Characterisation of the HSV-1 DNA Packaging Protein Encoded by the UL25 Gene

Herpes simplex virus type 1 (HSV-1) DNA replication results in the formation of head- to-tail concatemers which are cleaved into genome size units and packaged into the procapsid in the nuclei of virus-infected cells. The procapsid is a spherical structure with icosahedral symmetry and contains an internal protein scaffold which is removed at the same time viral DNA is encapsidated. During the DNA packaging process the procapsid angularises and the DNA-containing capsid can subsequently mature into an infectious virion. The product of the HSV-1 UL25 gene is a minor component of the viral capsid and has been implicated in the HSV-1 DNA packaging process. The overall goal of this thesis was to investigate the role of the UL25 protein in the HSV-1 lytic cycle. Before a detailed study of this protein could be undertaken, a number of reagents had to be prepared, including potent UL25-specitlc antibodies. Therefore, an initial aim of the project was to express the UL25 protein in a variety of in vivo recombinant protein expression systems and to purify the soluble recombinant UL25 protein for use as an antigen in the production of UL25-specific monoclonal antibodies. Maltose binding protein (MBP)-tagged UL25 and polyhistidine (His)-tagged UL25 were expressed in Escherichia coli and recombinant baculovirus-infected Sf21 cells respectively. BALB/c mice were immunised with purified soluble MBP-UL25 fusion protein and given a final boost with purified soluble His-tagged UL25 protein. Twelve hybridoma cell lines secreting UL25-specific monoclonal antibodies were isolated. The monoclonal antibodies were characterised using Western blot, immunoprecipitation and immunofluorescence assays. From this analysis a monoclonal antibody that reacted strongly with the UL25 protein in each of the immunoassays was purified for use in subsequent experiments. In the absence of other HSV-1 proteins, UL25 localised predominantly to the cytoplasm of cells transiently expressing the protein. In cells infected with HSV-I, however, UL25 protein was concentrated in the nuclei at late times. To investigate whether the HSV-1 capsid shell proteins, VP5, VP23 and VP19C, were required for the nuclear localisation of UL25 in HSV-I-infected cells, the distribution of UL25 protein was examined in cells infected with HSV-1 mutants which fail to express these proteins. In non-complementing cells infected with VP23 or VPS null mutants, the distribution of UL25 protein was similar to the pattern in wild-type (wt) virus-infected cells indicating that neither VP23 nor VP5 were necessary for the nuclear localisation of the UL25 protein during HSV-1 infection. Since capsid assembly did not occur under these conditions (Desai et al., 1993), nuclear localisation of UL25 was independent of capsid assembly. The intracellular distribution of UL25 was also examined in non-complementing cells infected with a VP19C null mutant of HSV-1. However, this virus appeared to have an additional mutation, one which affected late viral protein production, and no conclusive results were obtained through the use of this virus. The localisation of the UL25 protein was therefore investigated in cells infected with ts2, a mutant of HSV-1 that contains a temperature sensitive (ts) lesion in the VP19C protein. In cells infected with ts2 at the non-permissive temperature (NPT), UL25 co-localised with the capsid shell proteins at the perinuclear region of cells with little, if any, UL25 protein observed in the nuclei. These findings suggested that the VP19C protein was necessary for the nuclear distribution of UL25 during wt HSV-1 infection. However, in cells infected with a ts2 marker rescuant at the NPT, UL25 remained localised to the perinuclear region while the capsid shell proteins were found in the nuclei. This result indicated that the altered intracellular distribution of UL25 in cells infected with ts2 at the NPT was not a consequence of the ts lesion in the VP19C protein. Furthermore, UL25 also localised to the perinuclear region of cells infected with HSV-1 A44, the parental syncytial strain of ts2, at the NPT. This virus formed syncytia to a greater extent in cells infected at the NPT compared to the permissive temperature and it is possible that the altered intracellular distribution of UL25 protein in cells infected with HSV-1 A44 at the NPT may have resulted from the formation of syncytia or from an aberrant interaction with a component of the HSV-1 tegument. The association of UL25 protein with the capsid was initially examined using the recombinant baculovirus expression system to obtain information about the copy number and the location of the UL25 protein in the capsid as well as its interaction with capsid shell proteins. The UL25 protein was incorporated into capsids generated in insect cells multiply infected with recombinant baculoviruses expressing the HSV-1 capsid shell, scaffolding and the UL25 proteins, suggesting that the UL25 protein can interact with capsids in the absence of other viral proteins. This finding is in agreement with earlier results of McNab et al. (1998). (Abstract shortened by ProQuest.)

Lipid Metabolism and HCV Infection

Chronic infection by hepatitis C virus (HCV) can lead to severe liver disease and is a global healthcare problem. The liver is highly metabolically active and one of its key functions is to control the balance of lipid throughout the body. A number of pathologies have been linked to the impact of HCV infection on liver metabolism. However, there is also growing evidence that hepatic metabolic processes contribute to the HCV life cycle. This review summarizes the relationship between lipid metabolism and key stages in the production of infectious HCV

Genome-to-genome analysis highlights the effect of the human innate and adaptive immune systems on the hepatitis C virus

Outcomes of hepatitis C virus (HCV) infection and treatment depend on viral and host genetic factors. Here we use human genome-wide genotyping arrays and new whole-genome HCV viral sequencing technologies to perform a systematic genome-to-genome study of 542 individuals who were chronically infected with HCV, predominantly genotype 3. We show that both alleles of genes encoding human leukocyte antigen molecules and genes encoding components of the interferon lambda innate immune system drive viral polymorphism. Additionally, we show that IFNL4 genotypes determine HCV viral load through a mechanism dependent on a specific amino acid residue in the HCV NS5A protein. These findings highlight the interplay between the innate immune system and the viral genome in HCV control

Bidirectional lipid droplet velocities are controlled by differential binding strengths of HCV Core DII protein

Host cell lipid droplets (LD) are essential in the hepatitis C virus (HCV) life cycle and are targeted by the viral capsid core protein. Core-coated LDs accumulate in the perinuclear region and facilitate viral particle assembly, but it is unclear how mobility of these LDs is directed by core. Herein we used two-photon fluorescence, differential interference contrast imaging, and coherent anti-Stokes Raman scattering microscopies, to reveal novel core-mediated changes to LD dynamics. Expression of core protein’s lipid binding domain II (DII-core) induced slower LD speeds, but did not affect directionality of movement on microtubules. Modulating the LD binding strength of DII-core further impacted LD mobility, revealing the temporal effects of LD-bound DII-core. These results for DII-core coated LDs support a model for core-mediated LD localization that involves core slowing down the rate of movement of LDs until localization at the perinuclear region is accomplished where LD movement ceases. The guided localization of LDs by HCV core protein not only is essential to the viral life cycle but also poses an interesting target for the development of antiviral strategies against HCV

Viral genome wide association study identifies novel hepatitis C virus polymorphisms associated with sofosbuvir treatment failure

Persistent hepatitis C virus (HCV) infection is a major cause of chronic liver disease, worldwide. With the development of direct-acting antivirals, treatment of chronically infected patients has become highly effective, although a subset of patients responds less well to therapy. Sofosbuvir is a common component of current de novo or salvage combination therapies, that targets the HCV NS5B polymerase. We use pre-treatment whole-genome sequences of HCV from 507 patients infected with HCV subtype 3a and treated with sofosbuvir containing regimens to detect viral polymorphisms associated with response to treatment. We find three common polymorphisms in non-targeted HCV NS2 and NS3 proteins are associated with reduced treatment response. These polymorphisms are enriched in post-treatment HCV sequences of patients unresponsive to treatment. They are also associated with lower reductions in viral load in the first week of therapy. Using in vitro short-term dose-response assays, these polymorphisms do not cause any reduction in sofosbuvir potency, suggesting an indirect mechanism of action in decreasing sofosbuvir efficacy. The identification of polymorphisms in NS2 and NS3 proteins associated with poor treatment outcomes emphasises the value of systematic genome-wide analyses of viruses in uncovering clinically relevant polymorphisms that impact treatment

Characterization of hepatitis C RNA-containing particles from human liver by density and size

Hepatitis C virus (HCV) particles found in vivo are heterogeneous in density and size, but their detailed characterization has been restricted by the low titre of HCV in human serum. Previously, our group has found that HCV circulates in blood in association with very-low-density lipoprotein (VLDL). Our aim in this study was to characterize HCV RNA-containing membranes and particles in human liver by both density and size and to identify the subcellular compartment(s) where the association with VLDL occurs. HCV was purified by density using iodixanol gradients and by size using gel filtration. Both positive-strand HCV RNA (present in virus particles) and negative-strand HCV RNA (an intermediate in virus replication) were found with densities below 1.08 g ml−1. Viral structural and non-structural proteins, host proteins ApoB, ApoE and caveolin-2, as well as cholesterol, triglyceride and phospholipids were also detected in these low density fractions. After fractionation by size with Superose gel filtration, HCV RNA and viral proteins co-fractionated with endoplasmic reticulum proteins and VLDL. Fractionation on Toyopearl, which separates particles with diameters up to 200 nm, showed that 78 % of HCV RNA from liver was >100 nm in size, with a positive-/negative-strand ratio of 6 : 1. Also, 8 % of HCV RNA was found in particles with diameters between 40 nm and 70 nm and a positive-/negative-strand ratio of 45 : 1. This HCV was associated with ApoB, ApoE and viral glycoprotein E2, similar to viral particles circulating in serum. Our results indicate that the association between HCV and VLDL occurs in the liver

Site-directed M2 proton channel inhibitors enable synergistic combination therapy for rimantadine-resistant pandemic influenza.

Pandemic influenza A virus (IAV) remains a significant threat to global health. Preparedness relies primarily upon a single class of neuraminidase (NA) targeted antivirals, against which resistance is steadily growing. The M2 proton channel is an alternative clinically proven antiviral target, yet a near-ubiquitous S31N polymorphism in M2 evokes resistance to licensed adamantane drugs. Hence, inhibitors capable of targeting N31 containing M2 (M2-N31) are highly desirable. Rational in silico design and in vitro screens delineated compounds favouring either lumenal or peripheral M2 binding, yielding effective M2-N31 inhibitors in both cases. Hits included adamantanes as well as novel compounds, with some showing low micromolar potency versus pandemic "swine" H1N1 influenza (Eng195) in culture. Interestingly, a published adamantane-based M2-N31 inhibitor rapidly selected a resistant V27A polymorphism (M2-A27/N31), whereas this was not the case for non-adamantane compounds. Nevertheless, combinations of adamantanes and novel compounds achieved synergistic antiviral effects, and the latter synergised with the neuraminidase inhibitor (NAi), Zanamivir. Thus, site-directed drug combinations show potential to rejuvenate M2 as an antiviral target whilst reducing the risk of drug resistance

Search for lepton-flavor violation at HERA

A search for lepton-flavor-violating interactions $e p \to \mu X$ and $e p\to \tau X$ has been performed with the ZEUS detector using the entire HERA I data sample, corresponding to an integrated luminosity of 130 pb^{-1}. The data were taken at center-of-mass energies, $\sqrt{s}$, of 300 and 318 GeV. No evidence of lepton-flavor violation was found, and constraints were derived on leptoquarks (LQs) that could mediate such interactions. For LQ masses below $\sqrt{s}$, limits were set on $\lambda_{eq_1} \sqrt{\beta_{\ell q}}$, where $\lambda_{eq_1}$ is the coupling of the LQ to an electron and a first-generation quark $q_1$, and $\beta_{\ell q}$ is the branching ratio of the LQ to the final-state lepton $\ell$ ($\mu$ or $\tau$) and a quark $q$. For LQ masses much larger than $\sqrt{s}$, limits were set on the four-fermion interaction term $\lambda_{e q_\alpha} \lambda_{\ell q_\beta} / M_{\mathrm{LQ}}^2$ for LQs that couple to an electron and a quark $q_\alpha$ and to a lepton $\ell$ and a quark $q_\beta$, where $\alpha$ and $\beta$ are quark generation indices. Some of the limits are also applicable to lepton-flavor-violating processes mediated by squarks in $R$-Parity-violating supersymmetric models. In some cases, especially when a higher-generation quark is involved and for the process $e p\to \tau X$, the ZEUS limits are the most stringent to date.Comment: 37 pages, 10 figures, Accepted by EPJC. References and 1 figure (Fig. 6) adde

The dependence of dijet production on photon virtuality in ep collisions at HERA

The dependence of dijet production on the virtuality of the exchanged photon, Q^2, has been studied by measuring dijet cross sections in the range 0 < Q^2 < 2000 GeV^2 with the ZEUS detector at HERA using an integrated luminosity of 38.6 pb^-1. Dijet cross sections were measured for jets with transverse energy E_T^jet > 7.5 and 6.5 GeV and pseudorapidities in the photon-proton centre-of-mass frame in the range -3 < eta^jet <0. The variable xg^obs, a measure of the photon momentum entering the hard process, was used to enhance the sensitivity of the measurement to the photon structure. The Q^2 dependence of the ratio of low- to high-xg^obs events was measured. Next-to-leading-order QCD predictions were found to generally underestimate the low-xg^obs contribution relative to that at high xg^obs. Monte Carlo models based on leading-logarithmic parton-showers, using a partonic structure for the photon which falls smoothly with increasing Q^2, provide a qualitative description of the data.Comment: 35 pages, 6 eps figures, submitted to Eur.Phys.J.

Beauty photoproduction measured using decays into muons in dijet events in ep collisions at s\sqrt{s}=318 GeV

The photoproduction of beauty quarks in events with two jets and a muon has been measured with the ZEUS detector at HERA using an integrated luminosity of 110 pb$^{- 1}$. The fraction of jets containing b quarks was extracted from the transverse momentum distribution of the muon relative to the closest jet. Differential cross sections for beauty production as a function of the transverse momentum and pseudorapidity of the muon, of the associated jet and of $x_{\gamma}^{jets}$, the fraction of the photon's momentum participating in the hard process, are compared with MC models and QCD predictions made at next-to-leading order. The latter give a good description of the data.Comment: 32 pages, 6 tables, 7 figures Table 6 and Figure 7 revised September 200