Among the lyssaviruses conservation of genome organization and a high degree of sequence similarity (11
) suggest rather similar mechanisms of virus replication. Nevertheless, different host tropism and pathogenicity among lyssaviruses may indicate the existence of diverse, host-adapted mechanisms of virus replication. A detailed molecular explanation for the distinct behavior of different lyssaviruses is lacking thus far.
One virus protein that has been identified as an important factor of pathogenicity in rabies virus infections is the RABV M protein (12
). Whereas previous studies compared attenuated and nonattenuated genotype 1 RABV, in the present study the M proteins of lyssaviruses with different host tropisms were compared to assess the contribution of M to virus replication and virus-host interactions.
In particular, the exchange of the viral M protein between genotype 1 RABV and European bat lyssaviruses EBLV-1 and EBLV-2 and possible impacts on the virus cycle were analyzed by introducing the EBLV M proteins into a genotype 1 virus genetic background. Since M is essential for virus assembly (31
), successful intergenotypic complementation was expected to occur only in case of highly conserved interactions during the processes of RNP recruitment and glycoprotein incorporation. Indeed, transcomplementation of NPgrL virus with the M and G genes deleted showed that infectious virus production was 10- and 100-fold decreased after complementation with EBLV-1 M and EBLV-2 M proteins, respectively (see Fig. ). These data indicated that the heterologous M proteins were not fully compatible, either in binding to the genotype 1 RNP and/or to the glycoprotein, or with RABV replication.
Whereas EBLV-1 M led to only 10-fold decreased infectious virus titers in the complementation experiments, autonomously replicating chimeric SAD EBLV-1 M was more severely affected in its ability to produce infectious virus than SAD EBLV-2 M. Obviously, in the recombinant virus, EBLV-2 M performed better in complementing RABV M functions than EBLV-1 M, although infectious virus titers after transcomplementation were lower than with EBLV-1 M. This discrepancy may be due to the multiple rounds of replication in the full-length clone which can affect both the level of RNA synthesis and virus assembly. In contrast, transcomplementation of minigenome RNPs that accumulated in the absence of M may be rather independent of such non-assembly functions of M. Indeed, previous transcomplementation of minigenomes with different genotype 1 M proteins led only to alterations in the transcript levels, whereas preaccumulated virus genomes remained constant, although complete chimeric RABV with the M proteins integrated in the genome exhibited clearly reduced genome accumulations (14
Comparable accumulation of vRNA as a replication product in SAD EBLV-1 M- and SAD EBLV-2 M-infected cells (Fig. ) excluded limiting concentrations of vRNA as the cause for the observed differences in the production of infectious virus. More likely, full-length RNPs accumulated in the infected cells as a consequence of a budding defect. Whereas intracellular accumulation of vRNA could explain reduced infectious virus production, this phenotype was also puzzling since dose-dependent stimulation of replication by M prior to RNP condensation (14
) was expected to be less efficient with increasing M sequence divergence. This would result in decreased replication product accumulation. Indeed, with increased infectious virus production in SAD EBLV-1Mpass, the ratio of vRNA and mRNA changed to the opposite, indicating increased transcription in the presence of the heterologous M protein (Fig. ). Further quantitative studies on mRNA and vRNA levels should shed more light on the regulation of RNA synthesis by the heterogenotypic M.
Intracellular accumulation of RABV RNPs or retention of almost complete virions could be a result of defects in late stages of virus assembly after RNP condensation by M. Indeed, examples for such late domain-defective RABV and other rhabdovirus species have been described (20
). Since the late domain PPEY motif in the M proteins of lyssaviruses is conserved, we did not expect a typical late domain-defective phenotype with virus accumulations at the surface of the infected cells. Defects in the assembly pathway could also lead to so-called skeleton-RNPs, which are condensed, M containing nonenveloped subviral structures that are observed after detergent treatment of enveloped VSV and RABV virions (5
). Moreover, accumulation of noncondensed RNPs as a result of defects in RNP recruitment by M may occur.
Ultrastructural analyzes revealed that beside budding stages at the cell membrane of all viruses electron dense condensed RNPs inside infected cells were only visible for nonmodified wild-type virus SAD L16 and in SAD RABV M-infected cells, both expressing the RABV M protein. These structures were membrane-enveloped and accumulated in cisternae of the degranulated and dilated rER (see Fig. ), indicating that budding events have occurred at the rER membrane. Accumulation of intracellular viruslike structures is a common phenomenon in RABV-infected cell cultures and even in primary neurons or neuronal tissues (22
). The lack of comparable structures in SAD EBLV-1 M- and SAD EBLV-2 M-infected cells, therefore, was surprising, in particular since budding of typical bullet shaped virus particles at the plasma membrane was observed (Fig. and Fig. ). Whereas the ability of the heterogenotypic M proteins to support bullet shaped virus particle formation at the plasma membrane showed that these M proteins were basically active in RNP recruitment and membrane envelopment, the decreased virus titers (Fig. ) strongly indicated that the efficiencies of particle formation were far below those of SAD RABV M.
Although the lack of intracellular budding in SAD EBLV-1 M and SAD EBLV-2 M could be a dose effect in a scenario in which recruitment of RNPs by M is a limiting factor, the efficiently budding SAD EBLV-1Mpass showed that even after increased infectious virus release (Fig. ), no intracellular budding events in the rER compartment were detectable. From these data we hypothesize that the RABV and EBLV-1 M proteins target different membranes for virus assembly.
Rapid increase of virus production within only three supernatant passages of SAD EBLV-1 M strongly indicated that adaptive mutations have occurred, either within the EBLV-1 M ORF or in an M interacting protein, making the heterogenotypic EBLV-1 M more compatible to the RABV backbone. Sequencing of SAD EBLV-1 M revealed that a single amino acid exchange at position 44 was evolved during the virus passages. Although no further mutations were detected in the nucleoprotein or in the cytoplasmic domain of the glycoprotein, it remains open whether the M44K mutation indeed contributed to the gain in virus production, and further experiments with regard to the role of the M44K mutation in virus assembly or other M-related functions are needed. Nevertheless, in addition to the detection of the M44K mutation as a thus-far exclusive mutation, the location of the mutation within the three-dimensional structure of lyssavirus M proteins, which was recently determined by Graham et al. (19
), is interesting. Although the region around amino acid position 44 was not found in the structure, it is clear that this presumable flexible region separates the globular domain of M from an N-terminal peptide that binds to a hydrophobic pocket within the globular domain during M self-association (Fig. ). Influences of the M44K mutation on self-association may have contributed to the gain in growth of SAD EBLV-1Mpass. However, based on the structural model, modifications in the interactions of M with the RABV ribonucleoprotein or with other viral structures cannot be excluded.
FIG. 12. Amino acid sequence comparison of the lyssavirus M proteins that were introduced into the recombinant viruses SAD RABVM, SAD EBLV-1 M, and SAD EBLV-2 M. Amino acids in EBLV-1 M and EBLV-2 M that diverged from RABV M are indicated by lowercase letters. (more ...)
Similar infectious virus titers in cell culture supernatants of SAD EBLV-1Mpass and SAD RABV M indicated that the intracellular accumulations of viruslike structures do not essentially contribute to the release of infectious virus, raising the question about their biological function. As shown by cross-sections of wild-type SAD L16 virions and intracellular viruslike structures in the degranulated rER, diameters and inner ultrastructure of the virus bodies were identical with extracellular virions (Fig. ), indicating that indeed membrane-enveloped, condensed RNPs were formed by budding into the rER lumen. Since RABV M is essential for RNP condensation (32
), these structures should contain all inner structural components of a virion. However, as evident from previous observations (22
), longitudinal sections demonstrated that the RNP structures were more variable in length than those that are typically observed in extracellular virions.
Whereas the intracellular viruslike structures most likely did not contribute to infectivity in cell cultures, a role in neurotropism and disease development cannot be ruled out. The observed accumulations of viruslike particles are commonly observed for the neurotropic lyssaviruses, and only few examples of nonlyssavirus rhabdoviruses with comparable accumulations exist. One example is the neurotropic Oita rhabdovirus that combines several characteristics of lyssa- and vesiculoviruses and was shown to produce enveloped viruslike structures in the ER (23
). Although no experimental data corroborate previous speculations about the role of the intracellular assembly structures in intraneuronal RNP transport (43
), this hypothesis is attractive in terms of intraneuronal long-distance transport of newly synthesized RNPs or viruslike structures without the need of budding to the extracellular space. This would also explain why in standard cell cultures the intracellular RNP accumulations do not contribute to the production of infectious virus.
Intracellular accumulations could also simply occur by budding at the ER membrane as a result of excess RABV M and G proteins in late stages of infection. M-RNP complexes at high intracellular concentrations could interact with ER membranes, and subsequent budding may occur. However, the lack of intracellular accumulations in SADEBLV-1Mpass-infected cells argues against a simple overproduction and unspecific budding at the rER membrane. SAD EBLV-1Mpass, which replicated efficiently, as demonstrated by growth curves identical to SAD RABV M (Fig. ), was efficiently replicating and infectious virus production was comparable to SAD RABV M, although the levels of intracellular viral RNAs (Fig. ) and protein levels (Fig. ) were slightly decreased. Whereas this could hint on a dose-dependent accumulation of intracellular viruslike structures, it is unlikely that the only 2.5-fold-decreased M protein level was causative for the complete lack of intracellular viruslike structures. Overproduction of M and G would also be expected at least in part of the SAD EBLV-1Mpass-infected cells. The complete lack of the intracellular assembly structures strongly indicates that the EBLV-1 M protein differs in its specificity either to cellular membranes or to membrane-associated proteins that are involved in virus budding.
Membrane binding by lyssavirus M proteins has not yet been addressed adequately thus far. Because positively charged amino acids are located in the N-terminal region of the lyssavirus M proteins, membrane binding is thought to occur similarly to vesicular stomatitis virus (VSV) M. For VSV M, it has been shown that the N terminus is required for membrane binding and, in addition, a second membrane-binding region in the globular domain of VSV M has been described (10
). Interestingly, amino acids within this second membrane binding region are also involved in binding of N-terminal residues during the process of self-association, which has been suggested to enhance membrane binding of both, VSV M and lyssavirus M proteins (19
). Since both positively charged N-terminal residues and those residues that are involved in M self-association are highly conserved (see Fig. ), it appears unlike that these motifs contribute to the different phenotypes of the EBLV-1 M and RABV M proteins in the assembly of intracellular viruslike structures. Thus, membrane specificity in lyssavirus budding may be determined by other regions or motifs within the lyssavirus M proteins.
Indeed, preliminary experiments with EBLV-1 also indicate a lack of intracellular viruslike structures (results not shown). A direct comparison to the results shown here, however, is difficult because of the lower replication levels of EBLV-1 compared to RABV. In particular, in view of low pathogenicity in experimental EBLV-1 infections of terrestrial mammals (1
), further experiments are required to assess whether the ability to form viruslike structures in the rER compartment correlates with the degree of neurovirulence or virus transmission.
With the intergenotypic comparison of M functions on an identical genetic background, we provide for the first time strong evidence that the matrix proteins of lyssaviruses exhibit diverse and genotype specific functions in the process of virus assembly. This may result in the use of different membranes for virus assembly. On the other hand, basal intraviral interactions that are required for virus assembly and release appear rather conserved, as indicated by the general possibility of intergenotypic complementation and fast selection of efficiently replicating SAD EBLV-1Mpass. A detailed analysis of SAD EBLV-1Mpass shall uncover the role of the M44K mutation within the M gene or other virus proteins that are important for lyssavirus assembly and release. This may also provide a new basis for the identification of virus protein interactions that regulate RNP condensation and particle assembly.