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Posttranslational modifications and secretion efficiency of immunogenic hepatitis B virus L protein deletion variants
© Niedre-Otomere et al; licensee BioMed Central Ltd. 2013
Received: 11 July 2012
Accepted: 20 February 2013
Published: 25 February 2013
Subviral particles of hepatitis B virus (HBV) composed of L protein deletion variants with the 48 N-terminal amino acids of preS joined to the N-terminus of S protein (1-48preS/S) induced broadly neutralizing antibodies after immunization of mice with a Semliki Forest virus vector. A practical limitation for use as vaccine is the suboptimal secretion of such particles. The role of the N-terminal preS myristoylation in the cellular retention of full-length L protein is described controversially in the literature and the relation of these data to the truncated L protein was unknown. Thus, we studied the effect of preS myristoylation signal suppression on 1-48preS/S secretion efficiency, glycosylation and subcellular distribution.
The findings are that 1-48preS/S is secreted, and that removal of the N-terminal myristoylation signal in its G2A variant reduced secretion slightly, but significantly. The glycosylation pattern of 1-48preS/S was not affected by the removal of the myristoylation signal (G2A mutant) but was different than natural L protein, whereby N4 of the preS and N3 of the S domain were ectopically glycosylated. This suggested cotranslational translocation of 1-48preS in contrast to natural L protein. The 1-48preS/S bearing a myristoylation signal was localized in a compact, perinuclear pattern with strong colocalization of preS and S epitopes, while the non-myristoylated mutants demonstrated a dispersed, granular cytoplasmic distribution with weaker colocalization.
The large deletion in 1-48preS/S in presence of the myristoylation site facilitated formation and secretion of protein particles with neutralizing preS1 epitopes at their surface and could be a useful feature for future hepatitis B vaccines.
Hepatitis B virus (HBV) contains three closely related transmembrane proteins in its lipid envelope. The S protein comprises the common C terminus of all three HBV surface (HBs) proteins and is the main structural component of the viral envelope. The nonessential M protein contains a preS2 N-terminal extension of 55 amino acids (aa), while the essential L protein has a further N terminal extension of 108 or 119 aa, termed preS1 . The preS1 domain contains at its N-terminus an essential attachment site of HBV to infection-susceptible cells  while the preS2 sequence seems to have no essential function in the viral life cycle beyond acting as a spacer in the L protein . HBs proteins are secreted from infected, transfected, or transduced cells as 20–22 nm spherical or filamentous, noninfectious lipoprotein particles.
During translation, the preS domain (i. e. preS1 + preS2) of L protein remains unglycosylated and in the cytosol whereas the S domain is partially N-glycosylated and assumes a topology at the endoplasmatic reticulum (ER) with at least two transmembrane passes . However, in a posttranslational maturation step approximately 50% of the preS domains are translocated to the lumen of secretory structures and appear later at the surface of the secreted HBV or subviral particles while the cytosolic domains remain within the particles [5–7]. Full length L protein alone in absence of the S protein is not secreted from transfected cells [8–10]. The retention of L protein depends on the cytosolic localization of preS1 , but N-terminal shortening of the preS sequence by more than 110 aa (or 98 aa in HBV genotype D) finally leads to its cotranslational translocation to the ER  governed by the signal peptide I  of the S domain, N-glycosylation of the residual preS sequence  and secretion of subviral particles.
The 48 aa N-terminal part of preS1 carries HBV-neutralizing epitopes [2, 13] while the C-terminal part does not . Furthermore the C-terminal part in its cytosolic orientation prevents secretion . These observations led to the design of expression vectors encoding the N-terminal part of preS1 linked with the S protein for the generation of potential HBV vaccines [16, 17]. Using replication-deficient Semliki Forest virus (rSFV) vectors, we could express the neutralization-relevant preS1 part linked to the S domain of HBs subviral particles and consequently use these vectors for immunization of mice . The influence of the large internal deletion in the L protein on its structure and topology was not yet known. Therefore, we analyzed the cellular localization, the N-glycosylation pattern of expressed 1-48preS/S protein variants, and the surface exposure of the shortened preS1 domain on the subviral particles.
Furthermore, secretion of the subviral particles from the transduced cells was found to be a limiting factor in our previous study. Therefore, we searched for ways to improve the secretion of the HBs particles. The N–terminus of the L protein is modified by myristate at Gly 2 of preS1 . Kuroki et al.  found that myristoylation alone did not cause retention, but Prange et al. reported the opposite . More recently, Abou-Jaoude et al.  studied the secretion and infectivity of hepatitis delta virus variants with and without N-terminal myristoylation of L and other HBV envelope proteins. They found no detectable influence of the myristoylation on the secretion of these viruses but could show that infectivity was lost after inactivation of the myristoylation signal in L protein. It was not clear how these findings could be applied to the context of the 1-48preS/S particles with a heavily truncated preS domain. Thus, it appeared useful to determine whether inactivation of the myristoylation signal would improve secretion of the 1-48preS/S protein and allow for surface exposure of the preS1 epitopes. Exchange of Gly2 to Ala (G2A) disrupts the myristoylation motif Met-Gly recognized by N-myristoyl transferase  and this mutation was introduced in an expression vector for generation of L protein deletion variants without a myristoylation signal.
Results and discussion
Secretion of L protein deletion variants
Intracellular, OD at 492 nm
L deletion variant
1-48preS/S, myr wt
1.10 ± 0.16
1.00 ± 0.12
1.08 ± 0.08
1.06 ± 0.09
G2A 1-48preS/S, myr-
0.52 ± 0.06
0.56 ± 0.09
0.69 ± 0.04
0.78 ± 0.04**
G2S 1-48preS/S, myr-
0.67 ± 0.08
0.43 ± 0.06
1.14 ± 0.09
0.60 ± 0.06**
1-48preS/S0, myr wt
0.17 ± 0.01
0.14 ± 0.01
0.72 ± 0.10
0.25 ± 0.07
The influence of myristoylation of L protein on secretion has been found to be different in various studies [20, 22] suggesting a potential ER retention . Thus, we studied the secretion of the 1-48preS/S0 variant by inactivation of the myristoylation site but particles were again not secreted (not shown). The N-glycosylation pattern of the S0 variant was unaltered and was obviously not related to secretion competence. Theoretically, the postulated glycosylation at N3 of the S domain could enhance secretion as shown for glycosylation of M protein , but this was not the case for the 1-48preS/S0 variant. Based on previous data  we conclude that the major determinant of effective secretion of wt and modified L protein subviral particles is the in-frame co-expressed S protein.
Surprisingly, presence of the myristoylation signal did not impair the secretion of 1-48preS/S particles but improved it slightly (Table 1). The somewhat better secretion of these particles with the myristoylation signal is also in agreement with reports that myristoylation supports virus-like particle formation of Lassa virus  and virus assembly of HIV . Although, myristoylation is reported to be dispensable for virion morphogenesis of HBV , its role and mechanisms in facilitating generation of subviral particles may be a subject of future investigation.
The authors thank I. Timofejeva, A. Ose and N. Gabrusheva for the excellent technical assistance, D. Pjanova for help with confocal microscopy images, K. Sasnauskas for the plasmids pFD Pr [13–59] and pFD Pr [13–59]S and H. Schaller for anti-preS1 polyvalent antibodies H863. This work was supported by The Latvian National Research Programme 2010 – 2013 “BIOMEDICINE”, and by the European Social Fund.
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