Virol J. 2013 Feb 25;10:63.

Posttranslational modifications and secretion efficiency of immunogenic hepatitis B virus L protein deletion variants.

Niedre-Otomere B, Bogdanova A, Bruvere R, Ose V, Gerlich WH, Pumpens P, Glebe D, Kozlovska T.

Biomedical Research and Study Centre, Ratsupites street 1, Riga, Latvia. baiba@biomed.lu.lv

 

Abstract

BACKGROUND: 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.

FINDINGS: 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.

CONCLUSIONS: 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.

PMID: 23442390

 

Supplements:

In our recent paper we have asked the question: „Which influence has removal of myristic acid (a saturated C14-fatty acid) at the aminoterminus of a shortened version of the large (L) protein of human Hepatitis B virus (HBV) on its secretion efficiency?” This question arised from our search for alternatives to the current vaccines against hepatitis B infections. We had previously developed viral vectors for expression of immunogenic HBV surface structures and could show strong development of broadly neutralizing antibodies in mice.1 The three HBV surface proteins (large L-, middle M- and small S-HBs proteins) share a common C-terminal S domain, but differ by lenghth due to two alternative aminoterminal extensions – the so called preS domains. We constructed recombinant nonreplicating Semliki Forest virus (a plus strand RNA virus) vectors which encode the essential parts for attachment of HBV to susceptible cells. One attachment site is within the 48 aminoterminal amino acids of Lprotein which were linked in the vector to the gene encoding the S domain (preS1-48/S). The second attachment site of HBV to liver cells is in the antigenic loop of the unmodified S protein which forms together with L protein secretable 20nm HBs antigen particles. The nonessential part of preS which inhibits secretion were deleted. Thus, we had obtained vectors expressing the two essential components of the HBV envelope for induction of neutralizing antibodies by experimental vaccines. The transduction with the viral vector strongly activates the innate immunity and directs the adaptive immune response to the Th1 arm.1

A limiting factor of the construct was the relatively low secretion efficiency of the preS1-48/S protein. We aimed to improve secretion of these particles, i. e. their efficient post-translational modifications, assembly and movement through the secretory pathway resulting in controlled release from mammalian cells. L protein carries at the very amino end the motif methionin-glycine which is a signal for removal of methionin and addition of myristic acid to glycine by the cellular enzyme N-myristoyltransferase. It has reported by Prange et al. that full lenghth L protein is not secreted from transfected cells, but replacement of the glycine in the myristic acid attachment site by another amino acid (so that the N-myristoyltransferase cannot recognize the site) makes the L protein secretable 2. Therefore, we hypothesised that exchange of the glycine in the myristic acid attachment site by another amino acid would improve secretion of our heavily shortened L protein as well. Contrary to our expectations, the inactivation of the myristic acid attachment site reduced secretion of our L protein deletion variants instead of improving it. Thus, presence of the myristoylation signal did not impair secretion of 1-48preS/S particles but even improved it.

Using confocal microscopy we could furthermore show that the myristoylation of the shortened L protein supported stronger co-localization with the co-expressed S protein, thereby possibly increasing assembly and secretion of the L-containing subviral particles. Unexpectedly, the shortened L protein was N-glycosylated not only at the known site asparagine 146 in the S domain, but at two additional sites: asparagine 4 in preS, and one other site. According to the current model of L protein topology3 at the membrane of secretory structures we attributed it to asparagine 3 in the S domain.

Importance:   We demonstrated that presence of myristoylation motif increased secretion of subviral HBs particles containing the shortened L protein, contradictory to what was expected, and had an effect on the localization of the protein in the host cell by anchoring the shortened L protein to the membranes of cellular secretory structures. Myristoylation is not necessary for assembly of HBV virions but necessary for their infectivity. The exact role of myristoylation in generation and immunogenicity of HBV subviral particles remains to be elucidated, but our findings may open up future investigations in this field.

Recent data from Thailand4 and Taiwan5 suggest that immunity aginst HBV is waining 20 years after vaccination in early childhood. Occult HBV infections are equally frequent in vaccinated and unvaccinated people4 and breakthroughs occur often.5 A potential new threat is the newly discovered HBV-like virus of tent-building bats the HBs proteins of which are able to mediate infection of human hepatocytes.6 Current hepatitis B vaccines contain only the small HBs protein, but recent research confirms that attachment and entry of HBV depends on the attachment of preS1-48 to the newly identified HBV receptor sodium taurocholate cotransporting polypeptide (NTCP) which is essential for the enterohepatic bile acid circulation7. PreS1 antibodies alone without concomittant anti-S are able to neutralize HBV infectivity.8 Thus, efficient induction of antibodies against preS1-48 appears more desirable than ever, and our study contributes to the development of corresponding vaccines.

Baiba Niedre-Otomere-fig1

Figure 1. Three HBV surface (HBs) proteins  – large (L), middle (M) and small (S) share a common C-terminal S domain, but differ by aminoterminal extensions, which are called preS domains. Three HBs proteins have  a common glycosylation site at Asn 146 in S domain. M protein is glycosylated at Asn 4 in preS2 domain, while L protein is glycosylated only at the common glycosylation site a Asn 146 shared by all three proteins. The truncated L protein (preS1-48/S) consists of 48 aminoterminal amino acids of L protein linked to S domain and contains both essential binding determinants of HBV to liver cells and thus components for induction of neutralizing antibodies. The postulated N-glycosylation sites for the preS1-48/S protein are marked with the green circle.

 

References:

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  2. Prange R, Clemen A, Streeck RE: Myristylation is involved in intracellular retention of hepatitis B virus envelope proteins. J Virol 1991, 65:3919-3923.
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