Pocket Protein p130/Rb2 Is Required for Efficient Herpes Simplex Virus Type 1 Gene Expression and Viral Replication

We have reported previously that herpes simplex virus type 1 (HSV-1) infection disrupts normal progression of the mammalian cell cycle, causing cells to enter a G(1)-like state. Infected cells were characterized by a decline in cyclin-dependent kinase 2 (CDK2) activities, loss of hyperphosphorylated retinoblastoma protein (pRb), accumulation of E2F-pocket protein complexes, and failure to initiate cellular DNA replication. In the present study, we investigated the role of the pocket proteins pRb, p107, and p130 in HSV-1-dependent cell cycle inhibition and cyclin kinase regulation by infecting murine 3T3 cells derived from wild-type (WT) mouse embryos or embryos with deletions of pRb (pRb(-/-)), p107 (p107(-/-)), p130 (p130(-/-)), or both p130 and p107 (p130(-/-)/p107(-/-)). With respect to CDK2 inhibition, viral protein accumulation, viral DNA replication, and progeny virus yield, WT, pRb(-/-), and p107(-/-) cells were essentially identical. In contrast, after infection of p130(-/-) cells, we observed no inhibition of CDK2 activity, a 5- to 6-h delay in accumulation of viral proteins, an impaired ability to form viral DNA replication compartments, and reduced viral DNA synthesis. As a result, progeny virus yield was reduced 2 logs compared to that in WT cells. Notably, p130(-/-)/p107(-/-) double-knockout cells had a virus replication phenotype intermediate between those of the p107(-/-) and p130(-/-) cells. We conclude from these studies that p130 is a key factor in regulating aspects of cell cycle progression, as well as the timely expression of viral genes and replication of viral DNA.     

EWSR1 Binds the Hepatitis C Virus cis-Acting Replication Element and Is Required for Efficient Viral Replication

The hepatitis C virus (HCV) genome contains numerous RNA elements that are required for its replication. Most of the identified RNA structures are located within the 5′ and 3′ untranslated regions (UTRs). One prominent RNA structure, termed the cis-acting replication element (CRE), is located within the NS5B coding region. Mutation of part of the CRE, the 5BSL3.2 stem-loop, impairs HCV RNA replication. This loop has been implicated in a kissing interaction with a complementary stem-loop structure in the 3′ UTR. Although it is clear that this interaction is required for viral replication, the function of the interaction, and its regulation are unknown. In order to gain insight into the CRE function, we isolated cellular proteins that preferentially bind the CRE and identified them using mass spectrometry. This approach identified EWSR1 as a CRE-binding protein. Silencing EWSR1 expression impairs HCV replication and infectious virus production but not translation. While EWRS1 is a shuttling protein that is extensively nuclear in hepatocytes, substantial amounts of EWSR1 localize to the cytosol in HCV-infected cells and colocalize with sites of HCV replication. A subset of EWRS1 translocates into detergent-resistant membrane fractions, which contain the viral replicase proteins, in cells with replicating HCV. EWSR1 directly binds the CRE, and this is dependent on the intact CRE structure. Finally, EWSR1 preferentially interacts with the CRE in the absence of the kissing interaction. This study implicates EWSR1 as a novel modulator of CRE function in HCV replication.     

The HEX1 Gene of Fusarium graminearum Is Required for Fungal Asexual Reproduction and Pathogenesis and for Efficient Viral RNA Accumulation of Fusarium graminearum Virus 1

The accumulation of viral RNA depends on many host cellular factors. The hexagonal peroxisome (Hex1) protein is a fungal protein that is highly expressed when the DK21 strain of Fusarium graminearum virus 1 (FgV1) infects its host, and Hex1 affects the accumulation of FgV1 RNA. The Hex1 protein is the major constituent of the Woronin body (WB), which is a peroxisome-derived electron-dense core organelle that seals the septal pore in response to hyphal wounding. To clarify the role of Hex1 and the WB in the relationship between FgV1 and Fusarium graminearum, we generated targeted gene deletion and overexpression mutants. Although neither HEX1 gene deletion nor overexpression substantially affected vegetative growth, both changes reduced the production of asexual spores and reduced virulence on wheat spikelets in the absence of FgV1 infection. However, the vegetative growth of deletion and overexpression mutants was increased and decreased, respectively, upon FgV1 infection compared to that of an FgV1-infected wild-type isolate. Viral RNA accumulation was significantly decreased in deletion mutants but was significantly increased in overexpression mutants compared to the viral RNA accumulation in the virus-infected wild-type control. Overall, these data indicate that the HEX1 gene plays a direct role in the asexual reproduction and virulence of F. graminearum and facilitates viral RNA accumulation in the FgV1-infected host fungus.     

The Varicella-Zoster Virus ORFS/L (ORF0) Gene Is Required for Efficient Viral Replication and Contains an Element Involved in DNA Cleavage

The genome of varicella-zoster virus (VZV), a human alphaherpesvirus, consists of two unique regions, unique long (U_L) and unique short (U_S), each of which is flanked by inverted repeats. During replication, four isomers of the viral DNA are generated which are distinguished by the relative orientations of U_L and U_S. VZV virions predominantly package two isomeric forms of the genome that have a fixed orientation of U_L. An open reading frame (ORF) of unknown function, ORFS/L, also referred to as ORF0, is located at the extreme terminus of U_L, directly adjacent to the a-like sequences, which are known to be involved in cleavage and packaging of viral DNA. We demonstrate here that the ORFS/L protein localizes to the Golgi network in infected and transfected cells. Furthermore, we were able to demonstrate that deletion of the predicted ORFS/L gene is lethal, while retention of the N-terminal 28 amino acid residues resulted in viable yet replication-impaired virus. The growth defect was only partially attributable to the expression of the ORFS/L product, suggesting that the 5′ region of ORFS/L contains a sequence element crucial for cleavage/packaging of viral DNA. Consequently, mutations introduced into the extreme 5′ terminus of ORFS/L resulted in a defect in DNA cleavage, indicating that the region is indeed involved in the processing of viral DNA. Since the sequence element has no counterpart at the other end of U_L, we concluded that our results can provide an explanation for the almost exclusive orientation of the U_L seen in packaged VZV DNA.Varicella-zoster virus ([VZV] Human Herpesvirus 3), is a highly cell-associated alphaherpesvirus that causes chicken pox (varicella) upon infection of naïve individuals (2). During primary infection, VZV is able to establish latency in cranial nerves, as well as dorsal root and autonomic ganglia, where it remains dormant until a reactivation event occurs (11). Reactivation of VZV occurs primarily in elderly or immunocompromised individuals and results in the development of shingles (herpes zoster), which is often associated with severe pain and postherpetic neuralgia (1).The VZV genome, the smallest among the human herpesviruses, is approximately 125 kbp in size and encodes at least 70 unique open reading frames (ORFs) (1). As has been reported for all alphaherpesviruses, the VZV genome consists of two unique regions, unique long (U_L) and unique short (U_S), each flanked by inverted repeat regions (TR_L, IR_L, TR_S, and IR_S) (9). In contrast to herpes simplex virus type 1 (HSV-1), the prototype alphaherpesvirus, VZV contains only very short repeats (88 bp) on either end of U_L, characteristic of members of the Varicellovirus genus (6). During alphaherpesvirus replication, four isomers of viral DNA are generated which can be distinguished by the orientation of U_L and U_S relative to each other. While all four possible isomers of HSV-1 DNA are packaged in virions as equimolar populations, virions produced by VZV and other varicelloviruses, such as equine herpesvirus type 1 (EHV-1), contain predominantly only two of the four possible isomeric forms of the genome (6, 10, 12, 15, 23). It was shown by Southern blot analysis of VZV virion DNA that inversion of the U_L region is rare and occurs in only approximately 5% of cases (6), which also may be attributed to a rare circular configuration of the genome within the virion (14). A previous report on EHV-1 suggested that inversion of the U_L region in infected cells is common but that packaging occurs in a directional manner (23). For both VZV and EHV-1, the reason for the more-or-less exclusive orientation of U_L within the virion still remains unknown.The organization of the VZV genome is similar to that of HSV-1, and over 90% of the VZV ORFs have counterparts in the HSV-1 genome (1, 13). One of the genes with a predicted HSV-1 homologue is ORFS/L, also referred to as ORF0. ORFS/L is predicted to encode a tail-anchored 157-amino-acid (aa) residue type 2 transmembrane protein and was discovered by Kemble and coworkers (13). The gene is located at the very beginning of U_L, directly adjacent to the a-like sequences that contain PacI and PacII sites crucial for the cleavage and packaging of concatameric VZV DNA (Fig. ​(Fig.1)1) (13, 20). Although no function has yet been attributed to the ORFS/L (ORF0) gene or its product, bioinformatic analysis of the VZV genome indicated that it represents a homologue of HSV-1 UL56 (RefSeq accession no. {"type":"entrez-nucleotide","attrs":{"text":"NC_001348","term_id":"9625875","term_text":"NC_001348"}}NC_001348) (7, 8). While UL56 is dispensable for HSV-1 replication in vitro, it plays an important role in pathogenicity in vivo (3, 21). Little is known about the molecular mechanism of UL56 function in the case of HSV-1, but UL56 orthologues are specified by most members of the Alphaherpesvirinae subfamily (26). It was shown that the HSV-2 UL56 product localizes to the Golgi network and interacts with KIF1A, a kinesin motor protein, suggesting a role in vesicular trafficking (16, 17).Open in a separate windowFIG. 1.Overview of the VZV ORFS/L genomic region and the mutants generated. (A) Schematic representation of the VZV genome with a focus on the terminal region containing ORFS/L. Scale bars provide an accurate measure of the genome and the expanded region. (B) Overview of the mutants generated with mutations in the ORFS/L region. A cross indicates the deletion of the corresponding region. Black arrows indicate the loci of stop codon or HA tag insertion.A previous study of Kemble and coworkers also addressed the localization of the ORFS/L protein using a rabbit polyclonal antibody. It was reported that the ORFS/L product was found exclusively in the cytoplasm, which is contradictory to the findings for the HSV-2 orthologue and also to the localization of the ORFS/L protein based on in silico predictions from the primary sequence (13). ORFS/L of the P-Oka strain was recently shown to be unglycosylated but present in the virion (18). Furthermore, ORFS/L expression was detected in skin lesions of individuals, as well as neurons of dorsal root ganglia, during virus reactivation (13). In addition, the deletion of aa 29 to aa 157 of ORFS/L was shown to have an effect on viral replication in vitro and in vivo in the SCID-hu mouse model with thymus-liver implants. In this study, a virus-encoded luciferase reporter system was used to evaluate the growth properties of several bacterial artificial chromosome (BAC)-derived VZV mutants (28). However, it has remained unknown whether the observed growth defect is dependent on ORFS/L gene function or is due to the deletion of another critical sequence element.In this study, we sought to perform a systematic analysis of ORFS/L sequences. We were able to demonstrate that the ORFS/L protein localizes to the Golgi network in infected and transfected cells, providing further evidence for its predicted structure as a tail-anchored type 2 transmembrane protein and lending further support to the notion that it is the orthologue of HSV UL56. In addition, we showed that the ORFS/L gene product is important for efficient VZV replication in vitro. However, we also identified a 5′ region of the predicted ORFS/L that is essential for replication and plays a role in cleavage of viral DNA, as previously suggested by Davison and colleagues (6, 7). Since this essential region is not present at the opposite end of U_L, it could provide an explanation for the almost exclusive packaging in VZV virions of two viral DNA isomers with an invariable U_L orientation.     

The Pre-NH2-Terminal Domain of the Herpes Simplex Virus 1 DNA Polymerase Catalytic Subunit Is Required for Efficient Viral Replication

The catalytic subunit of herpes simplex virus 1 DNA polymerase (HSV-1 Pol) has been extensively studied; however, its full complement of functional domains has yet to be characterized. A crystal structure has revealed a previously uncharacterized pre-NH₂-terminal domain (residues 1 to 140) within HSV-1 Pol. Due to the conservation of the pre-NH₂-terminal domain within the herpesvirus Pol family and its location in the crystal structure, we hypothesized that this domain provides an important function during viral replication in the infected cell distinct from 5′-3′ polymerase activity. We identified three pre-NH₂-terminal Pol mutants that exhibited 5′-3′ polymerase activity indistinguishable from that of wild-type Pol in vitro: deletion mutants PolΔN43 and PolΔN52 that lack the extreme N-terminal 42 and 51 residues, respectively, and mutant PolA₆, in which a conserved motif at residues 44 to 49 was replaced with alanines. We constructed the corresponding pol mutant viruses and found that the polΔN43 mutant displayed replication kinetics similar to those of wild-type virus, while polΔN52 and polA₆ mutant virus infection resulted in an 8-fold defect in viral yield compared to that achieved with wild type and their respective rescued derivative viruses. Additionally, both polΔN52 and polA₆ viruses exhibited defects in viral DNA synthesis that correlated with the observed reduction in viral yield. These results strongly indicate that the conserved motif within the pre-NH₂-terminal domain is important for viral DNA synthesis and production of infectious virus and indicate a functional role for this domain.