Michael A. Whitt

RNA interference is an antiviral defence mechanism in Caenorhabditis elegans

RNA interference (RNAi) is an evolutionarily conserved sequence-specific post-transcriptional gene silencing mechanism that is well defined genetically in Caenorhabditis elegans. RNAi has been postulated to function as an adaptive antiviral immune mechanism in the worm, but there is no experimental evidence for this. Part of the limitation is that there are no known natural viral pathogens of C. elegans. Here we describe an infection model in C. elegans using the mammalian pathogen vesicular stomatitis virus (VSV) to study the role of RNAi in antiviral immunity. VSV infection is potentiated in cells derived from RNAi-defective worm mutants (rde-1; rde-4), leading to the production of infectious progeny virus, and is inhibited in mutants with an enhanced RNAi response (rrf-3; eri-1). Because the RNAi response occurs in the absence of exogenously added VSV small interfering RNAs, these results show that RNAi is activated during VSV infection and that RNAi is a genuine antiviral immune defence mechanism in the worm.

Rhabdoviruses and the Cellular Ubiquitin-Proteasome System: a Budding Interaction

ABSTRACT The matrix (M) proteins of vesicular stomatitis virus (VSV) and rabies virus (RV) play a key role in both assembly and budding of progeny virions. A PPPY motif (PY motif or late-budding domain) is conserved in the M proteins of VSV and RV. These PY motifs are important for virus budding and for mediating interactions with specific cellular proteins containing WW domains. The PY motif and flanking sequences of the M protein of VSV were used as bait to screen a mouse embryo cDNA library for cellular interactors. The mouse Nedd4 protein, a membrane-localized ubiquitin ligase containing multiple WW domains, was identified from this screen. Ubiquitin ligase Rsp5, the yeast homolog of Nedd4, was able to interact both physically and functionally with full-length VSV M protein in a PY-dependent manner. Indeed, the VSV M protein was multiubiquitinated by Rsp5 in an in vitro ubiquitination assay. To demonstrate further that ubiquitin may be involved in the budding process of rhabdoviruses, proteasome inhibitors (e.g., MG132) were used to decrease the level of free ubiquitin in VSV- and RV-infected cells. Viral titers measured from MG132-treated cells were reproducibly 10- to 20-fold lower than those measured from untreated control cells, suggesting that free ubiquitin is important for efficient virus budding. Last, release of a VSV PY mutant was not inhibited in the presence of MG132, signifying that the functional L domain of VSV is required for the inhibitory effect exhibited by MG132. These data suggest that the cellular ubiquitin-proteasome machinery is involved in the budding process of VSV and RV.

Mutations in the PPPY Motif of Vesicular Stomatitis Virus Matrix Protein Reduce Virus Budding by Inhibiting a Late Step in Virion Release

ABSTRACT The N terminus of the matrix (M) protein of vesicular stomatitis virus (VSV) and of other rhabdoviruses contains a highly conserved PPPY sequence (or PY motif) similar to the late (L) domains in the Gag proteins of some retroviruses. These L domains in retroviral Gag proteins are required for efficient release of virus particles. In this report, we show that mutations in the PPPY sequence of the VSV M protein reduce virus yield by blocking a late stage in virus budding. We also observed a delay in the ability of mutant viruses to cause inhibition of host gene expression compared to wild-type (WT) VSV. The effect of PY mutations on virus budding appears to be due to a block at a stage just prior to virion release, since electron microscopic examination of PPPA mutant-infected cells showed a large number of assembled virions at the plasma membrane trapped in the process of budding. Deletion of the glycoprotein (G) in addition to these mutations further reduced the virus yield to less than 1% of WT levels, and very few particles were assembled at the cell surface. This observation suggested that G protein aids in the initial stage of budding, presumably during the formation of the bud site. Overall, our results confirm that the PPPY sequence of the VSV M protein possesses L domain activity analogous to that of the retroviral Gag proteins.

Interaction of coxsackievirus B3 with the full length coxsackievirus-adenovirus receptor

Group B coxsackieviruses (CVB) utilize the coxsackievirus-adenovirus receptor (CAR) to recognize host cells. CAR is a membrane protein with two Ig-like extracellular domains (D1 and D2), a transmembrane domain and a cytoplasmic domain. The three-dimensional structure of coxsackievirus B3 (CVB3) in complex with full length human CAR and also with the D1D2 fragment of CAR were determined to ∼22 Å resolution using cryo-electron microscopy (cryo-EM). Pairs of transmembrane domains of CAR associate with each other in a detergent cloud that mimics a cellular plasma membrane. This is the first view of a virus–receptor interaction at this resolution that includes the transmembrane and cytoplasmic portion of the receptor. CAR binds with the distal end of domain D1 in the canyon of CVB3, similar to how other receptor molecules bind to entero- and rhinoviruses. The previously described interface of CAR with the adenovirus knob protein utilizes a side surface of D1.

A decade after the generation of a negative-sense RNA virus from cloned cDNA - what have we learned?

Since the first generation of a negative-sense RNA virus entirely from cloned cDNA in 1994, similar reverse genetics systems have been established for members of most genera of the Rhabdo- and Paramyxoviridae families, as well as for Ebola virus (Filoviridae). The generation of segmented negative-sense RNA viruses was technically more challenging and has lagged behind the recovery of nonsegmented viruses, primarily because of the difficulty of providing more than one genomic RNA segment. A member of the Bunyaviridae family (whose genome is composed of three RNA segments) was first generated from cloned cDNA in 1996, followed in 1999 by the production of influenza virus, which contains eight RNA segments. Thus, reverse genetics, or the de novo synthesis of negative-sense RNA viruses from cloned cDNA, has become a reliable laboratory method that can be used to study this large group of medically and economically important viruses. It provides a powerful tool for dissecting the virus life cycle, virus assembly, the role of viral proteins in pathogenicity and the interplay of viral proteins with components of the host cell immune response. Finally, reverse genetics has opened the way to develop live attenuated virus vaccines and vaccine vectors.

The Membrane-Proximal Stem Region of Vesicular Stomatitis Virus G Protein Confers Efficient Virus Assembly

ABSTRACT In this report, we show that the glycoprotein of vesicular stomatitis virus (VSV G) contains within its extracellular membrane-proximal stem (GS) a domain that is required for efficient VSV budding. To determine a minimal sequence in GS that provides for high-level virus assembly, we have generated a series of recombinant ΔG-VSVs which express chimeric glycoproteins having truncated stem sequences. The recombinant viruses having chimeras with 12 or more membrane-proximal residues of the G stem, and including the G protein transmembrane-cytoplasmic tail domains, produced near-wild-type levels of particles. In contrast, viruses encoding chimeras with shorter or no G-stem sequences produced ∼10- to 20-fold less. This budding domain when present in chimeric glycoproteins also promoted their incorporation into the VSV envelope. We suggest that the G-stem budding domain promotes virus release by inducing membrane curvature at sites where virus budding occurs or by recruiting condensed nucleocapsids to sites on the plasma membrane which are competent for efficient virus budding.

A phosphorylated 34-kDa protein and a subpopulation of polyhedrin are thiol linked to the carbohydrate layer surrounding a baculovirus occlusion body

Surrounding baculovirus occlusion bodies is an electron-dense layer reported to be composed of carbohydrate which we term calyx. Incubation of Autographa californica nuclear polyhedrosis virus occlusion bodies (AcMNPV OBs) with dilute alkaline saline (DAS) followed by centrifugation at 12,000 g resulted in the sedimentation of calyx material which contained pp34, residual polyhedrin (p32), and entrapped occluded virions (DAS P-12 fraction). Incubation of the DAS P-12 fraction with sodium dodecyl sulfate (SDS) resulted in solubilization of the entrapped virions and the majority of p32, while calyx material, pp34, and some p32 remained sedimentable at 12,000 g. Immunofluorescence microscopy of DAS-solubilized OBs using monoclonal antibody to pp34 and p32 revealed that both pp34 and p32 are closely associated with the calyx. When DAS P-12 fractions were resuspended in SDS and reducing agent, not only were the entrapped virions solubilized, but pp34 and the remaining p32 were also liberated, indicating that pp34 and a subpopulation of p32 are associated with the calyx via thiol linkages. Immunoblot analysis and peptide mapping demonstrated that pp34 is neither immunologically nor structurally related to p32. The kinetics of pp34 synthesis were also examined by immunoprecipitation of infected cell polypeptides using pp34-specific monoclonal antibody. pp34 was detected initially 15 hr postinfection (p.i.) and continued to be phosphorylated until 60-70 hr p.i. This study demonstrates that the AcMNPV calyx has a proteinaceous component and we propose that other occluded baculoviruses may also have a calyx-associated protein analogous to pp34.

Membrane fusion activity, oligomerization, and assembly of the rabies virus glycoprotein

The spike glycoprotein (G protein) of rabies virus (CVS strain) expressed in HeLa cells from cloned cDNA mediated membrane fusion after exposure to pHs of 6.1 or below. Chemical crosslinking showed that the rabies G protein, like the vesicular stomatitis virus (VSV) G protein, could be crosslinked to dimers and trimers, indicating that rabies G protein is a trimer. However, unlike the VSV G protein, rabies G protein trimers were not stable to sedimentation in sucrose gradients, even at a mildly acidic pH which stabilizes the VSV G protein trimers. In addition, we report that the expressed rabies virus G protein was functional because it could assemble into VSV particles (tsO45) lacking VSV G protein and rescue infectivity. These VSV (rabies) pseudotypes were neutralized only by an antibody to the rabies G protein. We also examined the properties of a hybrid protein containing the extracellular domain of the rabies virus glycoprotein and the transmembrane and cytoplasmic domains of the VSV G protein. This protein was transported to the cell surface and could be crosslinked to form dimers and trimers, but had little or no detectable membrane fusion activity. The lack of fusion activity was paradoxical because the hybrid protein could rescue VSV infectivity, although the titers were lower than those obtained with the wild-type rabies G protein.

Identification of Two Additional Translation Products from the Matrix (M) Gene That Contribute to Vesicular Stomatitis Virus Cytopathology

ABSTRACT The matrix (M) protein of vesicular stomatitis virus (VSV) is a multifunctional protein that is responsible for condensation of the ribonucleocapsid core during virus assembly and also plays a critical role in virus budding. The M protein is also responsible for most of the cytopathic effects (CPE) observed in infected cells. VSV CPE include inhibition of host gene expression, disablement of nucleocytoplasmic transport, and disruption of the host cytoskeleton, which results in rounding of infected cells. In this report, we show that the VSV M gene codes for two additional polypeptides, which we have named M2 and M3. These proteins are synthesized from downstream methionines in the same open reading frame as the M protein (which we refer to here as M1) and lack the first 32 (M2) or 50 (M3) amino acids of M1. Infection of cells with a recombinant virus that does not express M2 and M3 (M33,51A) resulted in a delay in cell rounding, but virus yield was not affected. Transient expression of M2 and M3 alone caused cell rounding similar to that with the full-length M1 protein, suggesting that the cell-rounding function of the M protein does not require the N-terminal 50 amino acids. To determine if M2 and M3 were sufficient for VSV-mediated CPE, both M2 and M3 were expressed from a separate cistron in a VSV mutant background that readily establishes persistent infections and that normally lacks CPE. Infection of cells with the recombinant virus that expressed M2 and M3 resulted in cell rounding indistinguishable from that with the wild-type recombinant virus. These results suggest that M2 and M3 are important for cell rounding and may play an important role in viral cytopathogenesis. To our knowledge, this is first report of the multiple coding capacities of a rhabdovirus matrix gene.

The Membrane-Proximal Domain of Vesicular Stomatitis Virus G Protein Functions as a Membrane Fusion Potentiator and Can Induce Hemifusion

ABSTRACT Recently we showed that the membrane-proximal stem region of the vesicular stomatitis virus (VSV) G protein ectodomain (G stem [GS]), together with the transmembrane and cytoplasmic domains, was sufficient to mediate efficient VSV budding (C. S. Robison and M. A. Whitt, J. Virol. 74:2239-2246, 2000). Here, we show that GS can also potentiate the membrane fusion activity of heterologous viral fusion proteins when GS is coexpressed with those proteins. For some fusion proteins, there was as much as a 40-fold increase in syncytium formation when GS was coexpressed compared to that seen when the fusion protein was expressed alone. Fusion potentiation by GS was not protein specific, since it occurred with both pH-dependent as well as pH-independent fusion proteins. Using a recombinant vesicular stomatitis virus encoding GS that contained an N-terminal hemagglutinin (HA) tag (GSHA virus), we found that the GSHA virus bound to cells as well as the wild-type virus did at pH 7.0; however, the GSHA virus was noninfectious. Analysis of cells expressing GSHA in a three-color membrane fusion assay revealed that GSHA could induce lipid mixing but not cytoplasmic mixing, indicating that GS can induce hemifusion. Treatment of GSHA virus-bound cells with the membrane-destabilizing drug chlorpromazine rescued the hemifusion block and allowed entry and subsequent replication of GSHA virus, demonstrating that GS-mediated hemifusion was a functional intermediate in the membrane fusion pathway. Using a series of truncation mutants, we also determined that only 14 residues of GS, together with the VSV G transmembrane and cytoplasmic tail, were sufficient for fusion potentiation. To our knowledge, this is the first report which shows that a small domain of one viral glycoprotein can promote the fusion activity of other, unrelated viral glycoproteins.