The Hepatitis E Virus Open Reading Frame 3 Product Interacts with Microtubules and Interferes with Their Dynamics

Hepatitis E virus (HEV) is the causative agent of hepatitis E, a major form of viral hepatitis in developing countries. The open reading frame 3 (ORF3) of HEV encodes a phosphoprotein with a molecular mass of approximately 13 kDa (hereinafter called vp13). vp13 is essential for establishing HEV infections in animals, yet its exact functions are still obscure. Our current study found evidence showing interaction between vp13 and microtubules. Live-cell confocal fluorescence microscopy revealed both filamentous and punctate distribution patterns of vp13 in cells transfected with recombinant ORF3 reporter plasmids. The filamentous pattern of vp13 was altered by a microtubule-destabilizing drug. The vp13 expression led to elevation of acetylated α-tubulin, indicating increased microtubule stability. Its association with microtubules was further supported by its presence in microtubule-containing pellets in microtubule isolation assays. Exposure of these pellets to a high-salt buffer caused release of the vp13 to the supernatant, suggesting an electrostatic interaction. Inclusion of ATP and GTP in the lysis buffer during microtubule isolation also disrupted the interaction, indicating its sensitivity to the nucleotides. Further assays showed that motor proteins are needed for the vp13 association with the microtubules because disruption of dynein function abolished the vp13 filamentous pattern. Analysis of ORF3 deletion constructs found that both of the N-terminal hydrophobic domains of vp13 are needed for the interaction. Thus, our findings suggest that the vp13 interaction with microtubules might be needed for establishment of an HEV infection.The hepatitis E virus (HEV), the sole member of the genus Hepevirus, is a single-strand positive-sense RNA virus that is the causative agent in endemics and epidemics of acute human hepatitis in many parts of the world (5). Transmitted mainly from contaminated water through the fecal-oral route, HEV infection causes a fulminant form of hepatitis that has a mortality rate of up to 20% in pregnant women (28). HEV infection is considered zoonotic. Swine and chicken HEV strains have been found in the United States (11, 23). A swine strain can infect chimpanzees under experimental conditions, and a human strain that is genetically similar to the swine strain can experimentally infect pigs (22). Direct evidence of the zoonotic nature of HEV infection has been provided in reports of a series of cases of HEV infection in people who ate undercooked deer meat 6 to 7 weeks before the onset of the disease (19, 33, 39). HEV RNA recovered from the leftover deer meat was found to be identical in nucleotide sequence to the HEV RNA recovered from the individuals who became ill (31).The HEV genome is approximately 7.2 kb in length and consists of three open reading frames (ORFs) (32). ORF1 encodes a nonstructural polyprotein that includes the RNA-dependent RNA polymerase. ORF2 encodes the capsid protein, the major structural protein in virion. ORF3 encodes a phosphoprotein that was found to be essential for establishing an HEV infection in macaques and pigs under experimental conditions (9, 12). It has been reported that ORF3 translation initiates at the third in-frame AUG codon, which lies 23 bases downstream of the ORF1 termination codon (10, 12). Propagation of HEV and studies of virus replication still rely upon nonhuman primates due to the lack of an effective cell culture system. As a result, functional study of the ORF3 product in HEV biology and infection is limited.The phosphoprotein encoded by HEV ORF3 has a molecular mass of approximately 13 kDa (hereinafter called vp13) (32). The exact functions of vp13 in HEV infection remain unknown although the findings of a number of studies have shown that it plays a role in cellular signaling pathways (13, 17, 24, 34-36, 40). During subcellular fractionation of COS-7 cells transfected with a vp13-expressing plasmid, vp13 was found to partition with the cytoskeletal fraction (40). Deletion of the N-terminal hydrophobic domain of vp13 abolished the association with the cytoskeleton fraction. The vp13-binding proteins in the cytoskeleton and the nature of this interaction are not known.In this study, we found that the HEV ORF3 product localizes to microtubules and interferes with their dynamics. The filamentous pattern of vp13 distribution in the cell was abolished by a microtubule-destabilizing drug. vp13 led to elevation of acetylated α-tubulin. These results suggested that vp13 interaction with the microtubules might facilitate HEV infection. We further studied the nature of the vp13-microtubule interaction.     

Release of Genotype 1 Hepatitis E Virus from Cultured Hepatoma and Polarized Intestinal Cells Depends on Open Reading Frame 3 Protein and Requires an Intact PXXP Motif

Hepatitis E virus genotype 1 strain Sar55 replicated in subcloned Caco-2 intestinal cells and Huh7 hepatoma cells that had been transfected with in vitro transcribed viral genomes, and hepatitis E virions were released into the culture medium of both cell lines. Virus egress from cells depended on open reading frame 3 (ORF3) protein, and a proline-rich sequence in ORF3 was important for egress from cultured cells and for infection of macaques. Both intracellular ORF3 protein accumulation and virus release occurred at the apical membrane of polarized Caco-2 cells. ORF3 protein and lipids were intimately associated with virus particles produced in either cell line; ORF2 epitopes were masked in these particles and could not be immunoprecipitated with anti-ORF2.Hepatitis E virus (HEV) remains enigmatic in spite of recent advances (see references 7 and 16 for reviews). HEV is a major cause of acute hepatitis in numerous developing countries, but hepatitis E is infrequently detected in industrialized countries even though seroprevalence rates of anti-HEV as high as 20% in these countries have been reported. Although hepatitis E normally is a self-limited acute disease, recent studies have identified it as an emerging cause of chronic hepatitis in immunocompromised patients. Whereas contaminated drinking water is the source of most infections in developing countries, the sources in industrialized countries are not fully evaluated, but many, if not most, infections appear linked to eating undercooked meat, especially pork. These differences in epidemiology may reflect the fact that most infections in developing countries are caused by genotypes 1 and 2 while those in industrialized countries are mainly due to genotypes 3 and 4.HEV was initially classified as a calicivirus, but subsequent sequence analysis suggested that it was more closely related to the enveloped rubella virus. However, although HEV may be associated with lipids under some conditions (22), HEV virions do not possess an envelope. Four genotypes of HEV that infect humans have been identified (4). Genotypes 1 and 2 infect primates exclusively, whereas genotypes 3 and 4 are zoonotic and commonly also infect swine and rarely other nonprimates. Recent identification of a strain infecting farmed rabbits in China suggests that other reservoirs may exist (32).The capsid protein encoded by open reading frame 2 (ORF2) is able to form infectious virus particles, but these particles remain cell associated. The crystal structure of a truncated recombinant protein has been solved, but the size of the protein in mature virions is unknown (11, 15, 28, 31). The virus is not cytopathic, and it is unclear how it gets out of cells.The 7.2-kb genome of HEV is a capped mRNA that contains three ORFs that encode proteins involved in replication (ORF1), a capsid protein (ORF2), and a small protein of only 113 to 114 amino acids (ORF3). All but the 5′ terminus of ORF3 is overlapped by ORF2, and both proteins are translated from the same bicistronic subgenomic RNA (10). When overexpressed in cell culture, ORF2 is glycosylated, and ORF3 is phosphorylated (26); this phosphorylated ORF3 protein binds to nonglycosylated ORF2 protein in cell culture, but phosphorylation is not required for infection of macaques (9). The virus has been exceedingly difficult to propagate in cell culture, but recently Okamoto and colleagues reported the successful adaptation of both a genotype 3 and a genotype 4 strain to efficient growth in cultures of PLC/PRF/5 hepatoma or A549 lung cells (23, 24).The tiny ORF3 protein is particularly intriguing because it has a significant impact on virus propagation through mechanisms that have yet to be defined. Data from experiments performed with overexpressed ORF3 protein have suggested that, among other things, ORF3 may interact with cellular proteins, including signaling proteins containing Src homology 3 domains (14), bikunin (27), hemopexin (21), and microtubule proteins (13), and it may function to modulate the acute-phase disease response (3), protect cells from mitochondrial depolarization (18), and enhance expression of glycolytic pathway enzymes (17). Yet within transfected hepatoma cells in culture, virions of an ORF3 null mutant of genotype 1 were assembled in the absence of ORF3 protein and were infectious for naïve hepatoma cells (6) although this same ORF3 null mutant was unable to mount a detectable infection in rhesus monkeys (8). Also, swine transfected with genotype 3 mutant genomes encoding a truncated ORF3 protein did not get infected, indicating that an intact ORF3 protein is needed for infectivity in vivo (12). This lack of infectivity in vivo is possibly explained by the recent demonstration that the ORF3 protein of genotype 3 virus is important for export of virions out of cultured cells in vitro (30); however, this dependence on ORF3 for virion egress has not been confirmed in vivo or for strains of the other three genotypes.The four major genotypes of human HEV appear to segregate naturally into two distinct groups. One group contains genotype 1 and 2 strains that lack a zoonotic component and are spread mainly via contaminated water; in contrast, the second group contains genotype 3 and 4 strains which are able to cross species boundaries and are zoonotic since humans have been infected as a result of eating undercooked meat (16, 25). The molecular basis for the two groupings is unknown, and much more extensive comparative analyses are required to determine which variables are epidemiologically relevant. Here, for lack of an efficient cell culture system for genotype 1 or 2 strains, we have utilized an infectious cDNA clone of a genotype 1 strain in order to explore the role of the ORF3 protein in this group.     

NTPase and 5′ to 3′ RNA Duplex-Unwinding Activities of the Hepatitis E Virus Helicase Domain

Hepatitis E virus (HEV) is a causative agent of acute hepatitis, and it is the sole member of the genus Hepevirus in the family Hepeviridae. The open reading frame 1 (ORF1) protein of HEV encodes nonstructural polyprotein with putative domains for methyltransferase, cysteine protease, helicase and RNA-dependent RNA polymerase. It is not yet known whether ORF1 functions as a single protein with multiple domains or is processed to form separate functional units. On the basis of amino acid conserved motifs, HEV helicase has been grouped into helicase superfamily 1 (SF-1). In order to examine the RNA helicase activity of the NTPase/helicase domain of HEV, the region (amino acids 960 to 1204) was cloned and expressed as histidine-tagged protein in Escherichia coli (HEV Hel) and purified. HEV Hel exhibited NTPase and RNA unwinding activities. Enzyme hydrolyzed all rNTPs efficiently, dATP and dCTP with moderate efficiency, while it showed less hydrolysis of dGTP and dTTP. Enzyme showed unwinding of only RNA duplexes with 5′ overhangs showing 5′-to-3′ polarity. We also expressed and purified two HEV Hel mutants. Helicase mutant I, with substitution in the nucleotide-binding motif I (GKS to GAS), showed 30% ATPase activity. Helicase mutant II, with substitutions in the Mg²⁺ binding motif II (DEAP to AAAP), showed 50% ATPase activity. Both mutants completely lost ability to unwind RNA duplexes with 5′ overhangs. These findings represent the first report demonstrating NTPase/RNA helicase activity of the helicase domain of HEV ORF1.Viruses with single-strand positive-sense RNA genomes represent the largest class of viruses, which includes numerous pathogens of humans, plants, and animals. In these viruses, RNA replication occurs through negative-strand RNA intermediate, which may also act as the template for synthesis of subgenomic RNAs in some viruses. During replication, various nonstructural proteins remain associated with the viral polymerase in a small compartmentalized replisome. Most of the other accessory proteins are obtained from the cellular machinery.Helicase seems to be essential for RNA replication by many positive-sense RNA viruses (19). Many positive-strand RNA viruses encode their own RNA helicases and besides RNA-dependent RNA polymerase, helicase is the most conserved viral sequence in these viruses. It has been shown by direct mutagenesis studies in poliovirus (26, 39), alphaviruses (31), brome mosaic virus (2, 41), nidoviruses (40), and flaviviruses (15) that helicase functions are essential for viral replication. In addition, it may be involved in RNA translocation, genome packaging, protection of RNA at the replication center, modulating RNA-protein interactions, etc.Helicases are classified into six superfamilies, SF-1 to SF-6 (11, 35), and can be classified further into subfamilies, A (3′→5′) or B (5′→3′) depending on their unwinding directionality. Classic helicases (exhibiting both NTPase and unwinding activities) are referred to as subtype α, while translocases (with no unwinding activity) are referred to as subtype β (35). SF-1 and SF-2 constitute largest of these superfamilies with seven signature motifs (I, Ia, II, III, IV, V, and VI), which form core of the enzyme. Although these motifs are not comparable between SF-1 and SF-2, universal features of core domains include (i) conserved residues involved in binding and hydrolysis of the NTP and (ii) an arginine finger that plays a key role in energy coupling.Hepatitis E virus (HEV) is a nonenveloped virus in the genus Hepevirus of the family Hepeviridae. Hepatitis E is an important public health disease in many developing countries and is also endemic in some industrialized countries (8). Infection by HEV has a known association with increased mortality during pregnancy (22, 23). HEV has a positive-sense RNA genome of ∼7.2 kb, consisting of a 5′ noncoding region (5′NCR) of 27 to 35 nucleotides (nt), followed by three open reading frames (ORFs)—ORF1, ORF2, and ORF3—and a 3′NCR of 65 to 74 nt, ending with a poly(A) tail of variable length (37). The 5′ end has m⁷G cap (18). ORF1 is known to encode for the viral nonstructural polyprotein with a proposed molecular mass of ∼186 kDa (3). Based on protein sequence homology, the ORF1 polyprotein is proposed to contain four putative domains indicative of methyltransferase, papain-like cysteine protease, RNA helicase (Hel), and RNA-dependent RNA polymerase (RdRp) (24). ORF2 encodes the major structural protein (capsid protein), which has N-terminal signal peptide and three glycosylation sites and is translocated across the endoplasmic reticulum (ER). ORF2 protein associates with the 5′ end of the viral RNA, suggesting its regulatory role in the virus replication (36, 37, 44, 45). ORF3 encodes a protein which gets phosphorylated by the cellular mitogen activated protein kinase and is associated with cellular membranes and cytoskeleton fractions (43).HEV belongs to an “alpha-like” supergroup of positive-sense single-stranded RNA (+ssRNA) viruses with conserved motifs of replication-related proteins in the ORF1, with typical signature sequences homologous with the other members of the family (11, 12, 13). ORF1 of HEV encodes additional domains such as the Y domain, papainlike protease, “proline-rich hinge,” and the X domain. Methyltransferase (25), RdRp (1), and X domain (binding to poly-ADP-ribose) (9) in ORF1 have been characterized, whereas the functions of the other domains are yet to be identified. Intracellularly expressed RdRp localizes itself in the ER membranes (30), suggesting that HEV replicates probably in ER in the cytosolic compartment of the cells. It is still unknown whether ORF1 polyprotein undergoes cleavages to form separate functional units of the replication machinery or functions as a single protein with multiple functional domains.The putative RNA helicase of HEV contains all of the seven conserved segments typical of the SF-1 helicase (12, 13). Putative SF-1 helicases are extremely widespread among +ssRNA viruses. Based on sequence comparisons, such helicases have been identified in a variety of plant virus families, as well as in animal viruses such as alphavirus, rubivirus, hepatitis E virus, and coronavirus (11). When compared to other +ssRNA viral helicases belonging to SF-1, HEV helicase showed the highest overall similarity with the helicase of beet necrotic yellow vein virus, a plant furovirus. HEV helicase was speculated to have N-terminal NTPase and C-terminal RNA-binding domains (24). A major obstacle in studying HEV replication has been lack of cell culture system. We report here experimental verification of the helicase activity of the recombinant helicase domain protein of HEV.     

Open Reading Frame 33 of a Gammaherpesvirus Encodes a Tegument Protein Essential for Virion Morphogenesis and Egress

Tegument is a unique structure of herpesvirus, which surrounds the capsid and interacts with the envelope. Morphogenesis of gammaherpesvirus is poorly understood due to lack of efficient lytic replication for Epstein-Barr virus and Kaposi''s sarcoma-associated herpesvirus/human herpesvirus 8, which are etiologically associated with several types of human malignancies. Murine gammaherpesvirus 68 (MHV-68) is genetically related to the human gammaherpesviruses and presents an excellent model for studying de novo lytic replication of gammaherpesviruses. MHV-68 open reading frame 33 (ORF33) is conserved among Alpha-, Beta-, and Gammaherpesvirinae subfamilies. However, the specific role of ORF33 in gammaherpesvirus replication has not yet been characterized. We describe here that ORF33 is a true late gene and encodes a tegument protein. By constructing an ORF33-null MHV-68 mutant, we demonstrated that ORF33 is not required for viral DNA replication, early and late gene expression, viral DNA packaging or capsid assembly but is required for virion morphogenesis and egress. Although the ORF33-null virus was deficient in release of infectious virions, partially tegumented capsids produced by the ORF33-null mutant accumulated in the cytoplasm, containing conserved capsid proteins, ORF52 tegument protein, but virtually no ORF45 tegument protein and the 65-kDa glycoprotein B. Finally, we found that the defect of ORF33-null MHV-68 could be rescued by providing ORF33 in trans or in an ORF33-null revertant virus. Taken together, our results indicate that ORF33 is a tegument protein required for viral lytic replication and functions in virion morphogenesis and egress.Gammaherpesviruses are associated with tumorigenesis. Like other herpesviruses, they are characterized as having two distinct stages in their life cycle: lytic replication and latency (15, 16, 18, 21, 54). Latency provides the viruses with advantages to escape host immune surveillance and to establish lifelong persistent infection and contributes to transformation and development of malignancies. However, it is through lytic replication that viruses propagate and transmit among hosts to maintain viral reservoirs. Both viral latency and lytic replication play important roles in tumorigenesis. The gammaherpesvirus subfamily includes Epstein-Barr virus (EBV), Kaposi''s sarcoma-associated herpesvirus (KSHV)/human herpesvirus 8 and murine gammaherpesvirus 68 (MHV-68), among others. EBV is associated with Burkitt''s lymphoma, nasopharyngeal carcinoma, Hodgkin''s disease, and lymphoproliferative diseases in immunodeficient patients (28). KSHV is etiologically linked with Kaposi''s sarcoma, primary effusion lymphoma, and multicentric Castleman''s disease (11-13, 22, 52). Neither in vivo nor in vitro studies of EBV and KSHV are convenient due to their propensity to establish latency in cell culture and their limited host ranges.MHV-68 is genetically related to these two human gammaherpesviruses, especially to KSHV, based on the alignment of their genomic sequences and other biological properties (55). As a natural pathogen of wild rodents, MHV-68 also infects laboratory mice (6, 40, 46) and replicates to a high titer in a variety of fibroblast and epithelial cell lines. These advantages make MHV-68 an excellent model for studying the lytic replication of gammaherpesviruses in vitro and certain aspects of virus-host interactions in vivo. In addition, the MHV-68 genome has been cloned as a bacterial artificial chromosome (BAC) that can propagate in Escherichia coli (1, 2, 36, 51), making it convenient to study the function of each open reading frame (ORF) by genetic methods. Exploring the functions of MHV-68 ORFs will likely shed light on the functions of their homologues in human gammaherpesviruses.Gammaherpesviral particles have a characteristic multilayered architecture. An infectious virion contains a double-stranded DNA genome, an icosahedral capsid shell, a thick, proteinaceous tegument compartment, and a lipid bilayer envelope spiked with glycoproteins (14, 30, 47, 49). As a unique structure of herpesviruses, the tegument plays important roles in multiple aspects of the viral life cycle, including virion assembly and egress (38, 48, 53), translocation of nucleocapsids into the nucleus, transactivation of viral immediate-early genes, and modulation of host cell gene expression, innate immunity, and signal transduction (9, 10, 23, 60). Some components of MHV-68 tegument have been identified by a mass spectrometric study (8), and the functions of some tegument proteins have been revealed, such as ORF45, ORF52, and ORF75c (7, 24, 29).MHV-68 ORF33 is conserved among Alpha-, Beta-, and Gammaherpesvirinae subfamilies. Its homologues include human herpes simplex virus type 1 (HSV-1) UL16, human herpes simplex virus type 2 (HSV-2) UL16, human cytomegalovirus (HCMV) UL94, EBV BGLF2, KSHV ORF33, and rhesus monkey rhadinovirus (RRV) ORF33. HSV-1 UL16 has been identified as a tegument protein and may function in viral DNA packaging, virion assembly, budding, and egress (5, 32, 35, 41, 44). HCMV UL94 is a virion associated protein and might function in virion assembly and budding (31, 57). EBV BGLF2, KSHV ORF33, and RRV ORF33 are also virion-associated proteins, but their functions are not clear (26, 43, 59). The mass spectrometric study of MHV-68 did not identify ORF33 as a virion component (8), although ORF33 is found to be essential for viral lytic replication by transposon mutagenesis of the MHV-68 genome cloned as a BAC (51). However, insertion of the 1.2-kbp Mu transposon in that study may influence the expression of ORFs approximate to ORF33. Consequently, the role ORF33 plays in viral replication needs to be confirmed, preferably through site-directed mutagenesis. Whether ORF33 is a tegument protein and the exact viral replication stage in which it functions also need to be investigated.We determined that MHV-68 ORF33 encodes a tegument protein and is expressed with true late kinetics. To explore the function of ORF33 in viral lytic phase, we used site-directed mutagenesis and generated an ORF33-null mutant, taking advantage of the MHV-68 BAC system. We showed that the ORF33-null mutant is capable of viral DNA replication, early and late gene expression, capsid assembly, and DNA packaging, but incapable of virion release. The defect of ORF33-null mutant can be rescued in trans by an ORF33 expression plasmid.     

Complementation of a Herpes Simplex Virus ICP0 Null Mutant by Varicella-Zoster Virus ORF61p

The herpes simplex virus (HSV) ICP0 protein acts to overcome intrinsic cellular defenses that repress viral α gene expression. In that vein, viruses that have mutations in ICP0''s RING finger or are deleted for the gene are sensitive to interferon, as they fail to direct degradation of promyelocytic leukemia protein (PML), a component of host nuclear domain 10s. While varicella-zoster virus is also insensitive to interferon, ORF61p, its ICP0 ortholog, failed to degrade PML. A recombinant virus with each coding region of the gene for ICP0 replaced with sequences encoding ORF61p was constructed. This virus was compared to an ICP0 deletion mutant and wild-type HSV. The recombinant degraded only Sp100 and not PML and grew to higher titers than its ICP0 null parental virus, but it was sensitive to interferon, like the virus from which it was derived. This analysis permitted us to compare the activities of ICP0 and ORF61p in identical backgrounds and revealed distinct biologic roles for these proteins.Alphaherpesviruses encode orthologs of the herpes simplex virus (HSV) α gene product ICP0. ICP0 is a nuclear phosphoprotein that behaves as a promiscuous activator of viral and cellular genes (7, 11, 28, 29). ICP0 also functions as an E3 ubiquitin ligase to target several host proteins for proteasomal degradation (4, 10, 11, 16, 26). Through this activity, ICP0 promotes degradation of components of nuclear domain 10 (ND10) bodies, including the promyelocytic leukemia protein (PML) and Sp100. These proteins are implicated in silencing of herpesvirus genomes (9, 10, 22, 34). Therefore, ICP0-mediated degradation of ND10 components may disrupt silencing of HSV genes to enable efficient gene expression. This hypothesis provides a plausible mechanistic explanation of how ICP0 induces gene activation.Introduction of DNA encoding the ICP0 orthologs from HSV, bovine herpesvirus, equine herpesvirus, and varicella-zoster virus (VZV) can also affect nuclear structures and proteins (27). In addition, and more specific to this report, ORF61p, the VZV ortholog, activates viral promoters and enhances infectivity of viral DNA like ICP0, the prototype for this gene family (24, 25). However, we have previously demonstrated two key biological differences between the HSV and VZV orthologs. We first showed that unlike ICP0, ORF61p is unable to complement depletion of BAG3, a host cochaperone protein. As a result, VZV is affected by silencing of BAG3 (15), whereas growth of HSV is altered only when ICP0 is not expressed (17). Furthermore, we have shown that while both proteins target components of ND10s, expression of ICP0 results in degradation of both PML and Sp100, whereas ORF61p specifically reduces Sp100 levels (16). These findings suggest that these proteins have evolved separately to provide different functions for virus replication.Virus mutants lacking the ICP0 gene have an increased particle-to-PFU ratio, a substantially lower yield, and decreased levels of α gene expression, in a multiplicity-of-infection (MOI)- and cell-type-dependent manner (2, 4, 8, 33). These mutants are also defective at degrading ND10 components (23). Depletion of PML and Sp100 accelerates virus gene expression and increases plaquing efficiency of HSV ICP0-defective viruses but has no effect on wild-type virus, suggesting that PML and Sp100 are components of an intrinsic anti-HSV defense mechanism that is counteracted by ICP0''s E3 ligase activity (9, 10). Interestingly, ICP0 null viruses are also hypersensitive to interferon (IFN) (26), a property that was suggested to be mediated via PML (3).To directly compare the activities of the two orthologs, we constructed an HSV mutant virus that expresses ORF61p in place of ICP0. The resulting chimeric virus only partially rescues the ICP0 null phenotype. Our studies emphasize the biological differences between ICP0 and ORF61p and shed light on the requirements for PML and Sp100 during infection.     

Identification of the Nuclear Export and Adjacent Nuclear Localization Signals for ORF45 of Kaposi's Sarcoma-Associated Herpesvirus

Open reading frame 45 (ORF45) of Kaposi''s sarcoma-associated herpesvirus 8 (KSHV) is an immediate-early phosphorylated tegument protein and has been shown to play important roles at both early and late stages of viral infection. Homologues of ORF45 exist only in gammaherpesviruses, and their homology is limited. These homologues differ in their protein lengths and subcellular localizations. We and others have reported that KSHV ORF45 is localized predominantly in the cytoplasm, whereas its homologue in murine herpesvirus 68 is localized exclusively in the nucleus. We observed that ORF45s of rhesus rhadinovirus and herpesvirus saimiri are found exclusively in the nucleus. As a first step toward understanding the mechanism underlying the distinct intracellular distribution of KSHV ORF45, we identified the signals that control its subcellular localization. We found that KSHV ORF45 accumulated rapidly in the nucleus in the presence of leptomycin B, an inhibitor of CRM1 (exportin 1)-dependent nuclear export, suggesting that it could shuttle between the nucleus and cytoplasm. Mutational analysis revealed that KSHV ORF45 contains a CRM1-dependent, leucine-rich-like nuclear export signal and an adjacent nuclear localization signal. Replacement of the key residues with alanines in these motifs of ORF45 disrupts its shuttling between the cytoplasm and nucleus. The resulting ORF45 mutants have restricted subcellular localizations, being found exclusively either in the cytoplasm or in the nucleus. Recombinant viruses were reconstituted by introduction of these mutations into KSHV bacterial artificial chromosome BAC36. The resultant viruses have distinct phenotypes. A mutant virus in which ORF45 is restricted to the cytoplasm behaves as an ORF45-null mutant and produces 5- to 10-fold fewer progeny viruses than the wild type. In contrast, mutants in which the ORF45 protein is mostly restricted to the nucleus produce numbers of progeny viruses similar to those produced by the wild type. These data suggest that the subcellular localization signals of ORF45 have important functional roles in KSHV lytic replication.Kaposi''s sarcoma-associated herpesvirus (KSHV) is a DNA tumor virus and the causative agent of several human cancers, including Kaposi''s sarcoma (KS), primary effusion lymphoma, and multicentric Castleman''s disease (3, 6). Like all herpesviruses, KSHV has two alternative life cycles, a latent and a lytic cycle. During latency, only a few viral genes are expressed, and no progeny viruses are produced. Under appropriate conditions, latent viral genomes are activated, initiate lytic replication, and express a full panel of viral genes, in a process that leads to viral assembly, release of progeny virus particles, and de novo infection of naïve cells (3, 6). KSHV establishes latent infection in the majority of infected cells in cases of KS, primary effusion lymphoma, and multicentric Castleman''s disease, but lytic replications occur in a small fraction. The recurrent and periodic lytic cycles of KSHV are believed to play critical roles in viral pathogenesis (6, 7).Open reading frame 45 (ORF45) is a KSHV-encoded gene product that plays a critical role in the viral lytic cycle. It is an immediate-early protein and is also present in viral particles as tegument protein (26, 27, 30). Disruption of ORF45 has no significant effect on overall viral lytic gene expression or DNA replication in BAC36-reconstituted 293T cells induced with both tetradecanoyl phorbol acetate (TPA) and sodium butyrate together, but the ORF45-null mutant produces 5- to 10-fold fewer progeny viruses than the wild type and the mutant virus has dramatically reduced infectivity, suggesting that ORF45 plays important roles at both early and late stages of viral infection (29). In addition to its roles as a tegument component, which are possibly involved in viral ingress and egress processes, KSHV ORF45 interacts with cellular proteins and modulates the cellular environment. At least two such functions have been described. First, KSHV ORF45 inhibits activation of interferon regulatory factor 7 (IRF-7) and therefore antagonizes the host innate antiviral response (28). Second, KSHV ORF45 interacts with p90 ribosomal kinase 1 and 2 (RSK1/RSK2) and modulates the extracellular signal-regulated kinase/RSK signaling pathway, which is known to play essential roles in KSHV reactivation and lytic replication (12). All of these data suggest that KSHV ORF45 is a multifunctional protein.ORF45 is unique to the gammaherpesviruses; it has no homologue in the alpha- or betaherpesviruses. ORF45 homologues have been identified as virion protein components in other gammaherpesviruses, such as Epstein-Barr virus (EBV), rhesus rhadinovirus (RRV), and murine herpesvirus 68 (MHV-68), suggesting that certain tegument functions of ORF45 are conserved (2, 11, 18). ORF45 homologues differ in protein length. KSHV ORF45 is the longest, at 407 amino acids (aa); RRV, EBV, MHV-68, and herpesvirus saimiri (HVS) have proteins of 353, 217, 206, and 257 aa, respectively. The limited homologies lie mostly at the amino- and carboxyl-terminal ends. The middle portion of KSHV ORF45 diverges from those of its homologues. The homologues differ in subcellular localization. We and others have reported previously that KSHV ORF45 is found predominantly in the cytoplasm (1, 21, 28, 30), whereas ORF45 of MHV-68 is found exclusively in the nucleus (9). Recently, we found KSHV ORF45 also present in the nuclei of BCBL-1 cells in what resembled viral replication compartments, suggesting that ORF45 could shuttle into the nucleus (12).Nucleocytoplasmic trafficking of proteins across the nuclear membrane occurs through nuclear pore complexes. Small molecules of up to approximately 9 nm in diameter, corresponding to a globular protein of approximately 40 to 60 kDa, can in principle enter or leave the nucleus by diffusion through nuclear pores (15, 17, 24). Large molecules are transported with the aid of a related family of transport factors, importins and exportins, which recognize nuclear localization sequence (NLS)-containing or nuclear export sequence (NES)-containing proteins (15, 17, 23). CRM1 (exportin 1) has been identified as a common export receptor that recognizes human immunodeficiency virus Rev-like leucine-rich NES sequences and is responsible for the export of such NES-containing proteins (4, 5, 19, 22). CRM1-dependent nuclear export is specifically inhibited by a pharmacological compound, leptomycin B (LMB), that interacts with CRM1 and thus blocks such NES-mediated protein export (4).To understand the mechanism underlying the distinct intracellular distribution of KSHV ORF45, we attempted to locate the signals that control its subcellular localization. In the research reported here, we identified a leucine-rich NES and an adjacent basic NLS in KSHV ORF45. We demonstrated that the regulated intracellular trafficking of ORF45, especially its translocation into the nucleus, is important for KSHV lytic replication.     

Borna Disease Virus Requires Cholesterol in both Cellular Membrane and Viral Envelope for Efficient Cell Entry

Borna disease virus (BDV), the prototypic member of the family Bornaviridae within the order Mononegavirales, provides an important model for the investigation of viral persistence within the central nervous system (CNS) and of associated brain disorders. BDV is highly neurotropic and enters its target cell via receptor-mediated endocytosis, a process mediated by the virus surface glycoprotein (G), but the cellular factors and pathways determining BDV cell tropism within the CNS remain mostly unknown. Cholesterol has been shown to influence viral infections via its effects on different viral processes, including replication, budding, and cell entry. In this work, we show that cell entry, but not replication and gene expression, of BDV was drastically inhibited by depletion of cellular cholesterol levels. BDV G-mediated attachment to BDV-susceptible cells was cholesterol independent, but G localized to lipid rafts (LR) at the plasma membrane. LR structure and function critically depend on cholesterol, and hence, compromised structural integrity and function of LR caused by cholesterol depletion likely inhibited the initial stages of BDV cell internalization. Furthermore, we also show that viral-envelope cholesterol is required for BDV infectivity.Borna disease virus (BDV) is an enveloped virus with a nonsegmented negative-strand RNA genome whose organization (3′-N-p10/P-M-G-L-5′) is characteristic of mononegaviruses (6, 28, 46, 48). However, based on its unique genetics and biological features, BDV is considered to be the prototypic member of a new virus family, Bornaviridae, within the order Mononegavirales (8, 28, 46, 49).BDV can infect a variety of cell types in cell culture but in vivo exhibits exquisite neurotropism and causes central nervous system (CNS) disease in different vertebrate species, which is frequently manifested in behavioral abnormalities (19, 33, 44, 53). Both host and viral factors contribute to a variable period of incubation and heterogeneity in the symptoms and pathology associated with BDV infection (14, 16, 29, 42, 44). BDV provides an important model for the investigation of both immune-mediated pathological events associated with virus-induced neurological disease and mechanisms whereby noncytolytic viruses induce neurodevelopmental and behavioral disturbances in the absence of inflammation (15, 18, 41). Moreover, serological data and molecular epidemiological studies suggest that BDV, or a BDV-like virus, can infect humans and that it might be associated with certain neuropsychiatric disorders (17, 24), which further underscores the interest in understanding the mechanisms underlying BDV persistence in the CNS and its effect on brain cell functions. The achievement of these goals will require the elucidation of the determinants of BDV cell tropism within the CNS.BDV enters its target cell via receptor-mediated endocytosis, a process in which the BDV G protein plays a central role (1, 5, 13, 14, 39). Cleavage of BDV G by the cellular protease furin generates two functional subunits: GP1 (GP_N), involved in virus interaction with a yet-unidentified cell surface receptor (1, 39), and GP2 (GP_C), which mediates a pH-dependent fusion event between viral and cellular membranes (13). However, a detailed characterization of cellular factors and pathways involved in BDV cell entry remains to be done.Besides cell surface molecules that serve as viral receptors, many other cell factors, including nonproteinaceous molecules, can influence cell entry by virus (52). In this regard, cholesterol, which plays a critical role in cellular homeostasis (55), has also been identified as a key factor required for productive infection by different viruses. Accordingly, cholesterol participates in a variety of processes in virus-infected cells, including fusion events between viral and cellular membranes (3), viral replication (23), and budding (35, 37), as well as maintenance of lipid rafts (LR) (12) as scaffold structures where the viral receptor and coreceptor associate (11, 26, 32, 36). LR are specialized microdomains within cellular membranes constituted principally of proteins, sphingolipids, and cholesterol. LR facilitate the close proximity and interaction of specific sets of proteins and contribute to different processes associated with virus multiplication (38). Cholesterol can also influence virus infection by contributing to the maintenance of the properties of the viral envelope required for virus particle infectivity (21, 54). Here, we show for the first time that cholesterol plays a critical role in BDV infection. Depletion of cellular cholesterol prior to, but not after, BDV cell entry prevented productive BDV infection, likely due to disruption of plasma membrane LR that appear to be the cell entry point for BDV. In addition, we document that cholesterol also plays an essential role in the properties of the BDV envelope required for virus particle infectivity.     

Divergent Evolution of Norovirus GII/4 by Genome Recombination from May 2006 to February 2009 in Japan

Norovirus GII/4 is a leading cause of acute viral gastroenteritis in humans. We examined here how the GII/4 virus evolves to generate and sustain new epidemics in humans, using 199 near-full-length GII/4 genome sequences and 11 genome segment clones from human stool specimens collected at 19 sites in Japan between May 2006 and February 2009. Phylogenetic studies demonstrated outbreaks of 7 monophyletic GII/4 subtypes, among which a single subtype, termed 2006b, had continually predominated. Phylogenetic-tree, bootscanning-plot, and informative-site analyses revealed that 4 of the 7 GII/4 subtypes were mosaics of recently prevalent GII/4 subtypes and 1 was made up of the GII/4 and GII/12 genotypes. Notably, single putative recombination breakpoints with the highest statistical significance were constantly located around the border of open reading frame 1 (ORF1) and ORF2 (P ≤ 0.000001), suggesting outgrowth of specific recombinant viruses in the outbreaks. The GII/4 subtypes had many unique amino acids at the time of their outbreaks, especially in the N-term, 3A-like, and capsid proteins. Unique amino acids in the capsids were preferentially positioned on the outer surface loops of the protruding P2 domain and more abundant in the dominant subtypes. These findings suggest that intersubtype genome recombination at the ORF1/2 boundary region is a common mechanism that realizes independent and concurrent changes on the virion surface and in viral replication proteins for the persistence of norovirus GII/4 in human populations.Norovirus (NoV) is a nonenveloped RNA virus that belongs to the family Caliciviridae and can cause acute gastroenteritis in humans. The NoV genome is a single-stranded, positive-sense, polyadenylated RNA that encodes three open reading frames, ORF1, ORF2, and ORF3 (68). ORF1 encodes a long polypeptide (∼200 kDa) that is cleaved in the cells by the viral proteinase (3C^pro) into six proteins (4). These proteins function in NoV replication in host cells (19). ORF2 encodes a viral capsid protein, VP1. The capsid gene evolved at a rate of 4.3 × 10⁻³ nucleotide substitutions/site/year (7), which is comparable to the substitution rates of the envelope and capsid genes of human immunodeficiency virus (30). The capsid protein of NoV consists of a shell (S) and two protruding (P) domains: P1 and P2 (47). The S domain is relatively conserved within the same genetic lineages of NoVs (38) and is responsible for the assembly of VP1 (6). The P1 subdomain is also relatively conserved (38) and has a role in enhancing the stability of virus particles (6). The P2 domain is positioned at the most exposed surface of the virus particle (47) and forms binding clefts for putative infection receptors, such as human histo-blood group antigens (HBGA) (8, 13, 14, 60). The P2 domain also contains epitopes for neutralizing antibodies (27, 33) and is consistently highly variable even within the same genetic lineage of NoVs (38). ORF3 encodes a VP2 protein that is suggested to be a minor structural component of virus particles (18) and to be responsible for the expression and stabilization of VP1 (5).Thus far, the NoVs found in nature are classified into five genogroups (GI to GV) and multiple genotypes on the basis of the phylogeny of capsid sequences (71). Among them, genogroup II genotype 4 (GII/4), which was present in humans in the mid-1970s (7), is now the leading cause of NoV-associated acute gastroenteritis in humans (54). The GII/4 is further subclassifiable into phylogenetically distinct subtypes (32, 38, 53). Notably, the emergence and spread of a new GII/4 subtype with multiple amino acid substitutions on the capsid surface are often associated with greater magnitudes of NoV epidemics (53, 54). In 2006 and 2007, a GII/4 subtype, termed 2006b, prevailed globally over preexisting GII/4 subtypes in association with increased numbers of nonbacterial acute gastroenteritis cases in many countries, including Japan (32, 38, 53). The 2006b subtype has multiple unique amino acid substitutions that occur most preferentially in the protruding subdomain of the capsid, the P2 subdomain (32, 38, 53). Together with information on human population immunity against NoV GII/4 subtypes (12, 32), it has been postulated that the accumulation of P2 mutations gives rise to antigenic drift and plays a key role in new epidemics of NoV GII/4 in humans (32, 38, 53).Genetic recombination is common in RNA viruses (67). In NoV, recombination was first suggested by the phylogenetic analysis of an NoV genome segment clone: a discordant branching order was noted with the trees of the 3D^pol and capsid coding regions (21). Subsequently, many studies have reported the phylogenetic discordance using sequences from various epidemic sites in different study periods (1, 10, 11, 16, 17, 22, 25, 40, 41, 44-46, 49, 51, 57, 63, 64, 66). These results suggest that genome recombination frequently occurs among distinct lineages of NoV variants in vivo. However, the studies were done primarily with direct sequencing data of the short genome portion, and information on the cloned genome segment or full-length genome sequences is very limited (21, 25). Therefore, we lack an overview of the structural and temporal dynamics of viral genomes during NoV epidemics, and it remains unclear whether NoV mosaicism plays a role in these events.To clarify these issues, we collected 199 near-full-length genome sequences of GII/4 from NoV outbreaks over three recent years in Japan, divided them into monophyletic subtypes, analyzed the temporal and geographical distribution of the subtypes, collected phylogenetic evidence for the viral genome mosaicism of the subtypes, identified putative recombination breakpoints in the genomes, and isolated mosaic genome segments from the stool specimens. We also performed computer-assisted sequence and structural analyses with the identified subtypes to address the relationship between the numbers of P2 domain mutations at the times of the outbreaks and the magnitudes of the epidemics. The obtained data suggest that intersubtype genome recombination at the ORF1/2 boundary region is common in the new GII/4 outbreaks and promotes the effective acquisition of mutation sets of heterogeneous capsid surface and viral replication proteins.     

Characterization of Lassa Virus Glycoprotein Oligomerization and Influence of Cholesterol on Virus Replication

Mature glycoprotein spikes are inserted in the Lassa virus envelope and consist of the distal subunit GP-1, the transmembrane-spanning subunit GP-2, and the signal peptide, which originate from the precursor glycoprotein pre-GP-C by proteolytic processing. In this study, we analyzed the oligomeric structure of the viral surface glycoprotein. Chemical cross-linking studies of mature glycoprotein spikes from purified virus revealed the formation of trimers. Interestingly, sucrose density gradient analysis of cellularly expressed glycoprotein showed that in contrast to trimeric mature glycoprotein complexes, the noncleaved glycoprotein forms monomers and oligomers spanning a wide size range, indicating that maturation cleavage of GP by the cellular subtilase SKI-1/S1P is critical for formation of the correct oligomeric state. To shed light on a potential relation between cholesterol and GP trimer stability, we performed cholesterol depletion experiments. Although depletion of cholesterol had no effect on trimerization of the glycoprotein spike complex, our studies revealed that the cholesterol content of the viral envelope is important for the infectivity of Lassa virus. Analyses of the distribution of viral proteins in cholesterol-rich detergent-resistant membrane areas showed that Lassa virus buds from membrane areas other than those responsible for impaired infectivity due to cholesterol depletion of lipid rafts. Thus, derivation of the viral envelope from cholesterol-rich membrane areas is not a prerequisite for the impact of cholesterol on virus infectivity.Lassa virus (LASV) is a member of the family Arenaviridae, of which Lymphocytic choriomeningitis virus (LCMV) is the prototype. Arenaviruses comprise more than 20 species, divided into the Old World and New World virus complexes (19). The Old World arenaviruses include the human pathogenic LASV strains, Lujo virus, which was first identified in late 2008 and is associated with an unprecedented high case fatality rate in humans, the nonhuman pathogenic Ippy, Mobala, and Mopeia viruses, and the recently described Kodoko virus (10, 30, 49). The New World virus complex contains, among others, the South American hemorrhagic fever-causing viruses Junín virus, Machupo virus, Guanarito virus, Sabiá virus, and the recently discovered Chapare virus (22).Arenaviruses contain a bisegmented single-stranded RNA genome encoding the polymerase L, matrix protein Z, nucleoprotein NP, and glycoprotein GP. The bipartite ribonucleoprotein of LASV is surrounded by a lipid envelope derived from the plasma membrane of the host cell. The matrix protein Z has been identified as a major budding factor, which lines the interior of the viral lipid membrane, in which GP spikes are inserted (61, 75). The glycoprotein is synthesized as precursor protein pre-GP-C and is cotranslationally cleaved by signal peptidase into GP-C and the signal peptide, which exhibits unusual length, stability, and topology (3, 27, 28, 33, 70, 87). Moreover, the arenaviral signal peptide functions as trans-acting maturation factor (2, 26, 33). After processing by signal peptidase, GP-C of both New World and Old World arenaviruses is cleaved by the cellular subtilase subtilisin kexin isozyme-1/site-1 protease (SKI-1/S1P) into the distal subunit GP-1 and the membrane-anchored subunit GP-2 within the secretory pathway (5, 52, 63). For LCMV, it has been shown that GP-1 subunits are linked to each other by disulfide bonds and are noncovalently connected to GP-2 subunits (14, 24, 31). GP-1 is responsible for binding to the host cell receptor, while GP-2 mediates fusion between the virus envelope and the endosomal membrane at low pH due to a bipartite fusion peptide near the amino terminus (24, 36, 44). Sequence analysis of the LCMV GP-2 ectodomain revealed two heptad repeats that most likely form amphipathic helices important for this process (34, 86).In general, viral class I fusion proteins have triplets of α-helical structures in common, which contain heptad repeats (47, 73). In contrast, class II fusion proteins are characterized by β-sheets that form dimers in the prefusion status and trimers in the postfusion status (43). The class III fusion proteins are trimers that, unlike class I fusion proteins, were not proteolytically processed N-terminally of the fusion peptide, resulting in a fusion-active membrane-anchored subunit (39, 62). Previous studies with LCMV described a tetrameric organization of the glycoprotein spikes (14), while more recent data using a bacterially expressed truncated ectodomain of the LCMV GP-2 subunit pointed toward a trimeric spike structure (31). Due to these conflicting data regarding the oligomerization status of LCMV GP, it remains unclear to which class of fusion proteins the arenaviral glycoproteins belong.The state of oligomerization and the correct conformation of viral glycoproteins are crucial for membrane fusion during virus entry. The early steps of infection have been shown for several viruses to be dependent on the cholesterol content of the participating membranes (i.e., either the virus envelope or the host cell membrane) (4, 9, 15, 20, 21, 23, 40, 42, 53, 56, 76, 78, 79). In fact, it has been shown previously that entry of both LASV and LCMV is susceptible to cholesterol depletion of the target host cell membrane using methyl-β-cyclodextrin (MβCD) treatment (64, 71). Moreover, cholesterol not only plays an important role in the early steps during entry in the viral life cycle but also is critical in the virus assembly and release process. Several viruses of various families, including influenza virus, human immunodeficiency virus type 1 (HIV-1), measles virus, and Ebola virus, use the ordered environment of lipid raft microdomains. Due to their high levels of glycosphingolipids and cholesterol, these domains are characterized by insolubility in nonionic detergents under cold conditions (60, 72). Recent observations have suggested that budding of the New World arenavirus Junin virus occurs from detergent-soluble membrane areas (1). Assembly and release from distinct membrane microdomains that are detergent soluble have also been described for vesicular stomatitis virus (VSV) (12, 38, 68). At present, however, it is not known whether LASV requires cholesterol in its viral envelope for successful virus entry or whether specific membrane microdomains are important for LASV assembly and release.In this study, we first investigated the oligomeric state of the premature and mature LASV glycoprotein complexes. Since it has been shown for several membrane proteins that the oligomerization and conformation are dependent on cholesterol (58, 59, 76, 78), we further analyzed the dependence of the cholesterol content of the virus envelope on glycoprotein oligomerization and virus infectivity. Finally, we characterized the lipid membrane areas from which LASV is released.     

Replacement of the Replication Factors of Porcine Circovirus (PCV) Type 2 with Those of PCV Type 1 Greatly Enhances Viral Replication In Vitro

Porcine circovirus type 1 (PCV1), originally isolated as a contaminant of PK-15 cells, is nonpathogenic, whereas porcine circovirus type 2 (PCV2) causes an economically important disease in pigs. To determine the factors affecting virus replication, we constructed chimeric viruses by swapping open reading frame 1 (ORF1) (rep) or the origin of replication (Ori) between PCV1 and PCV2 and compared the replication efficiencies of the chimeric viruses in PK-15 cells. The results showed that the replication factors of PCV1 and PCV2 are fully exchangeable and, most importantly, that both the Ori and rep of PCV1 enhance the virus replication efficiencies of the chimeric viruses with the PCV2 backbone.Porcine circovirus (PCV) is a single-stranded DNA virus in the family Circoviridae (34). Type 1 PCV (PCV1) was discovered in 1974 as a contaminant of porcine kidney cell line PK-15 and is nonpathogenic in pigs (31-33). Type 2 PCV (PCV2) was discovered in piglets with postweaning multisystemic wasting syndrome (PMWS) in the mid-1990s and causes porcine circovirus-associated disease (PCVAD) (1, 9, 10, 25). PCV1 and PCV2 have similar genomic organizations, with two major ambisense open reading frames (ORFs) (16). ORF1 (rep) encodes two viral replication-associated proteins, Rep and Rep′, by differential splicing (4, 6, 21, 22). The Rep and Rep′ proteins bind to specific sequences within the origin of replication (Ori) located in the intergenic region, and both are responsible for viral replication (5, 7, 8, 21, 23, 28, 29). ORF2 (cap) encodes the immunogenic capsid protein (Cap) (26). PCV1 and PCV2 share approximately 80%, 82%, and 62% nucleotide sequence identity in the Ori, rep, and cap, respectively (19).In vitro studies using a reporter gene-based assay system showed that the replication factors of PCV1 and PCV2 are functionally interchangeable (2-6, 22), although this finding has not yet been validated in a live infectious-virus system. We have previously shown that chimeras of PCV in which cap has been exchanged between PCV1 and PCV2 are infectious both in vitro and in vivo (15), and an inactivated vaccine based on the PCV1-PCV2 cap (PCV1-cap2) chimera is used in the vaccination program against PCVAD (13, 15, 18, 27).PCV1 replicates more efficiently than PCV2 in PK-15 cells (14, 15); thus, we hypothesized that the Ori or rep is directly responsible for the differences in replication efficiencies. The objectives of this study were to demonstrate that the Ori and rep are interchangeable between PCV1 and PCV2 in a live-virus system and to determine the effects of swapped heterologous replication factors on virus replication efficiency in vitro.     

Virus Entry via the Alternative Coreceptors CCR3 and FPRL1 Differs by Human Immunodeficiency Virus Type 1 Subtype

Human immunodeficiency virus type 1 (HIV-1) infects target cells by binding to CD4 and a chemokine receptor, most commonly CCR5. CXCR4 is a frequent alternative coreceptor (CoR) in subtype B and D HIV-1 infection, but the importance of many other alternative CoRs remains elusive. We have analyzed HIV-1 envelope (Env) proteins from 66 individuals infected with the major subtypes of HIV-1 to determine if virus entry into highly permissive NP-2 cell lines expressing most known alternative CoRs differed by HIV-1 subtype. We also performed linear regression analysis to determine if virus entry via the major CoR CCR5 correlated with use of any alternative CoR and if this correlation differed by subtype. Virus pseudotyped with subtype B Env showed robust entry via CCR3 that was highly correlated with CCR5 entry efficiency. By contrast, viruses pseudotyped with subtype A and C Env proteins were able to use the recently described alternative CoR FPRL1 more efficiently than CCR3, and use of FPRL1 was correlated with CCR5 entry. Subtype D Env was unable to use either CCR3 or FPRL1 efficiently, a unique pattern of alternative CoR use. These results suggest that each subtype of circulating HIV-1 may be subject to somewhat different selective pressures for Env-mediated entry into target cells and suggest that CCR3 may be used as a surrogate CoR by subtype B while FPRL1 may be used as a surrogate CoR by subtypes A and C. These data may provide insight into development of resistance to CCR5-targeted entry inhibitors and alternative entry pathways for each HIV-1 subtype.Human immunodeficiency virus type 1 (HIV-1) infects target cells by binding first to CD4 and then to a coreceptor (CoR), of which C-C chemokine receptor 5 (CCR5) is the most common (6, 53). CXCR4 is an additional CoR for up to 50% of subtype B and D HIV-1 isolates at very late stages of disease (4, 7, 28, 35). Many other seven-membrane-spanning G-protein-coupled receptors (GPCRs) have been identified as alternative CoRs when expressed on various target cell lines in vitro, including CCR1 (76, 79), CCR2b (24), CCR3 (3, 5, 17, 32, 60), CCR8 (18, 34, 38), GPR1 (27, 65), GPR15/BOB (22), CXCR5 (39), CXCR6/Bonzo/STRL33/TYMSTR (9, 22, 25, 45, 46), APJ (26), CMKLR1/ChemR23 (49, 62), FPLR1 (67, 68), RDC1 (66), and D6 (55). HIV-2 and simian immunodeficiency virus SIVmac isolates more frequently show expanded use of these alternative CoRs than HIV-1 isolates (12, 30, 51, 74), and evidence that alternative CoRs other than CXCR4 mediate infection of primary target cells by HIV-1 isolates is sparse (18, 30, 53, 81). Genetic deficiency in CCR5 expression is highly protective against HIV-1 transmission (21, 36), establishing CCR5 as the primary CoR. The importance of alternative CoRs other than CXCR4 has remained elusive despite many studies (1, 30, 70, 81). Expansion of CoR use from CCR5 to include CXCR4 is frequently associated with the ability to use additional alternative CoRs for viral entry (8, 16, 20, 63, 79) in most but not all studies (29, 33, 40, 77, 78). This finding suggests that the sequence changes in HIV-1 env required for use of CXCR4 as an additional or alternative CoR (14, 15, 31, 37, 41, 57) are likely to increase the potential to use other alternative CoRs.We have used the highly permissive NP-2/CD4 human glioma cell line developed by Soda et al. (69) to classify virus entry via the alternative CoRs CCR1, CCR3, CCR8, GPR1, CXCR6, APJ, CMKLR1/ChemR23, FPRL1, and CXCR4. Full-length molecular clones of 66 env genes from most prevalent HIV-1 subtypes were used to generate infectious virus pseudotypes expressing a luciferase reporter construct (19, 57). Two types of analysis were performed: the level of virus entry mediated by each alternative CoR and linear regression of entry mediated by CCR5 versus all other alternative CoRs. We thus were able to identify patterns of alternative CoR use that were subtype specific and to determine if use of any alternative CoR was correlated or independent of CCR5-mediated entry. The results obtained have implications for the evolution of env function, and the analyses revealed important differences between subtype B Env function and all other HIV-1 subtypes.     

Ectromelia Virus Inhibitor of Complement Enzymes Protects Intracellular Mature Virus and Infected Cells from Mouse Complement

Poxviruses produce complement regulatory proteins to subvert the host''s immune response. Similar to the human pathogen variola virus, ectromelia virus has a limited host range and provides a mouse model where the virus and the host''s immune response have coevolved. We previously demonstrated that multiple components (C3, C4, and factor B) of the classical and alternative pathways are required to survive ectromelia virus infection. Complement''s role in the innate and adaptive immune responses likely drove the evolution of a virus-encoded virulence factor that regulates complement activation. In this study, we characterized the ectromelia virus inhibitor of complement enzymes (EMICE). Recombinant EMICE regulated complement activation on the surface of CHO cells, and it protected complement-sensitive intracellular mature virions (IMV) from neutralization in vitro. It accomplished this by serving as a cofactor for the inactivation of C3b and C4b and by dissociating the catalytic domain of the classical pathway C3 convertase. Infected murine cells initiated synthesis of EMICE within 4 to 6 h postinoculation. The levels were sufficient in the supernatant to protect the IMV, upon release, from complement-mediated neutralization. EMICE on the surface of infected murine cells also reduced complement activation by the alternative pathway. In contrast, classical pathway activation by high-titer antibody overwhelmed EMICE''s regulatory capacity. These results suggest that EMICE''s role is early during infection when it counteracts the innate immune response. In summary, ectromelia virus produced EMICE within a few hours of an infection, and EMICE in turn decreased complement activation on IMV and infected cells.Poxviruses encode in their large double-stranded DNA genomes many factors that modify the immune system (30, 56). The analysis of these molecules has revealed a delicate balance between viral pathogenesis and the host''s immune response (2, 21, 31, 61). Variola, vaccinia, monkeypox, cowpox, and ectromelia (ECTV) viruses each produce an orthologous complement regulatory protein (poxviral inhibitor of complement enzymes [PICE]) that has structural and functional homology to host proteins (14, 29, 34, 38, 41, 45, 54). The loss of the regulatory protein resulted in smaller local lesions with vaccinia virus lacking the vaccinia virus complement control protein (VCP) (29) and in a greater local inflammatory response in the case of cowpox lacking the inflammation-modulatory protein (IMP; the cowpox virus PICE) (35, 45, 46). Additionally, the complete loss of the monkeypox virus inhibitor of complement enzymes (MOPICE) may account for part of the reduced mortality observed in the West African compared to Congo basin strains of monkeypox virus (12).The complement system consists of proteins on the cell surface and in blood that recognize and destroy invading pathogens and infected host cells (36, 52). Viruses protect themselves from the antiviral effects of complement activation in a variety of ways, including hijacking the host''s complement regulatory proteins or producing their own inhibitors (7, 8, 15, 20, 23). Another effective strategy is to incorporate the host''s complement regulators in the outermost viral membrane, which then protects the virus from complement attack (62). The extracellular enveloped virus (EEV) produced by poxviruses acquires a unique outer membrane derived from the Golgi complex or early endosomes that contain the protective host complement regulators (58, 62). Poxviruses have multiple infectious forms, and the most abundant, intracellular mature virions (IMV), are released when infected cells lyse (58). The IMV lacks the outermost membrane found on EEV and is sensitive to complement-mediated neutralization. The multiple strategies viruses have evolved to evade the complement system underscore its importance to innate and adaptive immunity (15, 36).The most well-characterized PICE is VCP (24-29, 34, 49, 50, 53, 55, 59, 60). Originally described as a secreted complement inhibitor (34), VCP also attaches to the surface of infected cells through an interaction with the viral membrane protein A56 that requires an unpaired N-terminal cysteine (26). This extra cysteine also adds to the potency of the inhibitor by forming function-enhancing dimers (41). VCP and the smallpox virus inhibitor of complement enzymes (SPICE) bind heparin in vitro, and this may facilitate cell surface interactions (24, 38, 50, 59). The coevolution of variola virus with its only natural host, humans, likely explains the enhanced activity against human complement observed with SPICE compared to the other PICEs (54, 64).Our recent work with ECTV, the causative agent of mousepox infection, demonstrated that the classical and alternative pathways of the complement system are required for host survival (48). The mouse-specific pathogen ECTV causes severe disease in most strains and has coevolved with its natural host, analogous to variola virus in humans (9). This close host-virus relationship is particularly important for evaluating the role of the complement system, given the species specificity of many complement proteins, receptors, and regulators (10, 47, 62). Additionally, the availability of complement-deficient mice permits dissection of the complement activation pathways involved. Naïve C57BL/6 mouse serum neutralizes the IMV of ECTV in vitro, predominately through opsonization (48). Maximal neutralization requires natural antibody, classical-pathway activation, and amplification by the alternative pathway. C3 deficiency in the normally resistant C57BL/6 strain results in acute mortality, similar to immunodeficiencies in important elements of the antiviral immune response, including CD8⁺ T cells (19, 32), natural killer cells (18, 51), and gamma interferon (33). During ECTV infection, the complement system acts in the first few hours and days to delay the spread of infection, resulting in lower levels of viremia and viral burden in tissues (48).This study characterized the PICE produced by ECTV, ectromelia virus inhibitor of complement enzymes (EMICE), and assessed its complement regulatory activity. Recombinant EMICE (rEMICE) decreased activation of both human and mouse complement. Murine cells produced EMICE at 4 to 6 h postinfection prior to the release of the majority of the complement-sensitive IMV from infected cells. rEMICE protected ECTV IMV from complement-mediated neutralization. Further, EMICE produced during natural infection inhibited complement deposition on infected cells by the alternative pathway. ECTV likely produces this abundance of EMICE to protect both the IMV and infected cells.