Johannes Löwer

APOBEC3 Proteins Inhibit Human LINE-1 Retrotransposition

The human cytidine deaminase family APOBEC3 represents a novel group of proteins in the field of innate defense mechanisms that has been shown to be active against a variety of retroviruses. Here we examined whether members of the APO-BEC3 family have an impact on retrotransposition of human long interspersed nuclear elements (LINE-1s or L1s). Using a retrotransposition reporter assay in HeLa cells, we demonstrate that in the presence of transiently transfected APOBEC3A, L1 retrotransposition frequency was reduced by up to 85%. Although APOBEC3G and -3H did not influence L1 retrotransposition notably, expression of APOBEC3B, -3C, and -3F inhibited transposition by ∼75%. Although reverse transcription of L1s occurs in the nucleus and APOBEC3 proteins are believed to act via DNA deamination during reverse transcription, activity against L1 retrotransposition was not correlated with nuclear localization of APOBEC3s. We demonstrate that APOBEC3C and APOBEC3B were endogenously expressed in HeLa cells. Accordingly, down-regulation of APOBEC3C by RNA interference enhanced L1 retrotransposition by ∼78%. Sequence analyses of de novo L1 retrotransposition events that occurred in the presence of overexpressed APOBEC3 proteins as well as the analyses of pre-existing endogenous L1 elements did not reveal an enhanced rate of G-to-A transitions, pointing to a mechanism independent of DNA deamination. This study presents evidence for a role of host-encoded APOBEC3 proteins in the regulation of L1 retrotransposition.

The non-autonomous retrotransposon SVA is trans-mobilized by the human LINE-1 protein machinery

SINE-VNTR-Alu (SVA) elements are non-autonomous, hominid-specific non-LTR retrotransposons and distinguished by their organization as composite mobile elements. They represent the evolutionarily youngest, currently active family of human non-LTR retrotransposons, and sporadically generate disease-causing insertions. Since preexisting, genomic SVA sequences are characterized by structural hallmarks of Long Interspersed Elements 1 (LINE-1, L1)-mediated retrotransposition, it has been hypothesized for several years that SVA elements are mobilized by the L1 protein machinery in trans. To test this hypothesis, we developed an SVA retrotransposition reporter assay in cell culture using three different human-specific SVA reporter elements. We demonstrate that SVA elements are mobilized in HeLa cells only in the presence of both L1-encoded proteins, ORF1p and ORF2p. SVA trans-mobilization rates exceeded pseudogene formation frequencies by 12- to 300-fold in HeLa-HA cells, indicating that SVA elements represent a preferred substrate for L1 proteins. Acquisition of an AluSp element increased the trans-mobilization frequency of the SVA reporter element by ~25-fold. Deletion of (CCCTCT)n repeats and Alu-like region of a canonical SVA reporter element caused significant attenuation of the SVA trans-mobilization rate. SVA de novo insertions were predominantly full-length, occurred preferentially in G+C-rich regions, and displayed all features of L1-mediated retrotransposition which are also observed in preexisting genomic SVA insertions.

Parvovirus B19 transmission by heat‐treated clotting factor concentrates

BACKGROUND: Human parvovirus B19 (B19) DNA can be frequently detected in plasma‐derived coagulation factor concentrates. The production of some clotting factor products includes heat treatment steps for virus inactivation, but the effectiveness of such steps for B19 inactivation is unclear. Moreover, detailed transmission case reports including DNA sequence analysis and quantification of B19 DNA from contaminated heat‐treated blood components have not been provided so far. Therefore, the correlation between B19 DNA in blood components and infectivity remains unclear.

A new cluster of hepatitis A infection in hemophiliacs traced to a contaminated plasma pool.

Recently, several clusters of hepatitis A have been observed among hemophiliacs linked to factor VIII concentrates treated for virus inactivation solely with the solvent/detergent (S/D) method, a procedure that does not affect nonenveloped viruses such as the hepatitis A virus (HAV). A new outbreak of hepatitis A in six hemophiliacs treated with the same lot of a factor VIII preparation occurred recently in Germany. The objective of the study was to clarify whether these diseases were caused by the administration of the S/D‐treated plasma product, rather than a community‐acquired infection. Polymerase chain reactions designed to detect HAV nucleic acid have been carried out in the implicated factor VIII lots, in the corresponding plasma pools, and in serum samples of four out of six infected individuals. The nucleic acid sequences were determined in samples that resulted in positive amplification products. HAV sequences were found in one of the two plasma pools used for manufacture of the incriminated product, in the incriminated lot itself, and in all recipient sera tested so far, although the latter were collected up to 7 weeks after the onset of jaundice. The sequences obtained were completely identical, revealing a unique HAV strain of genotype IA. This study provides conclusive evidence that hepatitis A can be transmitted by factor VIII concentrates treated solely by the S/D procedure for virus inactivation. This inactivation method is not effective against nonenveloped viruses. Since a number of hepatitis A transmission episodes have been described with such preparations during the past 10 years, their continued use seems to be questionable unless additional virus removal or inactivation steps are introduced to prevent the transmission of nonenveloped viruses. Molecular approaches again proved to be reliable tools for elucidating the chain of virus transmission. J. Med. Virol. 57:91–99, 1999.

HERV-K : The biologically most active human endogenous retrovirus family

The human genome contains a wide variety of endogenous retrovirus-like sequences. The human endogenous retrovirus type K (HERV-K) family comprises 30-50 members per haploid genome in humans and is highly conserved in Old World monkeys and apes. Some proviruses are displaying open reading frames (ORF) with coding capacity for viral particles. HERV-K sequences most likely code for the previously described human teratocarcinoma-derived virus (HTDV) and correlated expression of HERV-K Gag has been demonstrated by immunoelectron microscopy studies. Protease, but not yet reverse transcriptase (RT), enzymatic activity was demonstrated for recombinant HERV-K proteins. However, an ultrasensitive RT assay revealed specific polymerase activity associated with the HTDV particles. HERV-K transcription is specifically regulated by viral long terminal repeats and RNA is expressed at low steady-state levels in a variety of human tissues and tumours. In teratocarcinoma cell lines, HERV-K is highly expressed in a complex pattern showing full-length as well as subgenomic envelope (env) and two alternatively spliced small transcripts. The doubly spliced 1.8-kb mRNA codes for cORF protein which resembles Rev of HIV-1 and is located in the nucleolus. In addition, the cORF sequence acts as a leader and is essential for effective expression of glycosylated HERV-K Env protein. Although HERV-K sequences code for all necessary retroviral proteins, infectious particles could not yet be demonstrated. The putative implication of HERV sequences in pathophysiological processes, for example, testicular malignancies, remains to be elucidated.

Sensitivity of HCV core antigen and HCV RNA detection in the early infection phase

BACKGROUND : Various countries have introduced HCV NAT to exclude infectious donations collected during the preseroconversion window phase (PWP). For the same purpose, an ELISA has also been developed to detect HCV core antigen (cAg).

Human Teratocarcinomas Cultured in vitro Produce Unique Retrovirus-like Viruses

We have previously reported that among a series of human tumours investigated, only human teratocarcinoma cell lines derived from testicular tumours or pulmonary metastases of patients in Germany and the U.S.A. produced retrovirus-like particles spontaneously, albeit in low amounts. In a recent publication electron microscopical data suggested that the human teratocarcinoma-derived ( HTD ) particles were morphologically closely related, but not identical, to the type C retroviruses of animals. In this communication, the explantation of three human teratocarcinoma cell lines is briefly described. Evidence is presented that HTD particles (i) are synthesized only in a fraction of the epithelioid and differentiating cells; (ii) can be induced biochemically in a manner characteristic of retroviruses; (iii) either are not infectious or possess a peculiar host range; (iv) are immunologically unrelated to animal retrovirus strains; (v) possess an endogenous RNA-dependent DNA polymerase activity that can be banded at 1.16 g/ml in linear sucrose gradients. These results may be taken as suggestive evidence that HTD particles represent a novel group of unique retroviruses.

Recombinant Modified Vaccinia Virus Ankara Expressing the Hemagglutinin Gene Confers Protection against Homologous and Heterologous H5N1 Influenza Virus Infections in Macaques

BACKGROUND Highly pathogenic avian influenza viruses of the H5N1 subtype have been responsible for an increasing number of infections in humans since 2003. More than 60% of infected individuals die, and new infections are reported frequently. In light of the pandemic threat caused by these events, the rapid availability of safe and effective vaccines is desirable. Modified vaccinia virus Ankara (MVA) expressing the hemagglutinin (HA) gene of H5N1 viruses is a promising candidate vaccine that induced protective immunity against infection with homologous and heterologous H5N1 influenza virus in mice. METHODS In the present study, we evaluated a recombinant MVA vector expressing the HA gene of H5N1 influenza virus A/Vietnam/1194/04 (MVA-HA-VN/04) in nonhuman primates. Cynomolgus macaques were immunized twice and then were challenged with influenza virus A/Vietnam/1194/04 (clade 1) or A/Indonesia/5/05 (clade 2.1) to assess the level of protective immunity. RESULTS Immunization with MVA-HA-VN/04 induced (cross-reactive) antibodies and prevented virus replication in the upper and lower respiratory tract and the development of severe necrotizing bronchointerstitial pneumonia. CONCLUSION Therefore, MVA-HA-VN/04 is a promising vaccine candidate for the induction of protective immunity against highly pathogenic H5N1 avian influenza viruses in humans.

Serological response to human endogenous retrovirus K in melanoma patients correlates with survival probability.

A few years ago, reactivation of human endogenous retrovirus K (HERV-K) proviruses in melanoma was described. The expression of HERV-K proteins induces humoral immune responses. The aim of the present study was to elucidate the prognostic relevance of serological anti-HERV-K reactivity in melanoma patients. In a retrospective study, anti-HERV-K Gag and Env antibodies were detected in 51 of the 312 randomly selected and blinded sera from melanoma patients, but not in any of the 70 sera from healthy controls. Comparing serological HERV-K reactivity with established melanoma markers revealed a significant correlation (p = 0.018, Chi-square test) with the stage of disease classified according to the American Joint Committee on Cancer (AJCC). Anti-HERV-K reactivity was elevated in patients with acrolentiginous/mucosal/uveal melanoma (tumor subtypes developing at sun-protected sites) compared to patients with lentigo/nodular/superficial spreading melanoma (p = 0.011, Chi-square test). Patients with anti-HERV-K antibodies had a significantly decreased disease-specific overall survival (stage I-IV, p < 0.001; stage I-III, p = 0.005, log-rank test). Significantly, multivariate Cox regression analysis including prognostic markers in clinical use (e.g., AJCC stage, T-class, serum level of S100-beta) revealed serological HERV-K reactivity as an independent marker of reduced survival probability (p = 0.027) in melanoma patients with the early stages of the disease (AJCC I-III). This is the first report that the humoral anti-HERV-K immune response may provide additional prognostic information to that of established melanoma markers.

Parvovirus B19 DNA in plasma pools and plasma derivatives.

Human parvovirus B19 (B19) has been transmitted by various plasma‐derived medicinal products. The aim of this study was to determine the frequency and the level of B19 DNA contamination in plasma pools destined for fractionation and in a broad range of plasma derivatives. In addition, removal of B19 DNA by the manufacturing process was investigated in cases where corresponding samples from plasma pool and product were available.