mutant Gag polyproteins form capsids with slower kinetics than wild-type proteins, although unlikely given the detection of p27 during the chase period, was ruled out by Western blot analysis. Crude intracytoplasmic capsids were prepared from wild-type and

gp70+

p27 + OP20 +

Figure

3. Determination

of the Infectivity

of Mutant

Virions

Virus-containing culture medium was harvested from COS cells transfected with either wild-type or mutant T2111R55W DNA% and used to infect HeLa cells. Culture medium from these infected HeLa cells was assayed for RT activity at days I, 4, 6, 6, 10, and 12 postinfection. Wildtype infected cells (closed circles) show a rapid increase in RT activity 6-10 days after infection, reflecting the rapid spread of infectious viruses through the culture. In contrast, in mutant infected cells (open circles), no FtT activity above that detected with uninfected cells (closed squares) was observed. (Inset) Autoradiogram of radiolabeled, extracellular virion proteins (lane I, wild-type; lane 2, mutant T2111R55W). After the medium was harvested for infection of HeLa cells, the COS cells were pulse-labeled then chased for 24 hr, and released virions were pelleted from the culture medium and immunoprecipitated with anti-M-PMV antiserum.

capsids within 45 min (Rhee and Hunter, 1990). To compare the rate and extent of capsid formation in mutant and wild-type virus-infected cells, the mutant T21llR55W genome was introduced into HeLa ceils, and both cell populations and cell clones expressing the noninfectious mutant genome were selected as described previously (Rhee et al., 1990). B

A

Figure

4. lntracytoplasmic

Capsid

Formation

(A) HeLa cell lines, expressing either wild-type 123 or mutant l2111R55W genome, were pulse12345 6 7 8 910 123456 labeled with [3H]leucine, then chased in complete medium for the indicated periods. Cells Pr95+ were lysed, and assembled capsids were pelPr78-e leted by centriiugatiin. Radiolabeled Gag poly m+ proteins in the soluble (S) and pellet (P) fractions were immunoprecipitated with anti-p27 antiserum. In wild-type cells, Gag polyproteins p27 + can be detected in both soluble (lane 1) and pellet (lane 6) fractions after pulse-labeling. During the chase periods (lanes 2 and 7,0.5 hr SPSPSP chase; 3 and 6,1.5 hr; 4 and 9,3.0 hr; 5 and 10, soluble pellet 4.5 hr), a decreasing amount of Pm can be seen in the soluble fractions (lanes 2-5) with concomitant increases in the pellet fractions (lanes 7-10). In contrast, in mutant cells no Gag polyprotein can be detected in the pellet fraction after the 20 min pulse (lanes 1 and 2) nor after the chases (lanes 3 and 4, 1.5 hr chase; 5 and 6, 4.5 hr). (8) Western blot analysis of crude intracellular capsids prepared from lysates of HeLa cell lines, expressing either wild-type (lane 2) or mutant T2111R55W genome (lane 3). PrQ5 and Pr7w in the capsid pellet from wild-type cells can be detected with rabbit anti-p27 antiserum (lane 2) while no bands of virus-specific polyproteins can be seen in the mutant cells (lane 3). A cellular protein (arrowhead) that is nonspecifically cross-reactive with the anti-p27 antiserum can be seen in the uninfected HeLa cells (lane 1) and both sets of infected cells (lanes 2 and 3). wild-type

T21 llR55W

Morphogenesis 01

Mutant

of Mason-Pfizer

Monkey

Virus

mutant cells by pelleting 0.5% Triton X-100 lysates through a 30% sucrose cushion. The Gag polyproteins in the pellets were separated on a 10% polyacrylamide gel and immunoblotted with anti-p27 antibody after transfer to a nitrocellulose membrane (Figure 48). The typical bands of the gag-related polyproteins, PrS5 and Pr78sag, can be seen in the wild-type infected cells (lane 2) while no proteins are detected in the mutant cells (lane 3). This result supports the data obtained in the pulse-chase fractionation experiment and suggests that the mutant Gag polyproteins, containing two amino acid substitutions in the MA protein, are not preassembled into an intracytoplasmic capsid prior to release in virions. Mutant Capsids Ate Assembled at the Plasma Membrane through a Morphogenic Process Similar to That of Type C Retrovlruses Although the data described above suggested that T2111 R55W virions were assembling at the plasma membrane concomitant with budding rather than within the cytoplasm, it was still possible that detergent-sensitive intracytoplasmic capsids were being assembled. To distinguish between these two possibilities, electron microscopic studies of COS cells transfected with either wild-type or mutant pT2111R55W DNAs were performed (Figure 5). Typical fields of M-PMV-infected cells are shown in Figure 5A; preassembled capsids were observed scattered throughout the cytoplasm, and numerous intact capsids were found at the plasma membrane in the process of budding (Figure 58 in high magnification). In contrast, in the majority of mutant genome-expressing cells, no intracytoplasmic capsids were found (Figure 5C). Instead capsids could be observed assembling at the plasma membrane as they budded from the cell (Figures 5C and 50). This observation confirmed that the two amino acid substitutions in the MA protein in mutant T2111R55W convert the morphogenic process from that of a type D retrovirus to one of a type C virus. This morphogenic conversion in mutant virus assembly presumably results from inefficient arresting of mutant Gag polyproteins within the cytoplasm, thereby allowing individual Gag polyproteins to be transported to a site on the plasma membrane where they can self-assemble into membrane-associated capsids. In a small fraction of mutant virus-producing COS cells both plasma membrane-assembling capsids and accumulations of intracytoplasmic capsids were observed (Figure 5F). In these cells the rate of protein synthesis presumably exceeded the capacity for plasma membrane transport, allowing intracytoplasmic capsid assembly to occur. We have never observed intracytoplasmic assembly of the l2111R55W mutant proteins in HeLa cell clones, which express at a lower level. A Single Amino Acid Substitution in the MA Protein Is Sufficient to Alter the Site of M-PMV Capsid Assembly As T2111R55W contained two amino acid changes, we were therefore interested to determine whether either amino acid substitution by itself would be sufficient to modulate the site of capsid formation in M-PMV Since so-

dium bisulfite mutant l211, with a single isoleucine for threonine substitution at position 21, exhibited a wild-type phenotype (Rhee and Hunter, submitted), mutant R55W was constructed by use of oligonucleotide-directed mutagenesis. COS cells were transfected with the mutant genome and examined by electron microscopy (Figure 5E). As with the double mutant T21l/R55W, mutant viruses were found to assemble at the plasma membrane, and in a majority of the cells no preassembled capsids could be observed within the cytoplasm. Thus, the single substitution of a tryptophan residue for an arginine at position 55 in the MA protein is sufficient to modulate the site of virus assembly and to convert this type D virus to type C morphogenesis. Discussion In this article we describe a morphogenesis mutant of the prototype type D retrovirus M-PMV, a virus that normally preassembles immature capsids in the cytoplasm that are then transported to the plasma membrane. The single amino acid substitution of tryptophan for arginine in the MA protein of the M-PMV mutants (T21llR55W and R55W) we describe here alters the site of capsid assembly from a region within the cytoplasm to the inner surface of the plasma membrane, effectively converting its type D morphogenesis to that of a type C retrovirus. In mutant genome-transfected cells, capsid polyproteins are transported to and assemble at the membrane concurrently with virus budding, which is typical of the capsid assembly and budding processes of the mammalian leukemia viruses and lentiviruses. This dramatic phenotype demonstrates that there are no major differences in the capsid assembly process of the two morphogenic groups of retroviruses; type D capsid polyproteins clearly can efficiently assemble at both intracytoplasmic and plasma membrane locations. Moreover, it implies that retroviral capsid assembly can occur at whatever intracellular site the nascent precursor proteins are initially transported to, provided that they accumulate to a high enough concentration for protein-protein association and assembly to occur. This conclusion is supported by our observation that approximately 10% of COS cells transfected with mutant genomes of either T2111R55W or R55W show both intracytoplasmic capsids similar to those of wild-type M-PMV and membrane-associated assembling capsids (Figure 5F). However, in these cells the majority of budding structures resulted from Gag polyproteins assembling at the membrane; few preassembled capsids were found near the membrane and mutant capsids accumulated deep in the cytoplasm. Thus, Gag polyproteins with the arginine residue substitution in the MA protein can under certain conditions form capsids in the cytoplasm. Since this was not seen in HeLa cell clones expressing the mutant genomes, it is likely that in a small percentage of transfected COS cells the level of precursor protein expression was sufficiently high to overload the plasma membrane transport process and allow intracytoplasmic capsid assembly. The intracytoplasmic assembly of nonmyristylated simian

Figure 5. Electron Micrographs of COS Cells Expressing Wild-Type and Mutant Viral Genomes (A and B) Thin sections of wild-type genome-tranefected ceils; intracytoplaemic capsids (arrowhead) assembled within the cytoplasm and complete capsids can be seen budding (arrow) through the plasma membrane. (C-E) No preaeeembled, intracytoplaemic capeids were obeenred in the eections of COS cells producing mutant T2111R55W (C and D) and mutant R55W (E). Instead, capsids assembling at the plasma membrane with typical type C morphology can be eeen (arrow). (F) In sections of a small fraction of mutant T2ll/R55W-transfected COS cells, large accumulations of capeids in the cytoplasm (arrowhead) as well as assembling capsids at the membrane (arrow) can be seen. No budding of preaaaembled capsids could be seen. Magnifications: 30,000x (A, C, E), 37,000x (F), 78,OWx (B).

immunodeficiency virus lowing high level expression insect cells (Delchambre

Gag polyproteins

observed folfrom a baculovirus vector in et al., 1989) may represent an

analogous situation. Virions released from T21I/R55W-transfected

cells are

noninfectious. The basis for this lack of infectivity has not been defined at the present time; however, it may reflect the underrepresentation of viral glycoprotein within the vi-

rus particle. Both pulse-chase and immunoblot experiments (data not shown) have shown that the gp70-gp20

Morphogenesis

Mutant

83

of Mason-Pfizer

Monkey

Virus

complex is inefficiently incorporated into budding virions. The only mutation in these viral genomes is within the matrix protein coding region; thus it is possible that the changes within p10 (MA) abrogate a Gag-Env (TM) interaction that is necessary for efficient glycoprotein insertion into virions. This possibility is supported by our recent observation that a postbudding cleavage of the TM cytoplasmic domain, similar to that seen in murine leukemia virus (Karshin et al., 1977), is blocked in mutants with other mutations in the matrix protein (Rhee, Brody, and Hunter, unpublished data). Indeed, in T2111R55W virions released during a 12 hr chase (Figure 28, lane 3), approximately equal amounts of cleaved gp20 (TM) and uncleaved gp22 (TM) are found, indicating that this processing event is inefficient in this mutant also. An alternative reason for the abnormally low level of glycoprotein in mutant virions, which we cannot rule out at this time, is that the site of budding or the budding process itself is altered such that glycoproteins have reduced access to the extruded membrane. Future studies should allow us to delineate the basis of the defect in infectivity. The intracellular transport of proteins from their site of biosynthesis to their site of activity is a complex and highly regulated process in the cell. The (topogenic) signals in proteins that define their intracellular destination remain a central question in cell biology, and through molecular genetic studies some of these signals are being elucidated. The characteristics of secreted and membrane-spanning proteins that commit them to the secretory pathway have been described (Blobel, 1980; Blobel and Dobberstein, 1975; Palade, 1975; Sabatini et al., 1982), and sequences within the primary structure of proteins destined for the nucleus, mitochondria, and other organelles have been identified (Hase et al., 1984; Kalderon et al., 1984a, 1984b; Schreier et al., 1985). However, the intracellular trafficking of cytoplasmic proteins is not as well understood, even though it is likely that is also a highly directed and regulated process. Our previous studies on a mutant defective in myrlstylation of the M-PMV Gag polyproteins suggested that cotranslational addition of this fatty acid is required for the targeting of precursor molecules to the plasma membrane (Rhee and Hunter, 1987). Similar studies on the murine leukemia virus Gag precursors (Rein et al., 1988; Schultz and Rein, 1989) and on the src oncogene (Buss et al., 1988; Kamps et al., 1988; Pellman et al., 1985;

M-PMV MMN

i ~~~ELSQ~R-‘~QL-R~A-DLLTC

(1

(

I

I

I

Ii

II

I

I

I

1 MSVSGSKU2KLFJSCZQ~~G~S-SAI~QFL-IK-VS II I I

Resh, 1989) have also demonstrated that myrietylation is important for membrane targeting. Thus, studies of retrovirus capsid proteins may provide additional insights into general mechanisms of intracytoplasmic protein transport, since these viral components are synthesized as cytoplasmic proteins on free polysomes and are then transported to the inner face of the plasma membrane, where virus budding occurs. We interpret the results presented here as evidence for a specific, dominant targeting signal within the nascent Gag polyprotein that prevents transport of individual precursor molecules directly to the plasma membrane and instead directs them to a site for intracytoplasmic assembly. Electron micrographs of our previously described myristylation-defective mutants (Rhee and Hunter, 1987) and of additional mutants arrested in transport after assembly (Rhee and Hunter, submitted) suggest that this assembly site may be a specific, ribosome-free subcompartment of the cytoplasm. Since the R55W mutant Gag polyproteins are efficiently and rapidly transported to the inner surface of the plasma membrane, these nascent molecules must contain appropriate (plasma membrane) targeting information for this transport step, which is presumably equivalent to those in type C morphogenic viruses. While it is probable that the wild-type-M-PMV Gag polyproteins also possess this signal, we hypothesize that it is overriden by the dominant cytoplasmic targeting/retention signal present in individual precursor molecules. However, once capsids have been assembled at the cytoplasmic site, they must acquire or reexpress a signal that targets them specifically to the plasma membrane. We have shown previously that this is an intrinsic property of the assembled capsid and is independent of interactions with viral glycoprotein8 (Rhee et al., 1990). It remains to be determined whether the mutation described here disrupts a specific topogenic signal within the Gag polyprotein or merely induces a conformational change that results in altered transport. It is of interest, however, that the region within MA in which this mutation is located shows high sequence conservation when the matrix proteins of M-PMV (Sonigo et al., 1988) and mouse mammary tumor virus (Moore et al., 1987) are compared (Figure 8). A much higher level of sequence divergence exists when the MA protein of the type C Moloney murine leukemia virus (Shinnick et al., 1981) is compared with

Figure 8. Amino Acid Sequence Comparison

PWPQEc;rIDI&Wt"G DCFWY-IllIll.. 1.111 PWPEE~It5ZWKXVG RFN?PY-I I I r-7II !

of MA Protein of M-PMV and Amino Termini of gag Precursor Polyproteins of Mouse Mammarv Tumor Virus and Murine Leukemia Virus The predicted amino acid sequence of the MA protein of M-PMV (Sonigo et al., 1986) is aligned with the amino-terminal 100 amino acids of mouse mammary tumor virus (MMTV; Moore et al., 1987) and Maloney murine leukemiavirus (Mo-MuLV; Shinnick et al., 1981). The region in which the mutation described here

I 62 'IQVKIKVFSPGPHGHPCYI VIWEUXDPPPWWF-----

(asterisk) is located is displayed in the open box. Bars indicate identities; dots show conserved amino acid changes. The dashed lines are spaces introduced in the sequences to maximize homologies identical or conserved between M-PMV and MMTV

between

proteins.

In the boxed

region,

55.6%

of the residues

are identical

and 72.2%

are

Cell 84

those of the tvoe 6 and D viruses. It is temotina to soeculate therefore*that this short stretch of amino acids directly interacts with the host cell transport machinery. There is a growing awareness of the participation of socalled chaperone proteins in a variety of intracellular transport processes (Hemmingsen et al., 1988; Pelham, 1988; Reading et al., 1989). Many of these appear to be members of the heat shock family of proteins, and while none have been directly shown to mediate intracytoplasmic transport, one of the proteins, hsp90. associated with the cytoplasmic form of pp8VL (&ugge et al., 1983; Courtneidge and Bishop, 1982; Garber et al., 1985) is a member of this class. It seems likely then that different cellular proteins might be involved in directing wild-type and mutant Gag polyproteins to cytoplasmic versus plasma membrane locations and that the mutants described here, together with other transport mutants, will be valuable tools in dissecting the transport pathways that exist within eukaryotic cells. ExperImental Procedures In Vltm Mutegenesls Sodium bisulfite mutagenesis was carried out as previously described (Everett and Chambon, 1982; Shortle and Nathans, 1978) with some modification, The single-stranded DNA of MlSGAG, a mutagenesis vector containing the entire coding sequence of the MA protein (Rhee and Hunter, 1987) was mixed with linear, double-stranded replicative form DNA of M13.X.d11/94. a deletion clone of MlbGAG containing a 252 bp deletion within the MA protein coding domain (Rhee and Hunter, submitted). The mixture was treated with 3 M sodium bisulfite at 3pc for 1 hr. and the single-stranded region of the mutated heteroduplex was then filled in at 16% for 4 hr with 1.5 U of DNA polymerase I in the presence of all four deoxynucleoside triphosphates at a concentration of 100 pM each. Details will be described elsewhere (Rhee and Hunter, submitted). To substitute a tryptophan residue for the arginine at position 55 in the MA protein, oligonucleotide-directed mutagenesis was carried out on single-stranded M13.GAG DNA as previously described (Zoller and Smith, 1984) After mutagenesis, a0.8 kb Narl-Sstl fragment was excised from the replicative form of mutant phage and substituted for the wild-type fragment in an M-PMV expression vector, pSHRM15 (Rhw et al., 1990). This plasmid contains a nonpermuted infectious M-PMV genome, derived from the integrated provirus clone pMPMV6A/7 (Barker et al.. 1985) and a hygromycin resistance gene under control of the SV40 early promoter. The presence of the mutations was confirmed by dideoxy sequencing of the double-stranded DNA (Sanger et al., 1977). The mutant constructions were designated pT2111R55W and pR55W. Cells and Transfectlon A cell clone containing an integrated copy of the mutant proviral DNA (line T2111R55W) was established as described previously (Rhee et al., 1990). The mutant viral DNA pT2111R55W was linearized with Fspl and transfected into semiconfluent monolayers of HeLa cells by the calcium phosphate precipftation method (Graham and van der Eb, 1973; Stow and Wilkie, 1978). Resistant cell colonies were selected in medium containing 250 U/ml of the antibiotic hygromycin Band screened to determine whether they expressed viral structural proteins as previously described (Rhee and Hunter, 1987). For the transient expression of viral proteins in COS cells, the cells were transfected with either wild-type pSHRM15 or mutant DNA (10 ug/SO mm plate) by the modified calcium phosphate precipitation method as described by Chen and Okayama (1987).

Redlolebellnf~

and Immunopreclpitetlon

of Virus

PnXelns

Forty-eight hours after transfection COS cells were pulse-labeled for 20 min with pH]leucine (0.8 mCi/ml, 157 Ci/mmol; Du Pant) and chased for the indicated period in complete growth medium (Rhee and

Hunter, 1967). Cells were lysed in lysis buffer A (1% liriton X-100, 1% sodium deoxycholate, 0.15 M NaCf, 0.05 M Tris [pH 7.51) and cellassociated viral proteins were immunoprecipitated with goat anti-MPMV antiserum (Division of Cancer Cause and Prevention, National Cancer Institute) as previously described (Bradac and Hunter, 1984). Radiolabeled virus particles, which were released from the pulselabeled cells into the culture medium, were pelleted by centrifugation for 10 min at 80,000 rpm in a Beckman TLAIOO rotor at 4%. The virus pellet was lysed in lysis buffer B (0.1% SDS, 1% Briton X-100, 1% sodium deoxycholate, 0.15 M NaCI, 0.05 M Tris [pH 751) and virionassociated viral proteins were immunoprecipitated with antiserum against M-PMV proteins as described above. Virus polypeptides were separated with a 10% resolving gel by SDS-PAGE (Bradac and Hunter, 1984). The amount of [3H]leucinelabeled proteins was quantitated by counting the radioactivity of the band in a liquid scintillation counter (Wills et al., 1984).

Fractionation and Western

of Gag Polyprutein, Blot AM~~SS

Capsid

Pmperetion,

Gag polyproteins were fractionated into free and capsid-associated forms as described previously (Rhee and Hunter, 1990). Briefly, HeLa cell lines were pulse-labeled and chased as described above. Cells were lysed in 1 ml of Triton X-100 lysis buffer (0.5% Triton X-100, 0.25 M sucrose, 1.0 mM EDTA, 0.14 M NaCI, 10 mM Tris [pH 7.51) at room temperature for 1 hr, and the lysates were centrifuged at 80,000 rpm for 10 min in a Beckman TLAlOO rotor at 4OC. The supernatant fraction containing soluble Gag polyproteins was adjusted to the composition of lysis buffer B by addition of appropriate detergent and salt. The pellet fraction containing capsid-associated Gag polyproteins was resuspended in lysis buffer 8. Viral proteins were immunoprectpitated with rabbit anti-p27 antiserum and analyzed by SDS-PAGE as described above. lntracytoplasmic capsids were pelleted from the lysates of HeLa cell clones in Triton X-100 lysis buffer through a 30% sucrose cushion as previously described (Rhee and Hunter, 1987). Proteins in the pellets were separated by SDS-PAGE and immunoblotted with rabbit anti-p27 antiserum and 1251-labeled protein A (0.06 mCi/ml. 2-10 mCi/mg; Du Pont) as previously described (Rhee and Hunter, 1990).

Cell-Fme

Infection

and RT Assay

Infectivity of the mutant virions was determined by measuring the spread of RT-containing virus through the culture at various times postinfection. Culture fluids were harvested for 24 hr from COS cells that had been transfected with either wild-type or mutant proviral DNA. After clarification by centrifugation at 10.000 rpm for 10 min in a Beckman JA20 rotor at 4oC, the level of RT activity in the medium was measured. An equivalent amount of RT-containing medium was used to infect fresh HeLa cells. Virus infection was carried out in the presence of 2.0 ug/ml of Polybrene (hexadimethrine bromide; Sigma) at 3pc for 1.5 hr and maintained for an additional 22 hr in complete growth medium. Culture fluid from the cells was harvested on days 1. 4, 6. 8, 10, and 12 postinfection, and the assays for RT were carried out as described previously (Rhee and Hunter, lge7; Sacks et al., 1978) with the following modifications. A 7.5 pl portion of the disrupted virus was incubated at 3pc for 1 hr with 30 pl of the RT mixture, which contained poly(rA)-oligo(dT) as synthetic RNA template-DNA primer (Boehringer Mannheim Biochemicals) and 13H]TTP (57 Cilmmol; Amersham Corp.). The reaction was terminated with a final concentration of 50 mM sodium pyrophosphate on ice, and the mbtture was spotted onto an NA45 Anion exchange membrane (Schleicher & Schunell, Inc.). Membranes were air dried and washed thoroughly in 0.5 M phosphate buffer (pH 6.5). The extent of DNA synthesis by RT activities was measured by counting the radioactivities on the dried membrane in an aqueous scintillation cocktail (Budget Solve; Research Products Int. Corp.).

Electron

Ylcmscopy

COS cells transfectad with virus DNA were prepared for electron microscopy as described previously (Compans et al., 1966). Cells were fixed for 1 hr at room temperature with 1% gltiraldehyde and then washed in phosphate-buffered saline. After postfbration wkh 1% osmium tetroxide, cells were embedded in an epoxy-resin mixture, sectioned, and stained with uranyl acetate and lead nitrate. All preparations were examined in a Philips 301 electron microscope.

Morphogenesis 05

Mutant

of Mason-Pfizer

Monkey

Virus

Acknowledgments

Fenner, virology

We thank Eugene Arms and Lawrence Ft. Melson, UAB Comprehensive Cancer Center electron microscope core facility, for excellent technical assistance in the electron microscopic studies. This work was supported by Public Health Service grant CA 27634 from the National Cancer Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 16 USC Section 1734 solely to indicate this fact.

Fine, D., and Schochetman, G. (1976). Type D primate review. Cancer Res. 38. 31233139.

Received

F (1975). The classification 6, 1-12.

and nomenclature

Garber, E.

A single amino acid substitution within the matrix protein of a type D retrovirus converts its morphogenesis to that of a type C retrovirus.

Two different morphogenic processes of retroviral capsid assembly have been observed: the capsid is either assembled at the plasma membrane during the...
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