Sequence-function analysis of the Sendai virus L protein domain VI

The large (about 2200 amino acids) L polymerase protein of nonsegmented negative-strand RNA viruses (order Mononegavirales) has six conserved sequence regions (“domains”) postulated to constitute the specific enzymatic activities involved in viral mRNA synthesis, 5′-end capping, cap methylation, 3′ polyadenylation, and genomic RNA replication. Previous studies with vesicular stomatitis virus identified amino acid residues within the L protein domain VI required for mRNA cap methylation. In our recent study we analyzed four amino acid residues within domain VI of the Sendai virus L protein and our data indicated that there could be differences in L protein sequence requirements for cap methylation in two different families of Mononegavirales — rhabdoviruses and paramyxoviruses. In this study, we conducted a more comprehensive mutational analysis by targeting the entire SeV L protein domain VI, creating twenty-four L mutants, and testing these mutations for their effects on viral mRNA synthesis, cap methylation, viral genome replication and virus growth kinetics. Our analysis identified several residues required for successful cap methylation and virus replication and clearly showed the importance of the K-D-K-E tetrad and glycine-rich motif in the SeV cap methylation. This study is the first extensive sequence analysis of the L protein domain VI in the family Paramyxoviridae, and it confirms structural and functional similarity of this domain across different families of the order Mononegavirales.     

Identification of aromatic amino acid residues in conserved region VI of the large polymerase of vesicular stomatitis virus is essential for both guanine-N-7 and ribose 2'-O methyltransferases

Non-segmented negative-sense RNA viruses possess a unique mechanism for mRNA cap methylation. For vesicular stomatitis virus, conserved region VI in the large (L) polymerase protein catalyzes both guanine-N-7 (G-N-7) and ribose 2'-O (2'-O) methyltransferases, and the two methylases share a binding site for the methyl donor S-adenosyl-l-methionine. Unlike conventional mRNA cap methylation, the 2'-O methylation of VSV precedes subsequent G-N-7 methylation. In this study, we found that individual alanine substitutions in two conserved aromatic residues (Y1650 and F1691) in region VI of L protein abolished both G-N-7 and 2'-O methylation. However, replacement of one aromatic residue with another aromatic residue did not significantly affect the methyltransferase activities. Our studies provide genetic and biochemical evidence that conserved aromatic residues in region VI of L protein essential for both G-N-7 and 2'-O methylations. In combination with the structural prediction, our results suggest that these aromatic residues may participate in RNA recognition.     

An unconventional pathway of mRNA cap formation by vesiculoviruses

mRNAs of vesicular stomatitis virus (VSV), a prototype of nonsegmented negative strand (NNS) RNA viruses (e.g., rabies, measles, mumps, Ebola, and Borna disease viruses), possess the 5'-terminal cap structure identical to that of eukaryotic mRNAs, but the mechanism of mRNA cap formation is distinctly different from the latter. The elucidation of the unconventional capping of VSV mRNA remained elusive for three decades since the discovery of the cap structure in some viral and eukaryotic mRNAs in 1975. Only recently our biochemical studies revealed an unexpected strategy employed by vesiculoviruses (VSV and Chandipura virus, an emerging arbovirus) to generate the cap structure. This article summarizes the historical and current research that led to the discovery of the novel vesiculoviral mRNA capping reaction.     

Biochemical characterization of vesicular stomatitis virus-infected Aedes albopictus cells deprived of methionine

Aedes albopictus cells infected with vesicular stomatitis virus (VSV) and maintained in medium lacking methionine produced 1000-fold less infectious virus than cultures maintained in complete medium. Analysis of viral macromolecular synthesis in cells maintained in the presence of varying concentrations of methionine showed that the reduction in virus yield was directly correlated with a reduction in viral RNA and protein synthesis. Of the viral mRNA which was made in methionine-starved cells the majority was not polysome associated. In contrast, virtually all of the virus mRNA in cells maintained in complete medium was polysome associated. In vitro translation of those mRNAs from methionine-starved cells, which were not polysome associated, indicated that they could be translated in vitro as efficiently as polysome-associated virus mRNA but only if the methylation inhibitor, S-adenosylhomocysteine, was not present. These results strongly suggest that methionine starvation of A. albopictus cells inhibits VSV replication by preventing cap methylation of the viral mRNAs, and thus reducing the efficiency with which they are translanted.     

Translation of capped and uncapped VSV mRNAs in the presence of initiation factors from poliovirus-infected cells

Initiation factor preparations from poliovirus-infected cells have been shown to support the translation of poliovirus RNA in vitro, but they fail to stimulate initiation of translation of other mRNAs. In this study, the role of the m⁷G cap group on vesicular stomatitis virus (VSV) mRNAs has been evaluated with respect to the ability of initiation factors from poliovirus-infected cells to discriminate against its translation. A series of uncapped and/or unmethylated VSV mRNAs was prepared and their translation in vitro was analyzed. Each was compared with capped and methylated VSV mRNA and with poliovirus RNA for its recognition by initiation factors from uninfected or poliovirus-infected HeLa cells. The results show that the capped 5′ terminus of mRNA is not the sole basis for recognition and discrimination by poliovirus-altered initiation factors.     

Complete nucleotide sequence of the matrix protein mRNA of vesicular stomatitis virus (New Jersey serotype)

The complete nucleotide sequence of the mRNA of the matrix (M) protein of vesicular stomatitis virus [New Jersey serotype, VSV(NJ)] was derived from a cDNA clone and mRNA. The mRNA is 758 nucleotides long (excluding polyadenylic acid) and encodes a protein of 229 amino acids. The predicted amino acid sequence was compared with that of the corresponding protein of Indiana serotype [VSV(IND)] and a fish rhabdovirus, spring viremia of carp virus (SVCV). An amino acid identity of 62% was found between the M proteins of VSV(NJ) and VSV(IND) while only 24% was present between VSV(NJ) and SVCV. A highly basic NH2-terminal domain followed by a proline-proline-X-tyrosine sequence was present in all the three M polypeptides. Except for the L gene sequence, the complete nucleotide sequence of the four genes of VSV(NJ) are now known. The comparison of the amino acid sequences between the Indiana and New Jersey serotypes demonstrates a high degree of homology between these genes except for the phosphoprotein gene, NS.     

Persistence of vesicular stomatitis virus in interferon-treated cell cultures.

A significant difference was observed in the functional stability of the vesicular stomatitis virus (VSV) genome in mouse L cells and the RK-13 line of rabbit kidney cells pretreated with homologous interferon. By utilizing the ability of vaccinia to rescue VSV from the inhibitory effects of interferon in these cell lines, it was demonstrated that there was a loss of a rescuable form of VSV genome in RK-13 cells pretreated with interferon; superinfection with vaccinia 24 hr after VSV infection did not result in a significant increase in VSV yield. In contrast, significant rescue of VSV occurred in interferon-treated L cells superinfected with vaccinia as late as 72 hr after VSV infection.The present study also provides evidence that in interferon-treated RK-13 cells doubly infected with VSV and vaccinia there was a correlation of the rescuability of the VSV genome and its ability to direct RNA synthesis.     

Sequential disassembly of the cytoskeleton in BHK21 cells infected with vesicular stomatitis virus

The cytopathic effects of vesicular stomatitis virus (VSV) that result in the rounding of BHK21 cells have been studied. The results indicate that they are mediated by a sequential alteration in the distribution of the components of the cytoskeleton, an effect that requires the expression of the viral L protein. The constituents of the cytoskeleton of BHK21 cells were analyzed by fluorescence microscopy. Actin filaments were the first component to become disorganized, so that disassembly of stress fibers were detected 1 hr after infection. The distribution of microtubules and intermediate filaments was unchanged at 2 hr after infection; however, both these cytoskeletal elements exhibited an altered distribution at 3-4 hr after infection. Actinomycin D and cycloheximide did not cause the same effects as infection with VSV, suggesting that inhibition of host-cell gene expression was not responsible. However, viral gene expression was required, since cells infected with uv-irradiated VSV showed the same distribution of cytoskeletal constituents as mock-infected controls. Cells infected at 39.5 degrees (the nonpermissive temperature) with mutants of VSV temperature sensitive in the viral NS (ts G22), N(ts G41), M(ts 0 23), and G(ts 0 45) proteins showed the same changes in the cytoskeleton as those detected with wild-type virus. In contrast, cells infected with ts G11 (L-) showed the characteristic effect of VSV on the cytoskeleton when incubated at 34 degrees (the permissive temperature), but not when incubated at 39.5 degrees. The T-1026 R1 mutant of VSV, which has a much less dramatic effect on cell morphology than wild-type virus, also caused a less marked disruption of the cytoskeleton.     

SARS vaccine based on a replication-defective recombinant vesicular stomatitis virus is more potent than one based on a replication-competent vector

A SARS vaccine based on a live-attenuated vesicular stomatitis virus (VSV) recombinant expressing the SARS-CoV S protein provides long-term protection of immunized mice from SARS-CoV infection (Kapadia, S.U., Rose, J. K., Lamirande, E., Vogel, L., Subbarao, K., Roberts, A., 2005. Long-term protection from SARS coronavirus infection conferred by a single immunization with an attenuated VSV-based vaccine. Virology 340(2), 174-82.). Because it is difficult to obtain regulatory approval of vaccine based on live viruses, we constructed a replication-defective single-cycle VSV vector in which we replaced the VSV glycoprotein (G) gene with the SARS-CoV S gene. The virus was only able to infect cells when pseudotyped with the VSV G protein. We measured the effectiveness of immunization with the single-cycle vaccine in mice. We found that the vaccine given intramuscularly induced a neutralizing antibody response to SARS-CoV that was approximately ten-fold greater than that required for the protection from SARS-CoV infection, and significantly greater than that generated by the replication-competent vector expressing SARS-CoV S protein given by the same route. Our results, along with earlier studies showing potent induction of T-cell responses by single-cycle vectors, indicate that these vectors are excellent alternatives to live-attenuated VSV.     

Intracellular events in the replication of defective interfering particles of vesicular stomatitis virus.

During a synchronous infection with both wild-type vesicular stomatitis virus (VSV) and the MS-T defective interfering (DI) particle at levels of high interference, total RNA synthesis was both reduced by 75% and shut off early as compared to the VSV infection alone. In the interfered infection the production of both VSV intracellular nucleocapsids and progeny virus was suppressed by 90% while there was a dramatic increase in intracellular MS-T nucleocapsids. There was a linear accumulation of MS-T nucleocapsids for only 5 hr with the maximum rate of synthesis being observed at 3 hr postinfection. The MS-T nucleocapsids contained genome-length, single-stranded 19 S DI RNA and were 40% of the positive (+)- and 60% of the negative (?)-strand sense. Neither the (+)- nor (?)strand of MS-T nucleocapsid RNA contained poly(A) sequences since none of the RNA or its ribonuclease digestion products bound to oligo(dT)-cellulose. We can detect no methylation of the nucleocapsid RNA and the 5′-termini were not blocked since both the (+)- and (?)-strands initiated with pppA.     

Temporal regulation of the rate of vesicular stomatitis virus mRNA translation during infection of Chinese hamster ovary cells

Vesicular stomatitis virus (VSV) mRNAs on polysomes 3 hr after infection are not translated in vivo as efficiently as mRNAs 2 hr after infection. There are 14?, 12?, 11?, and 8-fold increases in the amounts of L, G, N, and M messengers, respectively, on polysomes between 2 and 3 hr after infection. However, there are only 3?, 5?, 3?, and 4-fold increases in the rates of synthesis of the respective proteins. The in vivo translational efficiences of these messengers, which is a measure of their capacity to act as template for protein synthesis, are therefore lower at 3 hr after infection than at 2 hr. Since the mRNAs isolated 3 hr after infection are as active in a wheat germ cell-free protein synthesizing system as the mRNAs isolated at 2 hr, the decreased efficiency of VSV translation in vivo at 3 hr is not due to changes in mRNA primary structure. The observed difference is more likely due to a reduced efficiency of the host cell translational system to translate VSV mRNA at 3 hr relative to 2 hr. It was found that at 2 hr after infection, the majority of VSV mRNA is not associated with polysomes, but at 3 hr, the majority of viral messages is polysome bound.     

Glycosylation is not required for the fusion activity of the G protein of vesicular stomatitis virus in insect cells

The gene encoding the complete glycoprotein of vesicular stomatitis virus (VSV, Indiana serotype G protein) with potential asparagine-linked glycans at amino acid residues 179 and 338 was inserted into a baculovirus transfer vector pAcYM1, derived from the nuclear polyhedrosis virus of Autographa californica (AcNPV). The gene was placed under the control of the AcNPV polyhedrin promotor and expressed by the derived recombinant viruses to high levels in Spodoptera frugiperda cell lines. The principal product was the glycosylated version of the G protein, although some alternative (including probable degradation) forms of the protein were also observed. Similar recombinant viruses were prepared with deletion of one, the other, or both glycosylation sites of the VSV G protein. All forms expressed VSV G protein derivatives and mediated cell fusion and the production of syncytia at low pH. The fusogenic properties of the VSV G protein expressed on the surface of insect cells was prevented using anti-VSV sera, or by elevating the pH above 6.2. A reduction of the pH to 5.5, or 5.0, accelerated the rate of syncytia formation.