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The mutation F123A ablated the ability of VPg to co-purify eIF4G, eIF4A, and PABP but did not affect the ability of NTAP-MNV VPg to co-purify eIF4E (Fig. 5), suggesting that the eIF4E and eIF4G binding regions on VPg are distinct

These data would suggest that the direct interaction of VPg with eIF4G largely determines the eIF4F binding capacity of the norovirus VPg protein.

some of which function directly in translation initiation, whereas others may contribute to the regulation of host cell translation by virus infection.

using the number of unique peptides identified for each protein (Table 1) as a semiquantitative indirect measure of protein abundance in the purified complex, it was also apparent that eIF4G was enriched in the complex with respect to the other proteins isolated.

we attempted

to identify

components of the complex that interacted with VPg directly and contribute to viral translation

Independent observations indicate that eIF4G phosphorylation is stimulated during norovirus replication in cell culture and that this phosphorylated form of eIF4G is associated with MNV VPg in infected cells (29). The impact and functional relevance of initiation factor phosphorylation on the norovirus life cycle is currently under study.

high affinity interaction

Noroviruses, members of the Caliciviridae family of small positive-strand RNA viruses, are a major cause of acute gastroenteritis

As these proteins were not identified in both experimental systems and the focus of this study was to determine the role of canonical initiation factors in VPg-dependent translation, these additional host cell proteins factors were not examined in more detail.

recombinant MNV was performed by infecting baby hamster kidney cells with fowlpox virus expressing T7 RNA polymerase followed by transfection of MNV cDNA expression constructs as described previously

Previous reports have also highlighted a potential association of the norovirus VPg protein with components of the eIF3 complex (28) and eIF4E, PABP, and eIF4G as well as the ribosomal protein S6 (29), although the functional relevance of these interactions has yet to be demonstrated. Here we describe the proteomic characterization of the murine norovirus translation initiation factor complex, demonstrating that VPg associates directly with the core components of the eIF4F complex and PABP. We further demonstrate that the interaction between eIF4G and VPg is essential for norovirus translation. Furthermore, we demonstrate that eIF4G is required for efficient virus replication in cell culture.

although these cells are not permissive to MNV infection due to the lack of a suitable receptor, robust MNV translation and replication occurs upon transfection of viral RNA into the cytoplasm (22).

It is worth noting, however, that in addition to disrupting electrostatic interactions, increasing sodium chloride concentrations stabilize hydrophobic interactions.

It is important to note that in both these approaches low levels of eIF4E remained (Fig. 8C) and 4E-BP1 expression in the MNV permissive cells did not completely block the eIF4E-4G interaction. Therefore, further studies on the role of eIF4E during the norovirus life cycle are clearly warranted, but it is worth noting that in addition to a direct role in cap binding for translation initiation, eIF4E plays numerous roles in the regulation of gene expression.

Noroviruses, members of the Caliciviridae family of small positive-strand RNA viruses, are a major cause of acute gastroenteritis in man (8) but have also been identified in a number of other species including dogs (9, 10), cats (11), sheep (12), and cattle (13

We recently analyzed the solution structure of the core domain of VPg using nuclear magnetic resonance (NMR) spectroscopy and found that the MNV VPg protein consists of a compact structured core formed by a pair of α-helices that is flanked by long, flexible N and C termini (35). The region we have identified as being involved in the direct interaction with eIF4G, namely the C-terminal domain, is disordered and, therefore, is likely to adopt a fixed structure only upon interaction with eIF4G.

Noroviruses, a major cause of gastroenteritis in man, have evolved a mechanism that relies on the interaction of translation initiation factors with the virus-encoded VPg protein covalently linked to the 5′ end of the viral RNA.

HSV takes advantage of the neuronal stress response to enter into a silent, latent state. To assist in the execution of this plan, HSV evolved a DNA sequence that allows itself to be suppressed in neurons and a mechanism to maintain an equilibrium between total suppression and potential to exit from the latent state.

translocated from satellite cells

The fundamental question is the identity of the mechanism by which a vigorously replicating virus, on entry into the body, is silenced in neurons harboring latent virus.

Thus, VP16, a virion protein brought into the cells during infection, recruits several cellular proteins, including LSD1, to derepress α gene promoters

In some neurons, the virus establishes a latent, silent state. In other neurons, the virus replicates, and it is most likely that the virus in these neurons is transmitted and replicates in other ganglionic cells.

Finally, in contrast to the events following entry of virus after retrograde transport from the periphery, the data suggest that reactivation does not trigger a stress response that leads to activation of REST

Thus, VP16, a virion protein brought into the cells during infection, recruits several cellular proteins, including LSD1, to derepress α gene promoters. One α gene product, infected cell protein 0 (ICP0), derepresses β and γ1 genes. Ultimately, the onset of viral DNA synthesis enables the expression of very late, or γ2, genes (4).

In contrast, simultaneous expression of all viral genes during reactivation from latency is likely to minimize yield, but the mission of the virus is to assemble enough viruses to reach the portal of egress from the body (e.g., mouth, genitals) rather than to overwhelm the host with infectious virus.

Between 5 and 24 h after excision, mRNAs representative of all viral gene kinetic groups increase 100-fold in amount. Viral DNA also increases in amount, indicating that viral proteins are made. At the same time, viral LAT and miRNA concentrations decrease at least 10-fold (34). It is convenient to define the initial phase lasting no more than 5 h as the preactivation phase and the remaining time interval as the activation phase.

The same concentration of HDAC inhibitor was ineffective in inducing the reactivation of R111 recombinant virus in ganglionic organ cultures maintained in medium containing anti-NGF antibody.

Selective degradation with RNase I demonstrates that GCXV has a single-stranded RNA genome.

reverse genetics system

no Illumina reads aligned to the segment 5 sequences from the other isolates, and no contigs from ACH27 exhibited significant similarity to the segment 5 sequences from the other isolates. Therefore, segment 5 appears to be absent from ACH27 and TR7094.

However, only four segments were assembled for ACH27 and TR7094 (genome size ∼10.6 kb). For these two isolates, the four assembled segments corresponded to the four largest segments assembled in the other isolates (Figure 1Figure 1, segments 1–4).

The phylogeny inferred from segment 5 is also inconsistent with those of the other segments. In addition to the absence of this segment in TR7094 and ACH27, this segment exhibited very low levels of nt divergence (0.4%–2.3%), with most sequence variations only present in a single isolate.

with each genome segment separately packaged into virions. Although multicomponent genomes are relatively common among RNA viruses that infect plants and fungi, this method of genome organization has not previously been seen in animal viruses (

RNA extracted from purified GCXV particles confirmed the presence of a segmented genome (

The mosquito pools for both ACH27 and TR7094 contained multiple viruses capable of replicating in mosquito cells, so we were unable to obtain pure cultures for these isolates (

Nuclease digestion assays (Figure 1Figure 1B), along with 5′ and 3′ rapid amplification of cDNA ends (RACE), confirmed that all five genome segments were single-stranded, positive-sense RNA (ssRNA+).

all five segments, (2) segments 1–4, and (3) segments 2–5

the number of plaques decreased more quickly than expected with dilution of the inoculant) (

segments 1–5 (passage 1) and segments 1–4 (passage 2)

Other members of the A3 family are believed to affect other exogenous viruses as well as endogenous retrovirus/retroelement movement within the genome. In particular, human A3A is a potent inhibitor of IAP and MusD and other retrotransposons such as LINE-1 and this inhibition is CDA-independent, at least in cultured cells [18]–[20]. A3A also inhibits adeno-associated virus replication, a nuclear-replicating parvovirus, via CDA-independent means [20]. In monocytes, A3A restricts HIV-1 infection and the decrease in A3A levels that occurs during monocyte-to-macrophage development is concomitant with increased susceptibility to HIV-1 infection [21]. A3A is not packaged into HIV virions and is thought to restrict infection by targeting incoming virus [22]–[24]. In contrast, A3A is packaged in human T-lymphotropic virus type-I virions and restricts infection, at least in transfected cells [25]. A3A preferentially deaminates cytidines that are in a TC motif [26].

To determine the level of transgene expression, we first isolated RNA from different tissues, including peripheral blood mononuclear cells (PBMCs), and performed reverse-transcribed real-time quantitative PCR (RT-qPCR). RNA from human H9 cultured cells and human and C57BL/6 mouse PBMCs served as controls. For each transgene, there was one high- (A3Ghigh, A3Ahigh) and one low- (A3Glow, A3Alow) expressing strain, defined by their relative expression in lymphoid tissues. The A3Ghigh strain expressed higher levels of the transgene than the endogenous mouse gene in spleen and thymus, but similar A3G levels in mouse and human PBMCs, while the A3Glow strain expressed approximately 10-fold lower levels in these tissues (Figure 1A). In contrast, the A3Ahigh strain expressed similar or lower levels than mouse A3; there was also about 2-fold lower expression of A3A in mouse PBMCs than in human PBMCs (Figure 1B). The A3Alow strain had very low but detectable levels of expression in several tissues. Since the β-actin regulatory region was used, transgene expression was seen in many tissues and in several at levels higher than endogenous mouse A3 (e.g. heart, brain and liver) (Figure 1A and 1B). We also performed western blots on different tissues from the 4 different mouse strains, using antiserum that detects both A3A and A3G. The relative protein expression levels were similar to that seen at the RNA level (Figure S1A and S1B).

We next determined if the in vivo-produced A3A and A3G proteins were functionally active. Extracts were prepared from primary splenocyte cultures and equal amounts (total protein concentration/volume) were incubated with FAM-labeled substrates containing the A3A- or A3G-preferred target sequence (S50-TTC and S50-CCC, respectively). As controls, we also performed these assays with extracts prepared from 293T cell lines transfected with A3A or A3G. Activity could be readily detected in transgenic mice expressing high levels of A3A or A3G. Further, in accord with the known specificity of the cytidine deaminases, extracts from the A3Ahigh mice deaminated the TTC- more efficiently than CCC-containing substrates, while those from A3Ghigh mice more efficiently deaminated the CCC substrate (Figure 2). For both A3Alow and A3G low, trace amounts of activity were detectable with the preferred substrates, while no activity was detectable with either endogenous mA3 or from mA3 knockout splenocytes. No deaminase activity was detected with WT mouse extracts, perhaps because the mouse protein has lower overall activity or expression. These data show that the transgenic mice expressed catalytically active human deaminases in these heterologous cells.

Humans have 7 APOBEC3 genes and determining how each specifically functions to inhibit retroviruses like HIV is complicated, because all 7 can be produced in a given cell type or tissue.

To overcome this limitation, we made transgenic mice that express two of the human proteins, APOBEC3A and APOBEC3G in mice that do not express their own APOBEC3. These mice were able to effectively block infection by several mouse retroviruses

We were unable to perform similar assays with in vivo produced MMTV, because the only cell-free virus in mice is found in milk and mammary tumors and we have not yet established breeding colonies of virus-infected human A3 transgenic mice.

A3G and A3A inhibit retrovirus infection by different means.

Two A3A and A3G mouse strains each were generated, expressing levels of these proteins within the range or at levels lower than that seen in human cells. This likely has relevance to what occurs in individual humans, where non-coding region polymorphisms in A3 genes alter expression levels and may influence progression to HIV-induced disease

An additional limitation of previous studies done on human A3 proteins is the reliance on transfecting constructs expressing A3 proteins, which may not reflect the endogenous levels of a protein expression found in vivo

Within the VF, viral core particles transcribe and release viral mRNAs that possess a dimethylated cap 1 structure at the 5′ terminus but lack a poly(A) tail (19).

Consistent with our findings, the authors noted that the rough endoplasmic reticulum (RER) made numerous contacts with VF, which they suggested may indicate a role for RER in the transport of newly synthesized viral proteins to the VF, as is the case for rubella virus (64).

To address this, we monitored protein expression levels of eIF4E, eIF4A1, and eIF4G over the course of an infection. As others have found (39), we were unable to detect any difference in the levels of total protein in mock versus infected cells from 0 to 20 h p.i. (Fig. 3C and data not shown). Together, these data suggest that cellular translation proteins are redistributed to the VF.

Most reovirus strains form filamentous VF through an association with stabilized microtubules. However, the T3D strain used in these experiments contains a temperature-sensitive mutation in the viral protein μ2 that prevents this association, resulting in the production of globular VF at 37°C (27, 28). Therefore, to evaluate if PMY labeling occurs within filamentous viral factories, we infected cells with the serotype 1 Lang (T1L) strain. As we found for T3D-infected cells, the PMY labeling localized to T1L VF at 18 h postinfection

It was unclear if this was a result of increased expression levels or as a consequence of redistribution of the proteins to the factories. To address this, we monitored protein expression levels of eIF4E, eIF4A1, and eIF4G over the course of an infection. As others have found (39), we were unable to detect any difference in the levels of total protein in mock versus infected cells from 0 to 20 h p.i. (Fig. 3C and data not shown). Together, these data suggest that cellular translation proteins are redistributed to the VF.

Our finding that σNS interacts with eIF3A and pS6R suggests that translational machinery is recruited to the factory by viral proteins. This is consistent with the finding of others that σNS cosediments with 40S and 60S ribosomes (62) and suggests that σNS is directly involved in viral translation.

FIG 3  Cellular translation initiation factors colocalize to viral factories. (A, B) CV-1 cells were infected with T3D or T1L at an MOI of 1. At 18 h p.i., RPM-labeled cells were coimmunostained for μNS and eIF4E (A) or eIF3A (B). Scale bars, 10 µm. (C) CV-1 cells were infected with T3D, MOI of 3, for the times indicated. Protein levels were assessed by immunoblotting. M = mock.

A variety of expression vectors (e.g., Escherichia coli, Pichia pastoris and DNA) have been used to express Pvs25 protein which has been administered alone or in combination with adjuvants

To date these studies suggest that the recombinant protein currently requires both not only linear, but conformation dependent epitopes, and a strong adjuvant to induce transmission-blocking antibodies.

Intranasal and intramuscular immunization with Baculovirus Dual Expression System-based Pvs25 vaccine substantially blocks Plasmodium vivax transmission

Recently, we have developed a new vaccine vector system based on the baculovirus Autographa californica nucleopolyhedrosis virus (AcNPV) termed the “Baculovirus Dual Expression System”, which drives expression of vaccine candidate antigens by a dual promoter that consists of tandemly arranged baculovirus-derived polyhedrin and mammalian-derived CMV promoters. It has been shown that AcNPV, an enveloped double-stranded DNA virus that naturally infects insects, possesses strong adjuvant properties that can activate dendritic cell-mediated innate immunity

Mucosal vaccines have several attractive features compared with parenteral vaccines (e.g., safety, cost-effectiveness and ease of administration), but studies on their use have been limited almost exclusively to protection against mucosally transmitted pathogens. We provide evidence that i.n. immunization is a feasible alternative for preventing malaria, which is transmitted through non-mucosal routes

These results are consistent with our previous work showing that intranasal immunization with the baculovirus-based vaccine induced strong systemic humoral immune responses with high titres of antigen-specific antibodies and conferred complete protection against malaria blood-stage challenge

which can induce immunological memory against heterologous antigens in a rodent model; however, it is precluded from clinical use due to its enterotoxicity and potential hazardous effects on olfactory nerves [22]. In contrast, a baculovirus-based delivery system may offer an attractive immunization method, as AcNPV exhibits low cytotoxicity and is incapable of replication in mammalian cells

The data described here adds to previously presented data showing the significant potential of the baculovirus dual expression system against the blood stages of the parasite

but also demonstrates clearly its ability to induce antibodies against the ookinete surface protein Pvs25, and to elicit a transmission-blocking immune response against the P. vivax isolates from endemic areas, and a transgenic rodent malaria parasite model in preliminary studies.

One was SMFA on peripheral blood from P. vivax infected patients.