Molecular genetic evidence that the hydrophobic anchors of glycoproteins E2 and E1 interact during assembly of alphaviruses

Abstract

Chimeric alphaviruses in which the 6K and glycoprotein E1 moieties of Sindbis virus are replaced with those of Ross River virus grow very poorly, but upon passage, adapted variants arise that grow >100 times better. We have sequenced the entire domain encoding the E2, 6K, and E1 proteins of a number of these adapted variants and found that most acquired two amino acid changes, which had cumulative effects. In three independent passage series, amino acid 380 of E2, which is in the transmembrane domain, was mutated from the original isoleucine to serine in two instances and to valine once. We have now changed this residue to seven others by site-directed mutagenesis and tested the effects of these mutations on the growth of both the chimera [SIN(RRE1)] and of parental Sindbis. These results indicate that the transmembrane domains of glycoproteins E2 and E1 of alphaviruses interact in a sequence-dependent manner and that this interaction is required for efficient budding and assembly of infectious virions.

FIG. 1.

Amino acid changes selected during passage of chimeric SIN(RRE1) virus. (A) Diagram of the genome organization of SIN(RR6KE1) with an expanded view of the glycoprotein region below. SIN sequences are shaded dark gray, and RR sequence is shaded light gray. Transmembrane domains are shaded with diagonal hatching; the “stem” region of E1 is shaded with wavy lines. Residue numbers where variants mapped are indicated below the open reading frame, and the parental amino acid in each case is shown above. aa, amino acids. (B) Amino acid changes found in eight independent passage series. In most cases the changes found were shown to be representative of the variant population by sequencing two independent clones and finding the change in each clone or by sequencing the PCR DNA prior to cloning. For the changes shown in parentheses, only a single clone was sequenced and it is uncertain whether these changes are representative of the revertant population. A limited number of other changes were also observed that were clonal, that is, found only in one of two sequenced clones and that may have arisen by PCR mutagenesis during cloning. These changes are not shown because their origin is uncertain and because, in any event, they do not represent the consensus sequence of the variant population.

FIG. 2.

Insertion in the 6K protein during passage of SIN(RRE1). The insertion into 6K that occurred in passage series 2 is shown. SIN sequences are shown in plain type; RR sequences are shown in boldface. The insert, shown in lowercase and italics, consists of nt 2594 to 2710 inclusive (117 nt) from SIN, corresponding to amino acids 306 to 344 of nsP2 and translated in the same frame as nsP2. The only change is that the last amino acid is Asp rather than Glu.

FIG. 3.

Growth curve of Ile-380 mutants. Constructs labeled Toto 54 (or Toto) contained different amino acids at position E2 and amino acid 380 in the SIN background; constructs labeled SINRRE1 (or RRE1) had I380 mutations in the chimeric background. BHK cells were transfected with RNA transcribed in vitro, and samples were removed at intervals and titered for released virus by plaque assay on monolayers of BHK cells.

FIG. 4.

Transmembrane anchor of E2. Sequences of the E2 anchor sequences for several alphaviruses are compared. AURA is Aura virus, SF is Semliki Forest virus, MID is Middelburg virus, BF is Barmah Forest virus, MAY is Mayaro virus, SAG is Sagiyama virus, ONN is O'Nyong-nyong virus, VEE is Venezuelan equine encephalitis virus, SES is Southern elephant seal louse virus, SPDV is salmon pancreas disease virus, and SLDV is rainbow trout sleeping disease virus.