Acidianus Filamentous Virus 1 (AFV1, Lipothrixviridae) is an enveloped filamentous virus that was characterized from a crenarchaeal host. It infects Acidianus species that thrive in the acidic hot springs (t>85 degrees C and pH<3) of Yellowstone National Park, USA. The AFV1 20.8 kb, linear, double-stranded DNA genome encodes 40 putative open reading frames, whose sequences generally show little similarity to other genes in the sequence databases. Because 3D structures are more conserved than sequences and hence more effective at revealing function, we set out to determine protein structures from putative AFV1 ORFS. The crystal structure of ORF157 reveals an alpha+beta protein with a novel fold, that remotely resembles the nucleotidyltransferase topology. In vitro, AFV1-157 displays a nuclease activity on linear dsDNA. Alanine substitution mutations demonstrated that E86 is essential to catalysis. AFV1-157 represents a novel class of nuclease, but its exact role in vivo remains to be determined.

IL-10 is an important anti-inflammatory molecule that can cause immunosuppression and long-term pathogen persistence during chronic infection of mice with viruses such as lymphocytic choriomeningitis virus. However, its specific role in immunity to acute viral infections is not fully understood. We found that IL-10 plays a detrimental role in host responses to acute influenza A virus since IL-10(-/-) mice had improved viral clearance and survival after infection compared to wild-type mice. Enhanced viral clearance in IL-10(-/-) mice was not correlated with increased CD4(+) or CD8(+) T cell recruitment into the lung but was correlated with increased pulmonary anti-influenza antibody titers, and this was dependent upon the presence of T cells, primarily CD4(+) T cells. In addition, virus-specific antibody produced during the early stages of infection in the respiratory tract of IL-10(-/-) but not wild-type mice was sufficient to mediate passive protection against viral challenge of naïve mice. Complement was necessary for this antibody-mediated passive protection, but FcR or neutrophil deficiency did not significantly influence viral clearance. Our results show that an absence of IL-10 at the time of primary infection leads to enhanced local virus-specific antibody production and thus, increased protection against influenza A virus infection.

Respiratory syncytial virus (RSV) is the main cause of bronchiolitis, the major cause of hospitalization in infancy. An ideal RSV vaccine would be effective in neonates, but the immune responses of infants differ markedly from those in adults, often showing a bias towards T helper 2 (Th2) responses and reduced interferon gamma (IFN-gamma) production. We have previously developed recombinant RSV vectors expressing the IFN-gamma and interleukin-4 (IL-4) that allow us to explore the role of these key Th1 and Th2 cytokines during infection. The aim of the current study was to explore whether immunomodulation of infant responses could enhance protection. Expression of IFN-gamma by a recombinant RSV (rRSV/IFN-gamma) vector attenuated primary viral replication in newborn mice without affecting the development of specific antibody or T cell responses. On challenge rRSV/IFN-gamma mice were protected from the exacerbated disease observed in mice primed with wild type RSV; however, anti-viral immunity was not enhanced. Conversely, expression of IL-4 by recombinant RSV did not affect virus replication in neonates but greatly enhanced Th2 immune responses on challenge without affecting weight loss. These studies demonstrate that it is possible to manipulate the infant immune responses using cytokine-expressing recombinant viruses and that neonatal deficiency in IFN-gamma responses may lead to enhanced disease during secondary infection.

Control of HIV-1 replication following non-sterilizing HIV-1 vaccination could be achieved by vaccine-elicited CD8(+) T cell mediated antiviral activity. To date, neither the functional nor the phenotypic profiles of CD8(+) T cells capable of this activity are clearly understood; consequently, little is known regarding the ability of vaccine strategies to elicit them. We used multiparameter flow cytometry and viable cell sorts from phenotypically-defined CD8(+) T cell subsets in combination with a highly standardized virus inhibition assay to evaluate CD8(+) T cell-mediated inhibition of viral replication. Here we show that vaccination against HIV-1 Env and Gag-Pol by DNA priming followed by rAd5 boosting elicited CD8(+) T cell mediated antiviral activity against several viruses with either lab-adapted or transmitted virus envelopes. As it did for chronically-infected virus controllers, this activity correlated with HIV-1 specific CD107a or MIP-1beta expression from HIV-1 specific T cells. Moreover, for vaccinees or virus controllers, purified memory CD8(+) T cells from a wide range of differentiation stages were capable of significantly inhibiting virus replication. Our data define attributes of an antiviral CD8(+) T cell response that may be optimized in the search for an efficacious HIV-1 vaccine.

The Epstein-Barr Virus (EBV) origin of plasmid replication (OriP) is required for episome stability during latent infection. Telomere repeat factor 2 (TRF2) binds directly to OriP and facilitates DNA replication and plasmid maintenance. Recent studies have found that TRF2 interacts with the DNA damage checkpoint protein Chk2. We show here that Chk2 plays an important role in regulating OriP plasmid stability, chromatin modifications, and replication timing. Depletion of Chk2 by siRNA leads to a reduction in DNA replication efficiency and a loss of OriP-dependent plasmid maintenance. This corresponds to a change in OriP replication timing and an increase in constitutive histone H3 acetylation. We show that Chk2 interacts with TRF2 in early G1/S phase of the cell cycle. We also show that Chk2 can phosphorylate TRF2 in vitro at a consensus acceptor site in the amino terminal basic domain of TRF2. TRF2 mutants with a serine to aspartic acid phospho-mimetic substitution mutation were reduced for their ability to recruit ORC and stimulate OriP replication. We suggest that Chk2 phosphorylation of TRF2 is important for coordinating ORC binding with chromatin remodeling during early S phase, and that a failure to execute these events leads to replication defects and plasmid instability.

Influenza viruses attach to cells via a sialic acid moiety (sialic acid receptor) that is alpha2-3 linked, or alpha2-6 linked, to galactose (alpha2-3SAL or alpha2-6SAL); sialic acid acts as a receptor for the virus. By lectin staining, we demonstrated that the alpha2-6SAL configuration is predominant in the respiratory tract of ferret including trachea, bronchi, and lung alveoli tissues. Recombinant wild type (rWt) influenza A/Solomon Island/3/06 (SI06) (H1N1) viruses were constructed to assess the impact of the hemagglutinin (HA) variations (amino acids 190 or 226) identified in natural variants on virus replication in the upper and lower respiratory tract of ferrets, virus antigenicity, and immunogenicity. A single amino acid change at residue 226 (from Gln to Arg) in the HA of SI06 resulted in the complete loss of binding to alpha2-6SAL, and concomitant loss of the virus's ability to replicate in the lower respiratory tract of ferrets. In contrast, the virus with Gln226 in the HA protein has receptor binding preference for alpha2-6SAL and replicates efficiently in the lungs. There was a good correlation between viral replication in the lungs of ferrets and disease symptoms. In addition, we also showed that the 190 and 226 residues impacted viral antigenicity and immunogenicity. Our data emphasizes the necessity of thoroughly assessing wild type influenza viruses for their suitability as reference strains and for carefully selecting the HA antigen for vaccine production during annual influenza vaccine evaluation processes.

Previously, RNA transcripts of cDNA clones of hepatitis C virus (HCV) genotypes 1a (H77, HCV-1 and HC-TN), 1b (HC-J4, Con1, and HCV-N) and 2a (HC-J6 and JFH1) were found to be infectious in chimpanzees. However, only JFH1 was infectious in human hepatoma Huh7 cells. We performed genetic analysis of HCV genotype 3a (S52) and 4a (ED43) prototype strains, and generated full-length consensus cDNA clones (pS52 and pED43). Transfection of Huh7.5 cells with RNA transcripts of these clones did not yield HCV Core expressing cells. However, intrahepatic transfection of chimpanzees resulted in robust infection with peak HCV RNA titers of approximately 5.5 log10 IU/mL. Genomic consensus sequences recovered from serum at time points of peak viral titers were identical to the parental plasmids. Both chimpanzees developed acute hepatitis with elevated liver enzymes and significant necro-inflammatory liver changes coinciding with detection of interferon-gamma secreting, intrahepatic T cells. However, onset and broadness of intrahepatic T-cell responses varied greatly in the two animals, with an early (week 4) multi-specific response in the ED43 infected animal (three weeks before first evidence of viral control), and a late (week 11) response with limited breadth in the S52 infected animal (without evidence of viral control). Autologous serum neutralizing antibodies were not detected during the acute infection in either animal. Both animals became persistently infected. In conclusion, we generated fully functional infectious cDNA clones of HCV genotypes 3a and 4a. Proof of functionality of all genes might further the development of recombinant cell culture systems for these important genotypes.

The first fully sequenced papillomavirus (PV) of marsupials, tentatively named Bettongia penicillata papillomavirus type 1 (BpPV1) was detected in papillomas from a woylie (Bettongia penicillata ogilbyi). The circular, dsDNA genome contains 7737 bp and encodes 7 open reading frames (ORFs): E6, E7, E1, E2, E4, L2 and L1 in typical PV conformation. BpPV1 is a close-to-root PV with L1 and L2 ORFs most similar to European hedgehog PV, and bandicoot papillomatosis carcinomatosis virus types 1 and 2 (BPCVs). It appears that the BPCVs arose by recombination between an ancient PV and an ancient polyomavirus more than 10 million years ago.

The role of epigenetic modifications in the regulation of gammaherpesvirus latency has been a subject of active study for more than 20 years. DNA methylation, associated with transcriptional silencing in mammalian genomes, has been shown to be an important mechanism in the transcriptional control of several key gammaherpesvirus genes. In particular, DNA methylation of the functionally conserved immediate-early replication and transcription activator (RTA) has been shown to regulate Epstein-Barr virus and Kaposi's Sarcoma-associated herpesvirus Rta expression. Here we demonstrate that the murine gammaherpesvirus (MHV68) homolog, encoded by gene 50, is also subject to direct repression by DNA methylation, both in vitro and in vivo. We observed that treatment of MHV68 latently B cell lines with a methyltransferase inhibitor induced virus reactivation. In addition, we show that methylation of the recently characterized distal gene 50 promoter represses activity in a murine macrophage cell line. To evaluate the role of de novo methyltransferases (DNMTs) in the establishment of these methylation marks, we infected mice in which conditional DNMT3a and DNMT3b alleles were selectively deleted in B lymphocytes. DNMT3A/DNMT3B-deficient B cells were phenotypically normal, displaying no obvious compromise in cell surface marker expression or antibody production in either naïve mice or in the context of non-viral and viral immunogens. However, mice lacking functional DNMT3a/3b in B cells exhibited hallmarks of deregulated MHV68 lytic replication, including increased splenomegaly and the presence of infectious virus in the spleen at day 18 following infection. In addition, total gene 50 transcripts were elevated in the spleens of these mice at day 18 - which correlated with hypomethylation of the distal gene 50 promoter. However, by day 42 post-infection aberrant virus replication was resolved and we observed wild-type frequencies of viral genome positive splenocytes in mice lacking functional DNMT3a/3b in B lymphocytes. The latter correlated with increased CpG methylation in the distal gene 50 promoter, which was restored to levels similar to littermate controls harboring functional DNMT3a/b alleles in B lymphocytes - suggesting the existence of an alternative mechanism for de novo methylation of the MHV68 genome. Importantly, this DNMT3a/DNMT3b-independent methylation appeared to be specifically targeted to the gene 50 promoter, as we observed that the promoters for MHV68 gene 72 (v-cyclin) and M11 (v-bcl2) remained hypomethylated at day 42 post-infection. Taken together these data provide the first evidence of the importance of DNA methylation in regulating gammaherpesvirus RTA/gene 50 transcription during virus infection in vivo, and provide insight into the hierarchy of host machinery required to establish this modification.

Measles virus (MV) causes transient severe immunosuppression in patients, which may lead to secondary viral and bacterial infections largely accounting for measles-related morbidity and mortality. MV is known to infect immune cells by using the human signaling lymphocyte activation molecule (SLAM, also called CD150) as a cellular receptor, but the mechanism by which MV causes immunosuppression is not well understood. We show that MV infection of SLAM knock-in mice, in which the V domain of mouse SLAM was replaced by the V domain of human SLAM, crossed with alpha/beta-interferon receptor knock-out mice reproduced many immunological alterations observed in human patients. These included lymphopenia, inhibition of T cell proliferation and antibody production, increased production of the Th2 cytokine interleukin (IL)-4 and the immunosuppressive cytokine IL-10, and suppression of contact hypersensitivity. Gross redistribution of lymphocytes among lymphoid tissues was not apparent in infected mice, nor was an increase of regulatory T cells. The numbers of lymphocytes in lymph nodes remained almost unchanged after MV infection despite enhanced apoptosis, suggesting that lymph nodes were replenished with lymphocytes from the peripheral blood, which may have contributed to the observed lymphopenia in the spleen. Blocking of IL-10 using an anti-IL10 receptor antibody ameliorated suppression of contact hypersensitivity in infected mice. These results indicate that SLAM knock-in mice lacking the expression of alpha/beta-interferon receptor serve as a useful small animal model to elucidate MV-induced immunosuppression.

Canine parvovirus (CPV) and its relative feline panleukopenia virus (FPV) bind the transferrin receptor type 1 (TfR) to infect their host cells, but show differences in the interactions with the feline and canine TfRs that determine viral host range and tissue tropism. We changed apical and protease-like domain residues by introducing point mutations and adding or removing glycosylation signals, and then examined the interactions of those mutant TfR with the capsids. Most substitutions had little effect on virus binding and uptake. However, mutations of several sites in the apical domain of the receptor either prevented binding to the capsids, or reduced the affinity of receptor binding to varying degrees. Glycans within the virus-binding face of the apical domain also controlled capsid binding. CPV, but not the related feline parvovirus, could use receptors containing a canine TfR-specific glycosylation to mediate efficient infection, while adding other N-linked glycosylation sites into the virus-binding face of the feline apical domain reduced or eliminated both binding and infection. Substitution of critical feline TfR residue 221 to every amino acid had effects on binding and infection which were significantly associated with the biochemical properties of the residue replaced. Receptors with reduced affinities mostly showed proportional changes in their ability to mediate infection. Testing feline TfR variants for their binding and uptake patterns in cells showed that low affinity versions bound fewer capsids and also differed in attachment to the cell surface and filopodia, but transport to the perinuclear endosome was similar.

It has previously been demonstrated that during human cytomegalovirus infection, the viral IE2 86 and IE2 40 proteins are both important for the expression of an early-late viral protein, UL84. Here, we show that expression of the UL84 protein is enhanced upon co-transfection with either IE2 86 or IE2 40, although IE2 40 appears to play a more important role. The UL84 protein levels are tightly linked to the amount of IE2 40 present, but this does not appear to be true for IE2 86. RNA remains constant for all corresponding proteins, indicating post-transcriptional regulation of UL84. The first 105 amino acids of UL84 are necessary and sufficient for this phenotype, and this region is also required for an interaction with IE2 86 and IE2 40. Treatment with proteasome inhibitors shows that UL84 exhibits some proteasome dependent degradation, and UL84 is not protected against this degradation when co-expressed with IE2 86 or IE2 40. UL84 also exhibits an inhibitory effect on IE2 86 and IE2 40 protein levels in these co-transfection assays. Further, we show that the amino acid sequence of UL84 is important for the enhancement governed by IE2 40. These results indicate that IE2 86, IE2 40 and UL84 serve to regulate protein expression in a post-transcriptional fashion, and that this regulation is independent of other viral proteins.

The SJPL cell line was reported and described as an immortalized porcine lung epithelial cell line suitable for influenza virus replication in Seo et al., J. Virol. 2001;75(19):9517-25 and has been available to researchers through ATCC (American Type Culture Collection) cell depositary services (deposit date: April 5, 2001; assigned accession number: PTA-3256) following a material transfer agreement (6)....

Leukocyte access into the central nervous system (CNS) parenchyma is tightly regulated by the blood brain barrier (BBB). Leukocyte migration through the endothelial cell wall into the perivascular space is well characterized; however, mechanisms regulating their penetration through the glia limitans into the parenchyma are less well studied and the role of monocytes relative to neutrophils is poorly defined. Acute viral encephalitis was thus induced in CCL2 deficient (CCL2(-/-)) mice to specifically abrogate monocyte recruitment. Impaired monocyte recruitment prolonged T cell retention in the perivascular space although no difference in overall CNS accumulation of CD4 or CD8 T cells was detected by flow cytometry. Delayed penetration to the CNS parenchyma was not associated with reduced or altered expression of either matrix metalloproteinases (MMP) or the T cell chemoattractants CXCL10 and CCL5. Nevertheless, decreased parenchymal leukocyte infiltration delayed T cell mediated control of virus replication as well as clinical disease. These data are the first to demonstrate that the rapid monocyte recruitment into the CNS during viral encephalitis is dispensable for T cell migration across the blood vessel endothelium. However, monocytes facilitate penetration through the glia limitans. Thus the rapid monocyte response to viral encephalitis constitutes an indirect antiviral pathway by aiding access of effector T cells to the site of viral infection.

During the transition from acute to chronic infection in HCV persistently infected individuals, cellular responses initiate within the first 6 months of primary infection and collapse thereafter whereas humoral responses activate later during the chronic phase. Whether and how this deviation of immune responses specifically influences HCV evolution is unknown. To determine the pattern of HCV evolution during this critical period, we conducted extensive sequence analysis on annual clonal hemigenomic sequences up to three years in six well-characterized subjects, using statistical methods that accounted for repeated measures. Significantly different evolutionary rates were observed in envelope versus non-envelope genes, with increasing rate of non-synonymous change (dN) in envelope and stable dN in non-envelope genes (p=0.006). The ratio of envelope to non-envelope non-synonymous rate increased from 2 in year 1 to 5 in years 2 and 3. Centripetal changes (reversions toward matching of worldwide subtype 1a consensus sequence) were frequently observed during the three year transition from acute infection to chronicity, even in the presence of nAb pressure. Remarkably, HVR1 sequences remained stable for up to 21 months in the absence of nAb pressure in one subject, followed by rapid changes that were temporally associated with the detection of nAb responses, strongly suggesting that HVR1 evolution is shaped by nAb pressure. These data provide the first systematic estimates of HCV evolutionary rates in multiple genes during early infection in vivo, and provide additional evidence for deterministic, rather than random, evolution of HCV.

Hepatitis C Virus is a leading cause of chronic liver disease. The identification and characterisation of key host cellular factors that play a role in the HCV replication cycle is important for the understanding of disease pathogenesis and the identification of novel anti-viral therapeutic targets. Gene expression profiling of JFH-1 infected Huh7 cells by microarray analysis was performed to identify host cellular genes that are transcriptionally regulated by infection. The expression of host genes involved in cellular defence mechanisms (apoptosis, proliferation and anti-oxidant responses), cellular metabolism (lipid and protein metabolism) and intracellular transport (vesicle trafficking and cytoskeleton regulation) was significantly altered by HCV infection. The gene expression patterns identified provide insight into the potential mechanisms that contribute to HCV associated pathogenesis. These include an increase in pro-inflammatory and pro-apoptotic signalling and a decrease in the anti-oxidant response pathways of the infected cell. To investigate whether any of the host genes regulated by infection were required by HCV during replication, siRNA silencing of host gene expression in HCV infected cells was performed. Decreasing the expression of host genes involved in lipid metabolism (TXNIP and CYP1A1) and intracellular transport (Rab33b and ABLIM3) reduced the replication and secretion of HCV indicating that they may be important factors for the virus replication cycle. These results show that major changes in the expression of many different genes, in target cells, may be crucial in determining the outcome of HCV infection.

The cytoplasmic domain of glycoprotein B (gB) from herpes simplex virus type 1 (HSV-1) is an important regulator of membrane fusion. C-terminal truncations of the cytoplasmic domain lead to either hyperfusion or fusion-null phenotypes. Currently, neither the structure of the cytoplasmic domain nor its mechanism of fusion regulation is known. Here we show, for the first time, that the full-length cytoplasmic domain of HSV-1 gB associates stably with lipid membranes, preferentially binding to membranes containing anionic headgroups. This interaction involves a large increase in helical content. But, the truncated cytoplasmic domains associated with the hyperfusion phenotype show a small increase in helical structure and a diminished association with lipid membranes whereas the one associated with the fusion-null phenotype shows no increase in helical structure and only a minimal association with lipid membranes. We hypothesize that stable binding to lipid membranes is an important part of the mechanism by which the cytoplasmic domain negatively regulates membrane fusion. Moreover, our experiments with truncated cytoplasmic domains point to two specific regions that are critical for membrane interactions. Taken together, our work provides several important new insights into the architecture of the cytoplasmic domain of HSV-1 gB and its interaction with lipid membranes.

Ranaviruses such as Frog Virus 3 (FV3; Iridoviridae) are increasingly prevalent pathogens infecting reptiles, amphibians and fish worldwide. Whereas studies in the frog Xenopus have revealed the critical involvement of CD8 T cell and antibody responses in host resistance to FV3, little is known about the role played by innate immunity to infection with this virus. We have investigated the occurrence, composition, activation status, and permissiveness to infection of peritoneal leukocytes (PLs) in Xenopus adults during FV3 infection by microscopy, flow cytometry and RT-PCR. The total number of PLs and the relative fraction of activated mononucleated macrophage-like cells significantly increase as early as 1 day post-infection (dpi), followed by NK cells at 3 dpi, before the peak of T cell response at 6 dpi. FV3 infection also induces a rapid up-regulation of pro-inflammatory genes including Arginase 1, IL-1beta and TNFalpha. Although PLs are susceptible to FV3 infection as evidenced by apoptotic cells, active FV3 transcription and the detection of viral particles by electron microscopy, the infection is weaker (fewer infectious particles), more transitory and involves a lower fraction (less than 1%) of PLs than the kidney, the main site of infection. However, viral DNA remains detectable in PLs for at least 3 weeks post-infection, past the point of viral clearance observed in the kidneys. This suggests that although PLs are actively involved in anti-FV3 immune responses, some of these cells can be permissive and harbor quiescent, asymptomatic FV3.

Profound type 1 interferon (IFN1)-dependent attrition of memory CD8 and CD4 T cells occurs early during many infections. It is dramatic 2-4 days following lymphocytic choriomeningitis virus (LCMV) infection of mice and can be elicited by the IFN-inducing Toll receptor agonist poly(I:C). We show that this attrition occurs in many organs, indicating that it is due to T cell loss rather than redistribution. This loss correlated with elevated intracellular staining of T cells ex vivo for activated caspases, but only with low levels of ex vivo staining with Annexin V, probably due to the rapid clearance of apoptotic cells in vivo. Instead, a high frequency of Annexin V-reactive CD8alpha(+) dendritic cells (DC), known to be highly phagocytic, accumulated in the spleen as the memory T cell populations disappeared. After short in vitro incubation, memory phenotype T cells isolated from LCMV-infected mice (day 3) or mice treated with poly(I:C) (12 hours) displayed substantial DNA fragmentation, as detected by TUNEL assay, when compared to T cells isolated from uninfected mice, indicating a role for apoptosis in the memory T cell attrition. This apoptosis of memory CD8 T cells early during LCMV infection was reduced in mice lacking the pro-apoptotic molecule Bim. Evidence is presented showing that high levels of T cell attrition, as found in young mice, correlates with reduced immunodomination by cross-reactive memory cells.