- Volume 95, Issue 4, 2014

The catalytic subunit of the herpes simplex virus 1 DNA polymerase (HSV-1 Pol) is essential for viral DNA synthesis and production of infectious virus in cell culture. While mutations that affect 5′–3′ polymerase activity have been evaluated in animal models of HSV-1 infection, mutations that affect other functions of HSV-1 Pol have not. In a previous report, we utilized bacterial artificial chromosome technology to generate defined HSV-1 pol mutants with lesions in the previously uncharacterized pre-NH2-terminal domain. We found that the extreme N-terminal 42 residues (deletion mutant polΔN43) were dispensable for replication in cell culture, while residues 44–49 (alanine-substitution mutant polA6) were required for efficient viral DNA synthesis and production of infectious virus. In this study, we sought to address the importance of these conserved elements in viral replication in a mouse corneal infection model. Mutant virus polΔN43 exhibited no meaningful defect in acute or latent infection despite strong conservation of residues 1–42 with HSV-2 Pol. The polA6 mutation caused a modest defect in replication at the site of inoculation, and was severely impaired for ganglionic replication, even at high inocula that permitted efficient corneal replication. Additionally, the polA6 mutation resulted in reduced latency establishment and subsequent reactivation. Moreover, we found that the polA6 replication defect in cultured cells was exacerbated in resting cells as compared to dividing cells. These results reveal an important role for the conserved motif at residues 44–49 of HSV-1 Pol for ganglionic viral replication.

Pigs can be severely harmed by influenza, and represent important reservoir hosts, in which new human pathogens such as the recent pandemic swine-origin H1N1 influenza A virus can arise by mutation and reassortment of genome segments. To obtain novel, safe influenza vaccines for pigs, and to investigate the antigen-specific immune response, we modified an established live-virus vaccine against Aujeszky’s disease of swine, pseudorabies virus (PrV) strain Bartha (PrV-Ba), to serve as vector for the expression of haemagglutinin (HA) of swine-origin H1N1 virus. To facilitate transgene insertion, the genome of PrV-Ba was cloned as a bacterial artificial chromosome. HA expression occurred under control of the human or murine cytomegalovirus immediate early promoters (P-HCMV, P-MCMV), but could be substantially enhanced by synthetic introns and adaptation of the codon usage to that of PrV. However, despite abundant expression, the heterologous glycoprotein was not detectably incorporated into mature PrV particles. Replication of HA-expressing PrV in cell culture was only slightly affected compared to that of the parental virus strain. A single immunization of pigs with the PrV vector expressing the codon-optimized HA gene under control of P-MCMV induced high levels of HA-specific antibodies. The vaccinated animals were protected from clinical signs after challenge with a related swine-origin H1N1 influenza A virus, and challenge virus shedding was significantly reduced.

Trim 5α was the first member of the tripartite motif (TRIM) family of proteins that was identified to potently restrict human immunodeficiency virus type 1 (HIV-1) replication. The breadth of antiretroviral activity of TRIM family members is an active area of investigation. In this study, we demonstrate that human Trim 37 possesses anti-HIV-1 activity. This antiretroviral activity and the manner in which it was displayed were implicated by (1) decreased viral replication upon Trim 37 transient overexpression in virus-producing cells, (2) correlation of the reduction of viral infectivity with Trim 37 virion incorporation, (3) increased HIV-1 replication during siRNA depletion of Trim 37 expression, and (4) reduction in viral DNA synthesis upon Trim 37 transient overexpression. Our findings provide the first demonstration, to our knowledge, of the potent antiviral activity of human Trim 37, and implicate an antiviral mechanism whereby Trim 37 interferes with viral DNA synthesis.

HIV-1 transcription depends on cellular transcription factors that bind to sequences in the long-terminal repeat (LTR) promoter. Each HIV-1 subtype has a specific LTR promoter configuration, and minor sequence changes in transcription factor binding sites (TFBSs) or their arrangement can influence transcriptional activity, virus replication and latency properties. Previously, we investigated the proviral latency properties of different HIV-1 subtypes in the SupT1 T cell line. Here, subtype-specific latency and replication properties were studied in primary PHA-activated T lymphocytes. No major differences in latency and replication capacity were measured among the HIV-1 subtypes. Subtype B and AE LTRs were studied in more detail with regard to a putative AP-1 binding site using luciferase reporter constructs. c-Jun, a member of the AP-1 transcription factor family, can activate both subtype B and AE LTRs, but the latter showed a stronger response, reflecting a closer match with the consensus AP-1 binding site. c-Jun overexpression enhanced Tat-mediated transcription of the viral LTR, but in the absence of Tat inhibited basal promoter activity. Thus, c-Jun can exert a positive or negative effect via the AP-1 binding site in the HIV-1 LTR promoter, depending on the presence or absence of Tat.