Herpes Simplex Virus Ribonucleotide Reductase DNA and Requirements for mRNA 3' End Formation

This study was undertaken to examine the organisation and structures of mRNAs mapping at the herpes simplex virus type 2 (HSV-2) ribonucleotide reductase locus. From comparisons between equivalent HSV-2 and HSV-1 nucleotide sequences, putative transcription control signals were identified, and functional analysis of certain control elements was performed. The positions of translated regions within the mRNAs encoding ribonucleotide reductase were identified as were conserved amino acid domains between viral and cellular reductases. During the course of these comparisons, a conserved DNA sequence, YGTGTTYY (Y = pyrimidine), located 30nuc downstream from the polyadenylation (AATAAA) signal of both viral and cellular genes, was identified. Functional tests showed that the YGTGTTYY signal was required for efficient formation of mRNA 3' termini. Arrangement of mRNAs and DNA Sequence Comparisons at the HSV-2 Ribonucleotide Reductase Locus. Four overlapping mRNAs have been identified at the ribonucleotide reductase locus. These mRNAs are unspliced and share common 5' and 3' termini. Two early mRNAs with sizes of 4.5kb and 1.2kb have a common 3' terminus and encode HSV-2 polypeptides Vmw138 and Vmw38 respectively which are almost certainly components of the viral ribonucleotide reductase. The other two mRNAs are late, 5' co-terminal species with sizes of 6.4kb and 1.7kb which appear to encode an identical 54,000 mol. wt. protein; the 6.4kb transcript is 3' co-terminal with the early mRNAs. As a consequence of this mRNA arrangement, the 5' termini of the 4.5kb and 1.2kb mRNAs and the 3' terminus of the 1.7kb mRNA are located within larger, overlapping transcripts. The genome region adjacent to the 6.4kb and 1.7kb mRNAs specifies three late transcripts which are synthesised in the opposite orientation; the 5' terminal region of one of these species overlaps with the 6.4kb and 1.7kb mRNAs. The 3' terminus of the late 1.7kb mRNA is located within the transcribed region of the 6. 4kb species. Previously, it has been proposed that read-through of HSV poly A sites is due to inefficient processing at certain 3' termini. The ability of sequences flanking the 1.7kb mRNA 3' terminus to produce functional mRNA was tested using a plasmid containing the bacterial chloramphenicol acetyltransferase (CAT) protein coding sequences fused to an HSV-2 immediate early promoter. CAT activities produced in HeLa cells by a plasmid carrying the 1.7kb mRNA 3' terminal sequences were comparable to activities obtained with a plasmid containing the 3' processing signals from a non-internal poly A site. Read-through of HSV poly A sites may therefore reflect a general reduction in mRNA 3' processing efficiency rather than differences in the 3' processing signals of individual genes. The HSV-1 transcripts equivalent to the 4.5kb and 1.2kb mRNAs have sizes of 5. 0kb and 1. 2kb and encode HSV-1 polypeptides Vmw136 and Vmw38 respectively. Nucleotide sequence comparisons between the 5' flanking regions of these equivalent HSV-2 and HSV-1 mRNAs have revealed the following homologies: 1) upstream from the 4.5kb and 5.0kb mRNA 5' termini, blocks of conserved sequences are present which resemble transcription control signals at the promoter regions of other genes; TATA box homologues, C-rich tracts and A+C-rich elements have been identified. (Abstract shortened by ProQuest.)

Inhibition of hepatitis C virus RNA replication by ISG15 does not require its conjugation to protein substrates by the HERC5 E3 ligase

Chronic infection of the liver by hepatitis C virus (HCV) induces a range of host factors including IFN-stimulated genes such as ISG15. ISG15 functions as an antiviral factor that limits virus replication. Previous studies have suggested that ISG15 could influence HCV replication in both a positive and a negative manner. In this report, we determined the effect of ISG15 on HCV RNA replication in two independent cell lines that support viral genome synthesis by inhibiting ISG15 expression through small interfering RNA, short-hairpin RNA and CRISPR/Cas9 gene knockout approaches. Our results demonstrated that ISG15 impairs HCV RNA replication in both the presence and absence of IFN stimulation, consistent with an antiviral role for ISG15 during HCV infection. ISG15 conjugation to protein substrates typically requires the E3 ligase, HERC5. Our results showed that the inhibitory effect of ISG15 on HCV RNA replication does not require its conjugation to substrates by HERC5

Australian postharvest technologies for fresh fruits and vegetables

General postharvest handling systems and technologies used in Australia for broccoli and other fruits and vegetables from harvest to wholesale market are presented to contrast with those in China. With production areas usually a considerable distance (e.g. 100 to 3500 km) from the major markets in the capital cities, appropriate systems and technologies are needed to minimise losses and maintain product integrity and quality while ensuring food safety. Aspects covered include field harvest (harvest aids, packing), packing houses (handling, grading, sorting, packing, cooling, pallet/unitisation), storage and transport (refrigeration, mixed loads), and wholesale markets

Comparative host genomics: how has human evolution affected our immune defence against hepatitis C virus?

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Modulation of triglyceride and cholesterol ester synthesis impairs assembly of infectious hepatitis C virus

In hepatitis C virus infection, replication of the viral genome and virion assembly are linked to cellular metabolic processes. In particular, lipid droplets, which store principally triacylglycerides (TAGs) and cholesterol esters (CEs), have been implicated in production of infectious virus. Here, we examine the effect on productive infection of triacsin C and YIC-C8-434, which inhibit synthesis of TAGs and CEs by targeting long-chain acyl-CoA synthetase and acyl-CoA:cholesterol acyltransferase, respectively. Our results present high resolution data on the acylglycerol and cholesterol ester species that were affected by the compounds. Moreover, triacsin C, which blocks both triglyceride and cholesterol ester synthesis, cleared most of the lipid droplets in cells. By contrast, YIC-C8-434, which only abrogates production of cholesterol esters, induced an increase in size of droplets. Although both compounds slightly reduced viral RNA synthesis, they significantly impaired assembly of infectious virions in infected cells. In the case of triacsin C, reduced stability of the viral core protein, which forms the virion nucleocapsid and is targeted to the surface of lipid droplets, correlated with lower virion assembly. In addition, the virus particles that were released from cells had reduced specific infectivity. YIC-C8-434 did not alter the association of core with lipid droplets but appeared to decrease production of infectious virus particles, suggesting a block in virion assembly. Thus, the compounds have antiviral properties, indicating that targeting synthesis of lipids stored in lipid droplets might be an option for therapeutic intervention in treating chronic hepatitis C virus infection

Hepatitis C virus NS5A targets the nucleosome assembly protein NAP1L1 to control the innate cellular response

Hepatitis C virus (HCV) is a single-stranded positive-sense RNA hepatotropic virus. Despite cellular defenses, HCV is able to replicate in hepatocytes and to establish a chronic infection that could lead to severe complications and hepatocellular carcinoma. An important player in subverting the host response to HCV infection is the viral non-structural protein NS5A that, in addition to its role in replication and assembly, targets several pathways involved in the cellular response to viral infection. Several unbiased screens identified the nucleosome-assembly protein 1-like 1 (NAP1L1) as an interaction partner of HCV NS5A. Here we confirm this interaction and map it to the C-terminus of NS5A of both genotype 1 and 2. NS5A sequesters NAP1L1 in the cytoplasm blocking its nuclear translocation. However, only NS5A from genotype 2 HCV, but not from genotype 1, targets NAP1L1 for proteosomal-mediated degradation. NAP1L1 is a nuclear chaperone involved in chromatin remodeling and we demonstrate the NAP1L1-dependent regulation of specific pathways involved in cellular responses to viral infection and cell survival. Among those we show that lack of NAP1L1 leads to a decrease of RELA protein levels and a strong defect of IRF3 TBK1/IKKϵ-mediated phosphorylation leading to inefficient RIG-I and TLR3 responses. Hence, HCV is able to modulate the host cell environment by targeting NAP1L1 through NS5A

Non cell autonomous upregulation of CDKN2 transcription linked to progression of chronic hepatitis C disease

Chronic hepatitis C virus infection (C-HC) is associated with higher mortality arising from hepatic and extrahepatic disease. This may be due to accelerated biological aging; however, studies in C-HC have thus far been based solely on telomere length as a biomarker of aging (BoA). In this study, we have evaluated CDKN2 locus transcripts as alternative BoAs in C-HC. Our results suggest that C-HC induces non-cell-autonomous senescence and accelerates biological aging. The CDKN2 locus may provide a link between C-HC and increased susceptibility to age-associated diseases and provides novel biomarkers for assessing its impact on aging processes in man

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

An interferon lambda 4-associated variant in the hepatitis C virus RNA polymerase affects viral replication in infected cells

Host IFNL4 haplotype status contributes to the development of chronic hepatitis C virus infection in individuals who are acutely infected with the virus. In silico studies revealed that specific amino acid variants at multiple sites on the HCV polyprotein correlate with functional single nucleotide polymorphisms (SNPs) in the IFNL4 locus. Thus, SNPs at the IFNL4 locus may select variants that influence virus replication and thereby outcome of infection. Here, we examine the most significantly IFNL4-associated amino acid variants that lie in the ‘Lambda (L) 2 loop’ of the HCV NS5B RNA polymerase. L2 loop variants were introduced into both sub-genomic replicon and full-length infectious clones of HCV and viral replication examined in the presence and absence of exogenous IFNλ4. Our data demonstrate that while mutation of NS5B L2 loop affects replication, individual IFNL4-associated variants have modest but consistent effects on replication both in the presence and absence of IFNλ4. Given the strong genetic association between these variants and IFNL4, these data suggest a nuanced effect of each individual position on viral replication, the combined effect of which might mediate resistance to the effects of IFNλ4

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