Vol. 64, No. 4

JOURNAL OF VIROLOGY, Apr. 1990, p. 1549-1555 0022-538X/90/041549-07$02.00/0 Copyright C) 1990, American Society for Microbiology

Expression Strategy of a Phlebovirus: Biogenesis of Proteins from the Rift Valley Fever Virus M Segment JOANN A. SUZICH,1 LAURA TORBORG KAKACH,2t AND MARC S. COLLETT'* Molecular Genetics, Inc., Minnetonka, Minnesota 55343,2 and Molecular Vaccines, Inc., 19 Firstfield Road, Gaithersburg, Maryland 208781 Received 2 May 1989/Accepted 15 December 1989

The middle (M) RNA segment of Rift Valley fever virus (RVFV) encodes four proteins: the major viral glycoproteins G2 and Gl, a 14-kilodalton (kDa) protein, and a 78-kDa protein. These proteins are derived from a single large open reading frame (ORF) present in the virus-complementary M-segment mRNA. We used recombinant vaccinia viruses in which sequences representing the M-segment ORF were engineered as a surrogate system to study phlebovirus protein expression. To investigate the translational initiation codon requirements for synthesis of these proteins, we constructed a series of vaccinia virus recombinants containing specific sequence changes which eliminated select ATG codons found in the region of the ORF preceding the mature glycoprotein-coding sequences (the preglycoprotein region). Examination of phleboviral proteins synthesized in cells infected with these vaccinia virus recombinants clearly showed that the first ATG of the ORF was required for the production of the 78-kDa protein, while synthesis of the 14-kDa protein was absolutely dependent on the second in-phase ATG codon. Efficient biosynthesis of glycoprotein G2 was shown to depend on one or more ATG codons within the preglycoprotein region, but not the first one of the ORF. Synthesis of about one-half of the total glycoprotein Gl was affected by the amino acid changes that eliminated ATG codons, while production of the remainder appeared to be independent of all ATG codons in the preglycoprotein region. These data indicated that the means for glycoprotein Gl biosynthesis was distinct from those of the other three M-segment gene products. The results presented herein suggest that a surprisingly complex expression strategy is employed by the RVFV M segment. Although the full nature of the mechanisms involved in the biogenesis of the four RVFV M-segment proteins remains unclear, it does involve the use of at least two (ATG codons 1 and 2), and likely more, distinct translation start sites within the same ORF to produce its complete complement of gene products.

ORF, giving rise to one or more polyprotein precursors of approximately 133 kDa that are cotranslationally processed to yield the mature proteins (33). Putative polyprotein precursors have not been observed in vivo, however. Furthermore, attempts to demonstrate in infected cells a precursorproduct relationship between the 78-kDa protein and the two proteins encompassed by the sequences represented in the 78-kDa protein (the 14-kDa protein and glycoprotein G2) have been unsuccessful (11). We have previously suggested that the 78-kDa protein and the 14-kDa protein arise by independent translational initiation events at ATG codons 1 and 2, respectively, of the ORF (11, 21). However, the translational mechanisms by which the major envelope glycoproteins G2 and Gl are produced remain obscure. The present study was undertaken to explore the biogenesis of these glycoproteins and to characterize more fully the expression strategy of this phlebovirus M segment. Using site-directed mutagenesis of potential translation start sites and recombinant vaccinia viruses to express the M-segment proteins, we showed that the expression strategy employed by the RVFV M segment is surprisingly complex. Not only does it involve multiple translational initiation events, but the biosynthetic pathways for the two major viral glycopro-

Rift Valley fever virus (RVFV) is a member of the Phlebovirus genus in the family Bunyaviridae. It possesses a genome consisting of three RNA segments designated L, M, and S (4, 31). The S segment encodes the viral nucleocapsid protein N and a nonstructural protein, NS, (28). Expression of the S-segment proteins of phleboviruses proceeds by an ambisense strategy: the nucleocapsid protein is synthesized from a subgenomic virus-complementary mRNA, while protein NSS is encoded by a subgenomic virus-sense mRNA (18, 26, 28). Little information is available regarding the L segment of phleboviruses; however, it is believed to contain information coding for the viral transcriptase protein and to employ a negative-sense coding strategy. Recently, these beliefs have been substantiated experimentally for members of the Bunyavirus genus (14; R. M. Elliott, personal communication). Expression of the phlebovirus M segment has also been shown to employ a negative-sense strategy. There appears to be a single mRNA for the RVFV M segment complementary to the genomic RNA (9). This mRNA possesses one large open reading frame (ORF) capable of encoding 133 kilodaltons (kDa) of protein (9, 12). Within this ORF, the coding sequences for the four M-segment proteins-the viral envelope glycoproteins G2 and Gl, a 78-kDa glycoprotein, and a 14-kDa nonglycosylated protein-have been positioned (12, 21). Data from in vitro studies have indicated that translation of the M-segment mRNA is initiated near the start of the

teins G2 and Gl appear to be distinct from one another. MATERIALS AND METHODS Antiserum reagents. Antiserum R900 was generated in rabbits against a synthetic peptide representing the amino acid sequences between methionine codons 2 and 3 of the RVFV M-segment ORF (21). Antisera to glycoprotein Gl and glycoprotein G2 were produced in rabbits immunized

* Corresponding author. t Present address: Plant Science Research, Inc., Minnetonka,

MN 55343.

1549

1550

SUZICH ET AL.

with bacterially produced Gl and G2 analog polypeptides, respectively, as previously described (20, 33). Site-directed mutagenesis and construction of recombinant vaccinia virus transfer vectors. In general, all recombinant DNA procedures were performed as described previously (22, 25). A series of mutations were introduced into the M-segment ORF changing progressively the second, third, fourth, and fifth in-phase methionine codons to nonmethionine codons. These were generated as follows. The shuttle plasmid pUC7 was prepared containing RVFV M-segment nucleotides 20 to 1092 inserted into the HindlIl site of pUC119 (34). pUC7 was transformed into Escherichia coli CJ236, a dut ung double mutant supplied with the MutaGene mutagenesis kit (Bio-Rad Laboratories, Richmond, Calif.), and single-stranded DNA was isolated (34). For mutagenesis of the second ATG codon, single-stranded pUC7 was annealed to a synthetic oligonucleotide having the sequence 5'-TAACCCAGAGCTCATTGAAGGAG-3'. This oligonucleotide encompasses M-segment nucleotides 125 to 146 and encodes a leucine (CTC) in place of the methionine (ATG) found in the wild-type sequence. Site-directed mutagenesis was done according to the directions supplied with the Muta-Gene kit. The plasmid containing the desired sequence changes, pUC72, was identified by restriction analysis with SstI, the mutation having created a new site for this restriction enzyme within the M-segment sequence. DNA sequencing provided final confirmation of the desired mutation. Mutant plasmid pUC72 was transformed into E. coli CJ236, and single-stranded DNA was isolated. To change methionine codon 3 of the M-segment ORF, sitedirected mutagenesis was carried out on single-stranded pUC72 using a synthetic oligonucleotide having the sequence 5'-GAGGAAGAGATTCCGGAGGAGC-3' representing M-segment nucleotides 165 to 186 with changes from the wild-type sequence at nucleotides 170 and 176. This oligonucleotide possesses an ATT codon for isoleucine in place of the wild-type ATG codon and an XmnI restriction site not found in the wild-type sequence. The mutant plasmid, pUC723, was identified by possession of the new XmnI site, and its structure was confirmed by DNA sequencing. Single-stranded DNA was isolated from CJ236 bacteria transformed with pUC723. A final mutagenesis reaction was done with a synthetic oligonucleotide having the sequence 5'-ACCATTGCAGGGATTGCAATTACAG-3' representing M-segment nucleotides 408 to 432 with two changes from the wild-type sequence. The change at nucleotide 413 eliminates methionine codon 4 of the M-segment ORF and destroys an NcoI restriction site; the sequence change at nucleotide 428 eliminates ATG codon 5 of the ORF. Both sequence changes introduce isoleucine codons in place of the methionine codons. Mutant plasmids were screened for the loss of the Ncol site. Subsequent DNA sequencing identified two different mutant constructs. pUC7234 possessed only the change at M-segment nucleotide 413, and pUC72345 contained both desired nucleotide changes. BamHI fragments of 310 base pairs containing M-segment nucleotides 20 to 302 and encompassing the engineered mutations in pUC72 and pUC723 were isolated and inserted into BamHI-digested pSP64-76. pSP64-76 is a previously described transcription plasmid possessing RVFV M-segment sequences 302 to 3767 (33). The resulting plasmids, pSP76-72 and pSP76-723, were then digested with Sall and EcoRI, and fragments of approximately 3.8 kilobases containing the entire M-segment ORF were isolated. These fragments were inserted into the SmaI site of the vaccinia

J. VIROL.

virus transfer vector pSC469 (20) to generate plasmids pSCRV72 and pSCRV723. DNA fragments containing RVFV M-segment nucleotides 20 to 568 were isolated from pUC7234 and pUC72345 by complete NdeI and partial BamHI restriction. These fragments were inserted into pSP64-76 that had been cut to completion with BamHI at RVFV M-segment nucleotide 302 and partially digested with NdeI at M-segment nucleotide 568. The resulting constructs, pSP76-7234 and pSP76-72345, were digested with Sall and EcoRI. Fragments of approximately 3.8 kilobases containing the entire M-segment ORF were isolated and cloned into pSC469 as described above to generate recombinant vaccinia virus transfer vectors pSCRV7234 and pSCRV72345. Vaccinia transfer vector pSCRV10 was constructed by cloning an MspI-EcoRI fragment isolated from pSP76-72345 and containing RVFV M-segment nucleotides 177 to 3767 into SmaI-restricted pSC469. Construction of recombinant RVFV-vaccinia viruses. The pSC469-RVFV recombinant transfer plasmids were transfected into BSC-40 cells infected with the WR strain of vaccinia virus to generate recombinant viruses 72, 723, 7234, 72345, and 10 by procedures previously described (6, 24). Recombinant RVFV-vaccinia viruses 7 and 8 have been described elsewhere (21). Radioimmunoprecipitations. Infection of BSC-40 cells with recombinant vaccinia viruses and radiolabeling of protein with [35S]methionine have been previously described (21). Cell lysates were prepared by boiling infected cells in buffer containing sodium dodecyl sulfate (SDS) and 2-mercaptoethanol (SDS buffer) (22). Lysates were cleared and used for immunoprecipitations as previously described (5). All precipitation experiments reported here were done in antibody excess as determined by serial immunoprecipitation analyses (10). Immune complexes were eluted from Pansorbin (Calbiochem-Behring, La Jolla, Calif.) by boiling in 0.5% SDS-50 mM Tris hydrochloride (pH 7.2) for 4 min. After removal of the bacteria by centrifugation, the supernatants were mixed with 2 x SDS sample buffer and boiled for 1 min. The radiolabeled, immunoprecipitated proteins were resolved by electrophoresis in SDS-containing polyacrylamide gels (23). The gels were fluorographed (7), dried, and exposed to X-ray film. For quantitation of radiolabeled proteins, regions of the dried gels corresponding to bands on exposed X-ray films were excised, and the amount of radioactivity in the gel slices was determined by liquid scintillation counting. RESULTS Biogenesis of 78- and 14-kDa proteins. The RVFV M segment has one large ORF with a coding capacity of 1,197 amino acids (1, 12). The coding sequences for the viral envelope glycoproteins G2 and Gl were positioned within this ORF as a result of protein and nucleic acid sequence determinations (12) (Fig. 1A). Using sequence-specific antibody reagents, two additional M-segment-encoded proteins were identified and localized, a 78-kDa protein and a 14-kDa protein (21, 33). The sequences represented in the 78-kDa protein initiate from methionine codon 1 of the ORF and encompass the sequences preceding the glycoprotein G2coding region (preglycoprotein region) as well as the G2coding sequences (Fig. 1). The sequences encoding the 14-kDa protein begin at methionine codon 2 of the ORF and include only sequences from the preglycoprotein region. This protein-coding sequence arrangement within the ORF

VOL. 64, 1990

RIFT VALLEY FEVER VIRUS EXPRESSION STRATEGY

A.

PRE

G2

481

GI

2092

3612

-_

I

I

I I I I

B.

3 A-AL-r-~~~ . -

6

---6

OW Di

IS.

-LT

I

no

I

7

ATG " (1)

8

I

ATG ATG a a (39) (52)

ATO ATO -(131X136) (154)

A ATG ATG AT

ATG ATG -A

ATG a

ATOa ATO a

-

ATO A

7 2 AT 79)r%

1551

ATO a

-

A a

72345

Aa

TG

D-

--- -

-

..dp

ATG ATO a Ak

mmlmm....

STG

7234

-

=

-40

ATG A

p

FIG. 1. RVFV M-segment sequences found in recombinant vaccinia viruses. (A) Schematic representation of the RVFV M segment. Nucleotide coordinates are given for beginning of the glycoprotein G2 (481)- and Gl (2092)-coding regions, the end of the ORF (3612), and the end of the genome (3885). The latter two numbers are corrected values relative to the original sequence (12) as an additional residue was identified at nucleotide position 3494 (1). The solid bars indicate the glycoprotein G2- and Gl-coding regions. Vertical marks indicate the in-phase methionine codons in the preglycoprotein region (PRE). The lollipop figures designate sites of the N-linked glycosylation recognition sequence: Asn-X-Thr/Ser. (B) Expanded view of the 5' M-segment sequences and in-phase methionine codons in the recombinant vaccinia viruses. The numbers in parentheses beneath the ATGs of virus 7 represent amino acid positions within the ORF and the first amino acid (residue 154) of mature glycoprotein G2.

suggests two possible mechanisms for the biogenesis of the

14-kDa protein. It could be generated by proteolytic processing of the 78-kDa protein or 78-kDa protein precursor initiated from methionine codon 1 of the ORF, or alternatively, it could arise from a translation product initiated from the second ATG of the ORF. Pulse-chase experiments have failed to demonstrate a precursor-product relationship between the 78- and the 14-kDa proteins (11). These results suggest, but certainly do not prove, that these two proteins arise by independent translational initiation events (11). To further investigate the biogenesis of these proteins, we exploited recombinant vaccinia viruses as a surrogate system to study RVFV M-segment gene expression. We have previously shown that a recombinant vaccinia virus containing the entire M-segment ORF (virus 7) correctly synthesized, processed, and transported all four M-segment gene products (21, 35). To explore the initiation codon requirements for production of the 78-kDa protein and the 14-kDa protein, we constructed two additional recombinant vaccinia viruses. Virus 8 contains sequences of the M-segment ORF beginning just before the second in-phase methionine codon and proceeding past the natural termination codon of the ORF (21) (Fig. 1B). Virus 72 is identical to virus 7 except methionine codon 2 of the ORF has been replaced with a leucine codon by site-directed mutagenesis techniques. In cells infected with virus 8, all M-segment proteins except the

78-kDa protein were synthesized (Fig. 2A). In some experiments, a very small amount of an apparent truncated "78-kDa" protein (ca. 74 kDa) was detected. Thus, the absence of methionine codon 1 in this virus eliminated 78-kDa protein production but had no effect on the biosynthesis of the 14-kDa protein. However, in cells infected with virus 72, absolutely no 14-kDa protein was found, while expression of the 78-kDa protein and glycoproteins G2 and Gl was apparently unaffected. These results strongly suggest that the 14-kDa protein is derived from a translation product initiated from the second in-phase methionine codon of the ORF, while production of the 78-kDa protein requires the first ATG codon. Translational initiation codon requirements for production of viral glycoproteins G2 and Gl. Although the above results describe the mode of production of the 78- and 14-kDa proteins, they do not address directly the biogenesis of glycoproteins G2 and Gl. They do indicate, however, that neither the first nor the second in-phase methionine codon of the M-segment ORF is absolutely required for the synthesis of glycoproteins G2 and Gl. This suggests that alternate biosynthetic options exist for these proteins. There are three additional in-phase methionine codons within the preglycoprotein region from which translation initiation could occur (Fig. 1). To assess the role of these potential translation start sites in the biogenesis of glycoproteins G2 and Gl, we

J. VIROL.

SUZICH ET AL.

1552

B.

A. G2 7 1 1697-

_"m

8

Gl 7

A G2

900 8

7

8

G1

68-

68

45-

45-

A

B , (Z1 5Is -iso .

900

7 72 7 72 7 72 11697-

Gl

G2

_

1166 97-

11697-

68-

68-

45-

45-

30-

l0O2 1-

302 1-

30-

14 14--

FIG. 2. RVFV M-segment proteins synthesized in the absence of methionine codons 1 or 2 of the M-segment ORF. BSC-40 cells were infected with recombinant vaccinia virus and labeled with [35S]methionine. Cell lysates were prepared in SDS lysis buffer, and proteins were immunoprecipitated with antiserum specific for glycoprotein G2 or Gl or for sequences encoded between ATG codons 1 and 2 of the M-segment ORF (antiserum 900). Immunoprecipitated proteins were analyzed on 10 to 18% gradient SDS-polyacrylamide gels. The numbers at the left indicate the molecular mass (in kilodaltons) of protein standards. (A) M-segment-specific proteins synthesized in cells infected with recombinant vaccinia virus 7 or 8. (B) M-segment-specific proteins synthesized in cells infected with recombinant virus 7 or 72.

generated recombinant vaccinia viruses similar to virus 72 but possessing additional site-specific mutations at the remaining internal methionine codons in the preglycoprotein region. In virus 723, methionine codons 2 and 3 of the M-segment ORF were changed to leucine and isoleucine, respectively. Viruses 7234 and 72345 contained the same mutations as virus 723 plus mutations which replaced ATG codon 4 (virus 7234) or ATG codons 4 and 5 (virus 72345) of the ORF with isoleucine codons. All viruses in this series maintained the first ATG codon of the ORF. These mutant viruses are schematically summarized in Fig. lB. Analysis of RVFV M-segment proteins produced in cells infected with these viruses is shown in Fig. 3. All the viruses directed the synthesis of the 78-kDa protein and glycoproteins G2 and Gl. However, the removal of the preglycoprotein region ATGs had an obvious negative effect on the biosynthesis of glycoprotein G2. Quantitative analyses of the data from this experiment revealed that with the elimination of the internal ATG codons, the levels of all three proteins actually decreased relative to their expression levels in virus 7-infected cells (Table 1). In virus 72345-infected cells, both the 78-kDa protein and glycoprotein Gl were expressed at about 50% of their wild-type levels, while synthesis of glycoprotein G2 was affected to a much greater extent, reduced to about 11% of the level found in virus 7-infected cells. The effect of the elimination of the internal preglycoprotein ATG codons on the production of the 78-kDa protein was unexpected. Experiments described above showed that the 78-kDa protein required the first ATG of the ORF for its synthesis (Fig. 2). However, the mutations eliminating the internal ATG codons changed amino acid residues within the 78-kDa protein and also within any putative precursor poly-

FIG. 3. Effect of the removal of potential internal translational initiation sites from the M-segment ORF on the biosynthesis of RVFV glycoproteins. Cleared SDS lysates were prepared from [35S]methionine-labeled cells infected with recombinant vaccinia virus 7, 72, 723, 7234, or 72345. Proteins were immunoprecipitated with antiserum specific for glycoprotein G2 or Gl. The precipitated proteins were resolved on SDS-10% polyacrylamide gels. The molecular masses (kilodaltons) of protein standards are indicated on the left. The protein band appearing in the anti-glycoprotein G2 panel at approximately 116 kDa is a nonspecific polypeptide unrelated to RVFV proteins.

peptide. Thus, one explanation for the decreased levels of the 78-kDa protein observed in the mutant virus-infected cells suggests that these amino acid changes cause an altered stability in, or processing of, a precursor polypeptide. To the extent this putative precursor was common to the envelope glycoproteins, this interpretation could also explain, in part, the decrease in the production by the mutant viruses of glycoproteins G2 and Gl. However, since we have been unable to detect M-segment-specific polyprotein precursors TABLE 1. RVFV protein expression in recombinant vaccinia virus-infected cells Virus

78-kDa

Relative expression levela G2

01

protein

7 8 72 723 7234 72345 10

1.0

0.65 0.85 0.48 0.44

1.0 1.3 0.48 0.46 0.23 0.11 0.07

1.0 1.0 0.66 0.72 0.44 0.49 0.44

a Cleared lysates were prepared from [35SJmethionine-labeled cells infected with the indicated recombinant vaccinia viruses. Proteins were immunoprecipitated from lysate aliquots under conditions of antibody excess with antiserum specific for glycoprotein G1, glycoprotein G2, or vaccinia virus proteins. Precipitated proteins were resolved on SDS-10% polyacrylamide gels. Regions corresponding to radiolabeled protein bands representing the RVFV 78-kDa protein and glycoproteins G2 and G1 and a vaccinia virusspecific 35-kDa protein were excised from the gels and counted. To control for any slight differences among the virus infections, counts in the RVFV-specific protein bands were divided by the counts in the vaccinia virus-specific 35-kDa protein band for each recombinant virus. The resulting values for RVFV proteins from virus 8 through virus 10 were divided into the value for the respective proteins from virus 7, the values for virus 7 then being normalized to 1.

VOL. 64, 1990

RIFT VALLEY FEVER VIRUS EXPRESSION STRATEGY

A.

G2

B. 48 b

2092

G1

--'

36t2 3665

Gl 4,

40

10

1553

10 A,1O

(

I 16-

97-

7SkD-

_

-

G2-

m- GI

02

72345 10

45-

hmm 30-

FIG. 4. RVFV glycoprotein expression in the absence of translational initiation codons in the preglycoprotein region of the M-segment ORF. (A) Schematic representation of the M-segment preglycoprotein region in recombinant vaccinia viruses 72345 and 10 (see the legend to Fig. 1 for definition of symbols). (B) Cleared [35S]methionine-labeled lysates of cells infected with virus 10 or 72345 were prepared in SDS lysis buffer, and proteins were immunoprecipitated with antiserum specific for glycoprotein G2 or Gl. The precipitated proteins were resolved on an SDS-10% polyacrylamide gel. The molecular masses (kilodaltons) of protein standards are indicated on the left.

in virus-infected cells, this hypothesis is difficult to investi-

DISCUSSION

gate further.

Alternatively, the quantitative protein expression data indicate that synthesis of glycoproteins G2 and G1 requires internal translation initiation sites within the preglycoprotein region. The dramatic reduction in glycoprotein G2 expression suggests a complete dependence on these translation start sites for its biogenesis. However, only a fraction of glycoprotein Gl synthesis may be explained by this mechanism. The ability of cells infected with virus 72345 to synthesize glycoprotein G1 at about 50% the wild-type (virus 7) level, while producing glycoprotein G2 at only 11%, suggests that an additional biosynthetic pathway exists for glycoprotein Gl. That ATG codon 1 of the ORF, which is present in virus 72345, is not involved in the biogenesis of glycoprotein Gl is suggested by the data presented for virus 8 (Fig. 2A; Table 1). However, in the context of virus 8, the involvement of the four remaining preglycoprotein translation start sites on glycoprotein G1 synthesis clouds this interpretation. Therefore, to evaluate the production of glycoprotein G1 in the absence of all methionine codons within the preglycoprotein region, we generated another recombinant vaccinia virus. Virus 10 possesses M-segment sequences beginning at amino acid 51 (just before the third methionine) and ending beyond the natural termination codon of the ORF. Virus 10 also contains the site-specific mutations eliminating methionine codons 3, 4, and 5 and so lacks all in-phase ATG codons preceding the start of the glycoprotein G2-coding sequences (Fig. 4A). RVFV Msegment protein expression in cells infected with either virus 10 or virus 72345 is shown in Fig. 4B. As expected for virus 10, no 78-kDa protein was produced in the absence of ATG codon 1. Further, only a very low level of glycoprotein G2, slightly below that produced in cells infected with virus 72345, was synthesized in these cells (Table 1). However, the virus 10-infected cells were able to synthesize glycoprotein G1 at the same level as cells infected with virus 72345 (Fig. 4B; Table 1). Apparently, a significant portion of glycoprotein G1 (about 50% of wild type) can be synthesized by a mechanism independent of all in-phase methionine codons in the preglycoprotein region of the M segment. may

The mRNA of the M segment of RVFV possesses a single large ORF which ultimately gives rise to four gene products. Conventional wisdom would predict the mature protein products to be derived from a polyprotein precursor by cotranslational and/or posttranslational proteolytic processing events. From our investigations, this appears to be only part of a more complicated mechanism employed for RVFV M-segment protein production. To dissect the actual gene expression strategy of this negative-sense phlebovirus RNA, we made extensive use of genetically engineered vaccinia viruses. We previously established this genetically manipulatable system as being useful and faithful in the synthesis, processing, and transport of RVFV M-segment-encoded polypeptides (21, 35). Here, we used this surrogate expression system in an attempt to establish the translational initiation codon requirements for each of the M-segment protein products. Data presented herein clearly show that ATG codon 1 of the ORF is required for 78-kDa protein synthesis and that in-phase ATG codon 2 is necessary for production of the 14-kDa protein. The results are less obvious regarding the translational start site requirements for biogenesis of envelope glycoproteins G2 and G1. ATG codon 1 of the ORF does not appear to be involved with the synthesis of glycoprotein G2. We were unable to demonstrate in pulse-chase experiments that products initiated from the first ATG codon (78 kDa) serve as precursors to glycoprotein G2 (11). Furthermore, the removal of ATG codon 1 of the ORF (as in virus 8) did not have a negative effect on glycoprotein G2 production. In fact, there was a slight increase in the relative level of glycoprotein G2 synthesis in the absence of this initiation site. However, glycoprotein G2 production did progressively decrease with the elimination of each of the internal methionine codons in the preglycoprotein region, until practically no glycoprotein G2 was made when all ATGs were changed. A conservative interpretation of these results is that glycoprotein G2 production involves translational initiation events at ATGs in the preglycoprotein region other than the first methionine codon. Synthesis of glyco-

1554

SUZICH ET AL.

protein Gl also was diminished with removal of the ATGs within the preglycoprotein region, although not nearly to the extent seen for glycoprotein G2. These observations may indicate that a portion of glycoprotein Gi (approximately 50%) was generated similarly to glycoprotein G2, as a result of translational initiations from these internal ATG codons. Interpretations of the data to this point suggest that the 78and 14-kDa proteins, glycoprotein G2, and a portion of glycoprotein Gi are generated from at least two primary polyprotein translation products initiated from ATGs within the preglycoprotein region. One putative precursor, starting from the first methionine codon and extending to the end of the ORF (1,197 amino acids), would serve as the precursor to the 78-kDaprotein. Our results do not preclude biogenesis of glycoprotein Gl from this same polyprotein. In fact, the parallel effect on the synthesis of the 78-kDa protein and glycoprotein Gl observed with the progressive elimination of ATG codons (Table 1) is consistent with a common precursor for these two proteins. However, the results with virus 10, in which 78-kDa protein synthesis was eliminated and glycoprotein Gl production was unaffected, may argue otherwise. In any event, the data do suggest that this putative precursor would contribute little to the biogenesis of glycoprotein G2. A second primary translation product, beginning from the second ATG and continuing to the ORF termination codon (1,159 amino acids), would ultimately yield the 14-kDa protein and glycoproteins G2 and Gl. The data further indicate that additional translation starts may occur from the remaining ATG codons in the preglycoprotein region, giving rise to the polyprotein precursors that could be processed to yield the viral envelope glycoproteins. Such precursors would also be expected to give rise to small polypeptides analogous to the 14-kDa protein derived from the preglycoprotein region. However, we have not attempted to verify the existence of these putative small protein products. The prediction that large polyprotein precursors are primary translation products of the M-segment ORF is supported by our previous results with a cell-free transcriptiontranslation system. In vitro translation of an M-segment mRNA-like transcript gave rise to polyprotein precursor species the size expected of a polypeptide encompassing the entire ORF (33). Recent analyses of this material on highresolution gels revealed it to be composed of multiple bands (unpublished data), consistent with the proposal of two or more primary polyprotein precursors. An unexpected finding from the present study was that the production of a portion of glycoprotein Gl (about 50%) appears to follow a biosynthetic pathway distinct from those discussed above. That is, a significant portion of glycoprotein Gl appears to be synthesized independently of the other three M-segment gene products. Although at this time we do not understand the exact mechanism(s) involved, several possibilities may be considered. For example, in the recombinant vaccinia virus-infected cells, a portion of glycoprotein Gl might be synthesized from a separate (subgenomic) mRNA. However, Northern analyses of RNA isolated from cells infected with each of the viruses described in this work revealed only a single species of M-segment-sized RNA which possessed RVFV preglycoprotein, glycoprotein G2, and glycoprotein Gl sequences (unpublished data). Thus, the mechanism for the independent production of glycoprotein Gl must occur at the posttranscriptional level. Among the translational mechanisms that might result in separate glycoprotein Gl production are initiation from a

J. VIROL.

non-ATG codon and internal translation initiation. Translation starts from ACG (2, 13, 15) and CTG (16, 30) codons have been reported in eukaryotic cell systems. The preglycoprotein region lacks any in-phase ACG codons, but there is a single in-phase CTG codon located between ATG codons 1 and 2 of the M-segment ORF (12). However, it is not likely that this codon is involved in the generation of glycoprotein Gl since its removal (in virus 10) had no effect on glycoprotein Gl synthesis. The apparent independent biogenesis of a portion of glycoprotein Gl may merely be a result of ribosomes initiating at the next available downstream ATG codon in the various engineered constructs. Whereas this might be a reasonable explanation for the observed consequences of ATG substitution for glycoprotein G2 synthesis, we consider it unlikely for glycoprotein Gl owing to its relatively efficient and quite constaht production among the different viruses. Thus, the independent glycoprotein Gl synthesis observed may be the result of translation initiation at a specific ATG (or alternate initiation codon) downstream of the preglycoprotein region. There are a number of viral (picornaviruses [19, 29]; paramyxovirus [27]; rhabdovirus [17]; hepadnavirus [8]) as well as cellular (human c-abl [3]; Drosophila melanogaster [32]) proteins which appear to be synthesized via internal translational initiation mechanisms. Clearly, additional experimentation is required to determine whether the RVFV glycoprotein Gl can be added to the growing list of proteins employing this expression strategy. The use of certainly two (the first and second ATGs), and probably additional, translation start sites within the same ORF to produce the full complement of RVFV M-segment gene products suggests that such a strategy must have some regulatory significance. This indeed appears to be the case for the use of ATG codons 1 and 2. Translational initiation from the first ATG codon appears to predetermine utilization of the N-linked glycosylation site within the preglycoprotein region of the ORF and, furthermore, precludes the proposed proteolytic cleavage at the preglycoprotein-glycoprotein G2 junction (20). Conversely, translation from ATG codon 2 results in the failure to use the preglycoprotein glycosylation site but does allow proteolytic cleavage at the amino terminus of mature glycoprotein G2 to yield the 14-kDa protein (20, 21). The putative proteolytic cleavage at this junction was found to be independent of glycosylation (20). Therefore, the amino acid sequence between ATG codons 1 and 2 is implicated in exerting a profound influence on subsequent protein glycosylation and proteolytic processing. We speculate that the presence or absence of these 37 amino acids affects the conformation, or possibly intracellular transit, of the resultant polypeptide. This effect in turn influences what subsequent modifications may take place on the polypeptide. Thus, use of the two-site translational initiation expression strategy serves as a mechanism for controlling posttranslational protein modifications. The present data suggest that the biosynthetic pathways for the two RVFV envelope glycoproteins differ, with a portion of glycoprotein Gl being produced independently of the other M-segment proteins. And although there are many unanswered questions raised by these results, the implications certainly have important biologic significance and consequences. However, speculation is reserved until additional data are obtained to further delineate the mechanisms involved.

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VOL. 64, 1990

ACKNOWLEDGMENTS This work was supported in part by U.S. Army Medical Research and Development Command research contract DAMD 17-85-C5226. The competent assistance in plasmid constructions by John Hansen is acknowledged. We are indebted to Ellan Welniak, Tom Molitor, and Michael Murtaugh of the College of Veterinary Medicine, University of Minnesota, St. Paul, for the use of their laboratories during a phase of this work. We also acknowledge the insightful reviews provided by referees of the manuscript.

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