Influenza A viruses lacking sialidase activity can undergo multiple cycles of replication in cell culture, eggs, or mice

Influenza A viruses possess both hemagglutinin (HA), which is responsible for binding to the terminal sialic acid of sialyloligosaccharides on the cell surface, and neuraminidase (NA), which contains sialidase activity that removes sialic acid from sialyloligosaccharides. Interplay between HA receptor-binding and NA receptor-destroying sialidase activity appears to be important for replication of the virus. Previous studies by others have shown that influenza A viruses lacking sialidase activity can undergo multiple cycles of replication if sialidase activity is provided exogenously. To investigate the sialidase requirement of influenza viruses further, we generated a series of sialidase-deficient mutants. Although their growth was less efficient than that of the parental NA-dependent virus, these viruses underwent multiple cycles of replication in cell culture, eggs, and mice. To understand the molecular basis of this viral growth adaptation in the absence of sialidase activity, we investigated changes in the HA receptor-binding affinity of the sialidase-deficient mutants. The results show that mutations around the HA receptor-binding pocket reduce the virus's affinity for cellular receptors, compensating for the loss of sialidase. Thus, sialidase activity is not absolutely required in the influenza A virus life cycle but appears to be necessary for efficient virus replication.     

What Happens Inside Lentivirus or Influenza Virus Infected Cells: Insights into Regulation of Cellular and Viral Protein Synthesis

Efficient manipulation of the regulatory mechanisms controlling host cell gene expression provides the means for productive infection by animal viruses. Upon infecting the host cell, viruses must: (i) bypass the cellular antiviral defense mechanisms to prevent the translational blocks imposed by the interferon pathway; and (ii) effectively “hijack” the host protein synthetic machinery into mass production of virion protein components. The multicomponent regulatory nature of cellular gene expression has provided the means of selecting for a diverse range of mechanisms utilized by animal viruses to ensure that replication efficiency is maintained throughout the virus life cycle. One important research component of the careful examination of gene regulation is those studies that focus on elucidating the mechanisms by which viruses control mRNA translation during host cell infection. Much of the work in our laboratory has focused on elucidating the strategies by which human immunodeficiency virus type 1 and influenza virus regulate protein synthesis during infection. Here we describe the ways in which these two distinctly different RNA viruses ensure the selective and efficient translation of their viral mRNAs in infected cells. These strategies include circumvention of the deleterious effects associated with activation of the interferon-induced protein kinase, PKR. Herein we describe our methodologies designed to elucidate the translational regulation in cells infected by these viruses. We conclude with a brief summary of new directions, utilizing these methods, taken toward understanding the translational control mechanisms imposed by these viral systems, and how our studies of virally infected cells have allowed us to identify growth-regulating components of normal, uninfected cells.     

Gene amplification and expression by RNA viruses and potential for further application to plant gene transfer

The great majority of plant viruses encapsidate messenger-sense ssRNA and have no natural DNA phase in their life cycle. Despite their RNA nature, essentially any desired change can be introduced into such genomes by using recombinant DNA techniques with suitably constructed, expressible viral cDNA clones. For some viruses such as brome mosaic virus, these methods have been used to define the sequences controlling RNA-directed genomic RNA replication and the expression of internal genes via subgenomic mRNAs. The results suggest a surprising degree of genetic flexibility, which appears to be reflected in the varied gene complements and genetic organizations of presumably related plant and animal RNA viruses sharing conserved replication genes. Foreign genes inserted in such RNA virus genomes can be amplified and expressed to a high level in transfected plant cells. In addition to the potential use of such viruses as episomal expression vectors, it should be possible to couple the viral pathways of RNA-dependent RNA synthesis to amplify and to further regulate the expression of genes transformed into plant chromosomes.     

The NB protein of influenza B virus is not necessary for virus replication in vitro

The NB protein of influenza B virus is thought to function as an ion channel and therefore would be expected to have an essential function in viral replication. Because direct evidence for its absolute requirement in the viral life cycle is lacking, we generated NB knockout viruses by reverse genetics and tested their growth properties both in vitro and in vivo. Mutants not expressing NB replicated as efficiently as the wild-type virus in cell culture, whereas in mice they showed restricted growth compared with findings for the wild-type virus. Thus, the NB protein is not essential for influenza B virus replication in cell culture but promotes efficient growth in mice.     

Identification and analysis of a retinoblastoma binding motif in the replication protein of a plant DNA virus: requirement for efficient viral DNA replication.

Geminiviruses are plant DNA viruses with small genomes whose replication, except for the viral replication protein (Rep), depends on host proteins and, in this respect, are analogous to animal DNA tumor viruses, e.g. SV40. The mechanism by which these animal viruses create a cellular environment permissive for viral DNA replication involves the binding of a virally encoded oncoprotein, through its LXCXE motif, to the retinoblastoma protein (Rb). We have identified such a LXCXE motif in the Rep protein of wheat dwarf geminivirus (WDV) and we show its functional importance during viral DNA replication. Using a yeast two-hybrid system we have demonstrated that WDV Rep forms stable complexes with p130Rbr2, a member of the Rb family of proteins, and single amino acid changes within the LXCXE motif abolish the ability of WDV Rep to bind to p130Rbr2. The LXCXE motif is conserved in other members of the same geminivirus subgroup. The presence of an intact Rb binding motif is required for efficient WDV DNA replication in cultured wheat cells, strongly suggesting that one of the functions of WDV Rep may be the linking between viral and cellular DNA replication cycles. Our results point to the existence of a Rb-like protein(s) in plant cells playing regulatory roles during the cell cycle.     

High-Resolution Functional Profiling of the Norovirus Genome

Human noroviruses (HuNoV) are a major cause of nonbacterial gastroenteritis worldwide, yet details of the life cycle and replication of HuNoV are relatively unknown due to the lack of an efficient cell culture system. Studies with murine norovirus (MNV), which can be propagated in permissive cells, have begun to probe different aspects of the norovirus life cycle; however, our understanding of the specific functions of the viral proteins lags far behind that of other RNA viruses. Genome-wide functional profiling by insertional mutagenesis can reveal protein domains essential for replication and can lead to generation of tagged viruses, which has not yet been achieved for noroviruses. Here, transposon-mediated insertional mutagenesis was used to create 5 libraries of mutagenized MNV infectious clones, each containing a 15-nucleotide sequence randomly inserted within a defined region of the genome. Infectious virus was recovered from each library and was subsequently passaged in cell culture to determine the effect of each insertion by insertion-specific fluorescent PCR profiling. Genome-wide profiling of over 2,000 insertions revealed essential protein domains and confirmed known functional motifs. As validation, several insertion sites were introduced into a wild-type clone, successfully allowing the recovery of infectious virus. Screening of a number of reporter proteins and epitope tags led to the generation of the first infectious epitope-tagged noroviruses carrying the FLAG epitope tag in either NS4 or VP2. Subsequent work confirmed that epitope-tagged fully infectious noroviruses may be of use in the dissection of the molecular interactions that occur within the viral replication complex.     

Why do human hepatitis viruses replicate so poorly in cell cultures?

The five viruses which classically cause hepatitis in man represent diverse families of viruses and share in common only a striking hepatotropism and substantial restrictions to replication in conventional cell cultures. Hepatitis A virus is unique among these viruses in that it is amenable to propagation in cell culture, but replication of this virus is much slower and less efficient than replication of other picornaviruses. This probably reflects less efficient cap-independent viral translation, as well as restrictions at other points in the replication cycle. We speculate that the significantly restricted replication of hepatitis viruses in cell culture reflects evolutionary forces controlling their transmission and propagation through human populations.     

In vitro investigation of HBV clinical isolates from Chinese patients reveals that genotype C isolates possess higher infectivity than genotype B isolates

Hepatitis B virus (HBV) genotype B and C are two major genotypes that are prevalent in Asia and differ in natural history and disease progression. The impact of HBV genotypes on viral replication and protein expression has been explored by the transfection of hepatoma cells with replication-competent HBV DNA, which mimics the later stages of the viral life cycle. However, the influence of HBV genotypes on the early events of viral infection remains undetermined, mainly due to the difficulties in obtaining sufficient infectious viral particles for infection assays. Here, we report that a high-titer HBV inoculum can be generated from the transient transfection-based cell model after optimizing transfection conditions and modifying the HBV-expressing construct. By performing in vitro infection assays using transiently transfected derived viruses, we found that clinical genotype C isolates possessed higher infectivity than genotype B isolates. Moreover, we identified a naturally occurring mutation sL21S in small hepatitis B surface protein, which markedly decreased the infectivity of HBV genotype C isolates, but not that of genotype B isolates. In summary, using infectious viral particles provided by the optimized transient transfection-based cell model, we have been able to investigate a wide range of HBV variants on viral infectivity, which may contribute to our understanding of the reasons for different clinical outcomes in HBV infections and the development of therapeutic drugs targeting the early stages of HBV life cycle.     

Visualization of viral assembly in the infected cell.

The study of the virus life cycle in infected cells is a methodological challenge due to the small size and diversity of the viral components. Recent developments on preservation of fine structure and molecular localization have provided a group of powerful methods with wide applications in cell biology and virology. Among the different electron microscopy (EM) techniques available to visualize viral assembly at the intracellular level, we will focus on conventional ultrathin sections, cryosections, and freeze-substitution. For obtaining molecular information associated to ultrastructure we have now a group of methods to detect viral proteins (immunogold labeling), as well as the viral genome, through the different techniques for detection of nucleic acids (the enzyme-gold approach, in situ hybridization, and elemental mapping). We will illustrate the applications of these methods with examples of viruses that exhibit different levels of structural complexity. These new approaches help to detect and identify viruses in clinical samples and to characterize the virus life cycle and the cellular components involved, to obtain data that could help for a therapeutic intervention, and to characterize virus-like particles that can be the basis of new and safe vaccines.     

Role of lipids in virus replication

Viruses intricately interact with and modulate cellular membranes at several stages of their replication, but much less is known about the role of viral lipids compared to proteins and nucleic acids. All animal viruses have to cross membranes for cell entry and exit, which occurs by membrane fusion (in enveloped viruses), by transient local disruption of membrane integrity, or by cell lysis. Furthermore, many viruses interact with cellular membrane compartments during their replication and often induce cytoplasmic membrane structures, in which genome replication and assembly occurs. Recent studies revealed details of membrane interaction, membrane bending, fission, and fusion for a number of viruses and unraveled the lipid composition of raft-dependent and -independent viruses. Alterations of membrane lipid composition can block viral release and entry, and certain lipids act as fusion inhibitors, suggesting a potential as antiviral drugs. Here, we review viral interactions with cellular membranes important for virus entry, cytoplasmic genome replication, and virus egress.     

Influenza A virus can undergo multiple cycles of replication without M2 ion channel activity

Ion channel proteins are common constituents of cells and have even been identified in some viruses. For example, the M2 protein of influenza A virus has proton ion channel activity that is thought to play an important role in viral replication. Because direct support for this function is lacking, we attempted to generate viruses with defective M2 ion channel activity. Unexpectedly, mutants with apparent loss of M2 ion channel activity by an in vitro assay replicated as efficiently as the wild-type virus in cell culture. We also generated a chimeric mutant containing an M2 protein whose transmembrane domain was replaced with that from the hemagglutinin glycoprotein. This virus replicated reasonably well in cell culture but showed no growth in mice. Finally, a mutant lacking both the transmembrane and cytoplasmic domains of M2 protein grew poorly in cell culture and showed no growth in mice. Thus, influenza A virus can undergo multiple cycles of replication without the M2 transmembrane domain responsible for ion channel activity, although this activity promotes efficient viral replication.     

DNA-tumor virus entry—From plasma membrane to the nucleus

DNA-tumor viruses comprise enveloped and non-enveloped agents that cause malignancies in a large variety of cell types and tissues by interfering with cell cycle control and immortalization. Those DNA-tumor viruses that replicate in the nucleus use cellular mechanisms to transport their genome and newly synthesized viral proteins into the nucleus. This requires cytoplasmic transport and nuclear import of their genome. Agents that employ this strategy include adenoviruses, hepadnaviruses, herpesviruses, and likely also papillomaviruses, and polyomaviruses, but not poxviruses which replicate in the cytoplasm. Here, we discuss how DNA-tumor viruses enter cells, take advantage of cytoplasmic transport, and import their DNA genome through the nuclear pore complex into the nucleus. Remarkably, nuclear import of incoming genomes does not necessarily follow the same pathways used by the structural proteins of the viruses during the replication and assembly phases of the viral life cycle. Understanding the mechanisms of DNA nuclear import can identify new pathways of cell regulation and anti-viral therapies.     

Viral and host proteins involved in picornavirus life cycle

Picornaviruses cause several diseases, not only in humans but also in various animal hosts. For instance, human enteroviruses can cause hand-foot-and-mouth disease, herpangina, myocarditis, acute flaccid paralysis, acute hemorrhagic conjunctivitis, severe neurological complications, including brainstem encephalitis, meningitis and poliomyelitis, and even death. The interaction between the virus and the host is important for viral replication, virulence and pathogenicity. This article reviews studies of the functions of viral and host factors that are involved in the life cycle of picornavirus. The interactions of viral capsid proteins with host cell receptors is discussed first, and the mechanisms by which the viral and host cell factors are involved in viral replication, viral translation and the switch from translation to RNA replication are then addressed. Understanding how cellular proteins interact with viral RNA or viral proteins, as well as the roles of each in viral infection, will provide insights for the design of novel antiviral agents based on these interactions.     

PLC-γ1 Signaling Plays a Subtype-Specific Role in Postbinding Cell Entry of Influenza A Virus

Host signaling pathways and cellular proteins play important roles in the influenza viral life cycle and can serve as antiviral targets. In this study, we report the engagement of host phosphoinositide-specific phospholipase γ1 (PLC-γ1) in mediating cell entry of influenza virus H1N1 but not H3N2 subtype. Both PLC-γ1-specific inhibitor and short hairpin RNA (shRNA) strongly suppress the replication of H1N1 but not H3N2 viruses in cell culture, suggesting that PLC-γ1 plays an important subtype-specific role in the influenza viral life cycle. Further analyses demonstrate that PLC-γ1 activation is required for viral postbinding cell entry. In addition, H1N1, but not H3N2, infection leads to the phosphorylation of PLC-γ1 at Ser 1248 immediately after infection and independent of viral replication. We have further shown that H1N1-induced PLC-γ1 activation is downstream of epidermal growth factor receptor (EGFR) signaling. Interestingly, both H1N1 and H3N2 infections activate EGFR, but only H1N1 infection leads to PLC-γ1 activation. Taking our findings together, we have identified for the first time the subtype-specific interplay of host PLC-γ1 signaling and H1N1 virus that is critical for viral uptake early in the infection. Our study provides novel insights into how virus interacts with the cellular signaling network by demonstrating that viral determinants can regulate how the host signaling pathways function in virally infected cells.     

Cell culture and infection system for hepatitis C virus

Hepatitis C virus (HCV) infection causes chronic liver disease and is a worldwide health problem. Despite ever-increasing demand for knowledge on viral replication and pathogenesis, detailed analysis has been hampered by a lack of efficient viral culture systems. We isolated HCV genotype 2a strain JFH-1 from a patient with fulminant hepatitis. This strain replicates efficiently in Huh7 cells. Efficient replication and secretion of recombinant viral particles can be obtained in cell culture by transfection of in vitro-transcribed full-length JFH-1 RNA into Huh7 cells. JFH-1 virus generated in cell culture is infectious for both naive Huh7 cells and chimpanzees. The efficiency of viral production and infectivity of generated virus is substantially improved with permissive cell lines. This protocol describes how to use this system, which provides a powerful tool for studying viral life cycle and for the construction of antiviral strategies and the development of effective vaccines. Viral particles can be obtained in 12 days with this protocol.