Acute, infectious diarrhea is the most common cause of morbidity and mortality among young children in developing countries; rotaviruses are the leading etiologic agents of severe diarrheal disease in infants and young children, causing an estimated 600,000 annual deaths globally. Although mortality due to rotavirus infections is much higher in developing than in developed countries, the frequencies of infection are remarkably similar. Because of the significant morbidity and mortality associated with rotavirus diarrhea, and because even advanced levels of hygiene seem unable to control the spread of rotavirus infections, there is an urgent need to develop effective vaccination and therapeutic strategies. Fundamental to these developments is a basic understanding of the molecular mechanisms by which rotaviruses interact with their host cells.
The rotavirus genome is composed of 11 segments of double-stranded RNA (dsRNA) and is encased in a capsid formed by three concentric layers of protein. The outermost layer, characteristic of infectious, triple-layered particles (TLPs), consists of two proteins, VP4 and VP7. VP4 is involved in receptor binding and cell penetration. The role of VP7 is less clear, although it has been recently shown that the protein interacts with cell-surface molecules at a postattachment step. After binding to the cell surface, the virus penetrates the plasma membrane and causes a productive infection of the cell. Penetration is enhanced by, and most probably depends on, trypsin treatment of the virus, which results in specific cleavage of VP4 into the polypeptides VP8 and VP5. The mechanism by which rotaviruses enter the cell has been controversial. Recently, we reported that neither clathrin-dependent endocytosis nor the caveolae uptake pathway play a significant role in rotavirus entry. We are interested in further characterizing the still largely undefined entry mechanism rotaviruses use to infect the cell. This knowledge should help us better understand cell entry mechanisms of nonenveloped viruses in general.
During or shortly after cell entry, the infecting virus uncoats, losing the two surface proteins and yielding a double-layered particle (DLP) that is transcriptionally active. The viral mRNAs contain 5′-methylated cap structures but lack the polyA tails characteristic of most cellular mRNAs. Instead, rotavirus mRNAs have at their 3′ end a consensus sequence (UGACC) that is conserved in all segments of the viral genome. The viral RNA transcripts direct the synthesis of six structural (VP1–4, VP6, and VP7) and six nonstructural (NSP1–6) proteins. Once a critical mass of viral proteins has been synthesized, the proteins accumulate into discrete, large cytoplasmic inclusions termed viroplasms, where the replication of the virus genome and assembly of transcriptionally active progeny DLPs are believed to take place. Once the DLPs are assembled, they bud across the membrane of the endoplasmic reticulum (ER) with the help of the nonstructural protein NSP4, acquiring a transient membrane envelope during this process. These enveloped particles contain, in addition to NSP4, the virus surface proteins VP4 and VP7. During the last step of rotavirus morphogenesis, the lipid envelope is lost by a largely unknown mechanism to yield mature, infectious TLPs. We are interested in characterizing the cellular and viral proteins that participate in this morphogenetic process.
Given that protein synthesis requires numerous components that cannot be encoded within viral genomes, viruses depend, as obligate intracellular parasites, on a cell's translation machinery to produce viral proteins. The successful amplification of viral genomes requires that viral mRNAs compete with cellular mRNAs for the host translation apparatus. Several viruses have developed remarkable strategies to ensure the efficient translation of their mRNAs while simultaneously inhibiting cellular protein synthesis. We are interested in characterizing the mechanisms that the virus uses to prevent the translation of cellular mRNAs while allowing, efficient translation of the viral mRNAs.
Early in the infection, the rotavirus takes over the host's translation machinery, shutting off the cell's protein synthesis; it has been proposed that the protein NSP3 plays an important role during this process. Recent experiments in our lab, in which we used RNA interference (RNAi) to silence the expression of NSP3 in virus-infected cells, indicated that this protein is not required for efficient translation of viral mRNAs and also suggested that NSP3 is only partially responsible for inhibiting cell protein synthesis. These experiments also suggested that NSP3 has other roles in virus replication. Despite the severe inhibition of cell protein synthesis induced by the virus, a small subset of cellular mRNAs continue to be translated. We are interested in determining the characteristics of these cellular mRNAs to learn about noncanonical translation strategies in mammalian cells.
Virus infections are certainly not the only stresses with which eukaryotic cells have to cope. Cells encounter a range of physiological and environmental conditions that require coordinated, adaptive expression of stress-response genes affecting survival, apoptosis, cell cycle progression, and differentiation. The ER integrates signals from throughout the cell to orchestrate these coordinated responses. It is the place where the folding of proteins destined for both intracellular organelles and the cell surface occurs. Accumulation of misfolded proteins causes ER stress and leads to the activation of the unfolded protein response (UPR).
The function of the UPR is to eliminate misfolded proteins in the ER by two mechanisms: (1) upregulating the expression of chaperone proteins and degradation factors to refold or eliminate misfolded proteins and (2) attenuating translation to reduce incoming protein traffic into the ER. Thus, an important function of the UPR is to protect the ER from stress by reducing the demand on the protein-folding machinery. Failure to alleviate ER stress leads to the activation of apoptotic pathways and cell death. Several viruses have been shown to induce ER stress and UPR signaling, while the pathogenesis of other viruses has been associated with the induction of ER stress. However, viruses that induce ER stress must face the consequences of activating the UPR. The general arrest of protein synthesis that takes place during the UPR must be dealt with to allow expression of viral and cellular proteins essential for the replication cycle of the virus, whereas overexpression of chaperones and regulation of the redox environment should be beneficial. Thus, it has been proposed that viruses might induce mechanisms that modulate the UPR, keeping the beneficial aspects while suppressing the deleterious ones. We have found that, during rotavirus infection, the mRNA levels of some chaperones such as grp78/BiP are increased and cell protein synthesis shuts down. These data suggest that rotavirus infection exerts a stress on the ER. We wish to establish directly whether rotaviruses induce an UPR and how it would be modulated during the viral cycle.
The recently described ubiquitous cellular defense mechanism known as RNAi has become a powerful and widely used tool for analyzing gene function. This evolutionarily conserved mechanism, triggered by dsRNA, specifically suppresses gene expression by selectively degrading mRNAs that match the sequence of the dsRNA triggering the response, without affecting the expression of other genes. During RNAi, 21– to 25–base-pair dsRNAs, known as short-interfering RNAs (siRNAs), serve as guides for the enzymatic cleavage of complementary RNAs, thus offering an exquisitely specific knockdown of the expression of a particular gene. The segmented nature of the genome of the viruses in the Reoviridae family makes them particularly amenable to analysis by RNAi. In the case of rotaviruses, each of the 11 segments of dsRNA is transcribed into a single mRNA; with exception of one bicistronic segment, each encodes a single protein. This makes it possible to silence the expression of individual genes without affecting, at least directly, the expression of the others. We recently silenced the expression of several rotavirus genes by RNAi, which has allowed us to explore directly the function of viral proteins in virus-infected cells. Of great interest is that, when examined in vivo in the context of a viral infection, the function of some of the viral proteins is as expected, based on previous data gathered from in vitro experiments or by transiently expressing individual viral genes in uninfected cells. Taking advantage of this new methodology, we plan to search for cellular proteins relevant to the efficient replication of the virus, by screening a retrovirus-based siRNA library that targets about one-third of the human genome.
Last updated September 2008
