Developing a next level integrated genomic surveillance: Advances in the molecular epidemiology of HIV in Germany.

Advances in the molecular epidemiological studies of the Human Immunodeficiency Virus (HIV) at the Robert Koch Institute (RKI) by laboratory and bioinformatic automation should allow the processing of larger numbers of samples and more comprehensive and faster data analysis in order to provide a higher resolution of the current HIV infection situation in near real-time and a better understanding of the dynamic of the German HIV epidemic. The early detection of the emergence and transmission of new HIV variants is important for the adaption of diagnostics and treatment guidelines. Likewise, the molecular epidemiological detection and characterization of spatially limited HIV outbreaks or rapidly growing sub-epidemics is of great importance in order to interrupt the transmission pathways by regionally adapting prevention strategies. These aims are becoming even more important in the context of the SARS-CoV2 pandemic and the Ukrainian refugee movement, which both have effects on the German HIV epidemic that should be monitored to identify starting points for targeted public health measures in a timely manner. To this end, a next level integrated genomic surveillance of HIV is to be established.

Transmitted drug resistance and subtype patterns of viruses from reported new HIV diagnoses in Germany, 2017-2020.

BACKGROUND: The transmission of resistant HIV variants jeopardizes the effective use of antiretrovirals for therapy and prophylaxis. Molecular surveillance of new HIV diagnoses with a focus on prevalence and type of resistance associated mutations and the subtype of circulating viruses is mandatory. METHOD: From 2017 to 2020, 11,527 new HIV diagnoses were reported in Germany to the Robert Koch Institute (RKI). Protease (PR) and reverse-transcriptase (RT) sequences were obtained from 4559 (39.6%) cases, and PR, RT and integrase (IN) sequences were obtained from 3097 (26.9%) cases. The sequences were analyzed with data from the national HIV reports. RESULTS: Among all cases in the analysis, the proportion of primary resistance was 4.3% for nucleoside reverse-transcriptase inhibitors (NRTIs), 9.2% for non-NRTI (NNRTIs), 3.3% for protease inhibitors (PIs) and 1.4% for integrase inhibitors (INIs). Dual-class resistance was highest for NRTIs/NNRTIs with 1.2%. There was no trend in the proportion of viruses resistant to drug classes. Most individual key mutations associated with relevant resistance had a prevalence below 1% including K65R (0.1%) and M184V (0.6%). A notable exception was K103NS, with a prevalence of 2.9% and a significant increase (pTrend=0.024) during 2017-2020. In this period, diagnoses of infections with HIV-1 subtype B were the most common at 58.7%, but its prevalence was declining (pTrend=0.049) while the frequency of minority subtypes (each < 1%) increased (pTrend=0.007). Subtype B was highest (75.6%) in men who have sex with men (MSM) and lowest in reported heterosexual transmissions (HETs, 22.6%). CONCLUSION: The percentage of primary resistance was high but at a stable level. A genotypic determination of resistance is therefore still required before the start of therapy. The subtype diversity of circulating HIV-1 is increasing.

The impact of regional socioeconomic deprivation on the timing of HIV diagnosis: a cross-sectional study in Germany.

BACKGROUND: HIV infections which are diagnosed at advanced stages are associated with significantly poorer health outcomes. In Germany, the proportion of persons living with HIV who are diagnosed at later stages has remained continuously high. This study examined the impact of regional socioeconomic deprivation on the timing of HIV diagnosis. METHODS: We used data from the national statutory notification of newly diagnosed HIV infections between 2011 and 2018 with further information on the timing of diagnosis determined by the BED-Capture-ELISA test (BED-CEIA) and diagnosing physicians. Data on regional socioeconomic deprivation were derived from the German Index of Socioeconomic Deprivation (GISD). Outcome measures were a non-recent infection based on the BED-CEIA result or an infection at the stage of AIDS. The effect of socioeconomic deprivation on the timing of diagnosis was analysed using multivariable Poisson regression models with cluster-robust error variance. RESULTS: Overall, 67.5% (n = 10,810) of the persons were diagnosed with a non-recent infection and 15.2% (n = 2746) with AIDS. The proportions were higher among persons with heterosexual contact compared to men who have sex with men (MSM) (76.8% non-recent and 14.9% AIDS vs. 61.7% non-recent and 11.4% AIDS). MSM living in highly deprived regions in the countryside (< 100 k residents) were more likely to have a non-recent infection (aPR: 1.16, 95% CI: 1.05-1.28) as well as AIDS (aPR: 1.41, 95% CI: 1.08-1.85) at the time of diagnosis compared to MSM in less deprived regions in the countryside. No differences were observed among MSM from towns (100 k ≤ 1 million residents) or major cities (≥ 1 million residents), and no differences overall in the heterosexual transmission group. CONCLUSIONS: An effect of socioeconomic deprivation on the timing of HIV diagnosis was found only in MSM from countryside regions. We suggest that efforts in promoting HIV awareness and regular HIV testing are increased for heterosexual persons irrespective of socioeconomic background, and for MSM with a focus on those living in deprived regions in the countryside.

Reconstruction of the Genetic History and the Current Spread of HIV-1 Subtype A in Germany.

HIV-1 non-B infections have been increasing in Europe for several years. In Germany, subtype A belongs to the most abundant non-B subtypes showing an increasing prevalence of 8.3% among new infections in 2016. Here we trace the origin and examine the current spread of the German HIV-1 subtype A epidemic. Bayesian coalescence and birth-death analyses were performed with 180 German HIV-1 pol sequences and 528 related and publicly available sequences to reconstruct the population dynamics and fluctuations for each of the transmission groups. Our reconstructions indicate two distinct sources of the German subtype A epidemic, with an Eastern European and an Eastern African lineage both cocirculating in the country. A total of 13 German-origin clusters were identified; among these, 6 clusters showed recent activity. Introductions leading to further countrywide spread originated predominantly from Eastern Africa when introduced before 2005. Since 2005, however, spreading introductions have occurred exclusively within the Eastern European clade. Moreover, we observed changes in the main route of subtype A transmission. The beginning of the German epidemic (1985 to 1995) was dominated by heterosexual transmission of the Eastern African lineage. Since 2005, transmissions among German men who have sex with men (MSM) have been increasing and have been associated with the Eastern European lineage. Infections among people who inject drugs dominated between 1998 and 2005. Our findings on HIV-1 subtype A infections provide new insights into the spread of this virus and extend the understanding of the HIV epidemic in Germany.IMPORTANCE HIV-1 subtype A is the second most prevalent subtype worldwide, with a high prevalence in Eastern Africa and Eastern Europe. However, an increase of non-B infections, including subtype A infections, has been observed in Germany and other European countries. There has simultaneously been an increased flow of refugees into Europe and especially into Germany, raising the question of whether the surge in non-B infections resulted from this increased immigration or whether German transmission chains are mainly involved. This study is the first comprehensive subtype A study from a western European country analyzing in detail its phylogenetic origin, the impact of various transmission routes, and its current spread. The results and conclusions presented provide new and substantial insights for virologists, epidemiologists, and the general public health sector. In this regard, they should be useful to those authorities responsible for developing public health intervention strategies to combat the further spread of HIV/AIDS.

National molecular surveillance of recently acquired HIV infections in Germany, 2013 to 2014.

To enable an up-to-date molecular analysis of human immunodeficiency virus (HIV) genotypes circulating in Germany we have established a surveillance system based on recently acquired HIV infections. New HIV infections are reported to the Robert Koch Institute as a statutory duty for anonymous notification. In 2013 and 2014, a dried serum spot (DSS) sample was received from 6,371 newly diagnosed HIV-cases; their analysis suggested that 1,797 samples originated from a recent infection. Of these, 809 were successfully genotyped in the pol region to identify transmitted drug resistance (TDR) mutations and to determine the HIV-1 subtype. Total TDR was 10.8%, comprising 4.3% with mono-resistance to nucleoside reverse transcriptase inhibitors (NRTIs), 2.6% to non-NRTIs, 3.0% to protease inhibitors and 0.6% and 0.2%, respectively, with dual- and triple-class resistances. HIV-1 subtype B was most prevalent with 77.0%. Non-B infections were identified more often in men and women with heterosexual transmission compared with intravenous drug users or men who have sex with men (79% and 76%, 33%, 12%; all p < 0.05). Non-B subtypes were also more frequently found in patients originating from countries other than Germany (46% vs 14%; p < 0.05) and in patients infected outside of Germany (63% vs 14%; p < 0.05).

Staufen-1 interacts with the human endogenous retrovirus family HERV-K(HML-2) rec and gag proteins and increases virion production.

The human endogenous retrovirus family HERV-K(HML-2) Rec protein is an RNA transport factor that enhances nuclear export of intron-containing retroviral transcripts. Using the yeast two-hybrid approach, we have newly identified human Staufen-1 as a Rec-interacting protein. The interaction was confirmed by coimmunoprecipitation experiments, and the relevant site in Staufen-1 has been mapped to double-stranded RNA binding domain 4 (RBD4). Staufen-1 is in several aspects functionally related to retroviral RNA transport proteins. It binds mRNAs and targets its ribonuclear cargo to polysomes for efficient translation. We observed an accumulation of Staufen-1 in the nucleus of Rec-expressing cells and colocalization in the nucleoli as well as in the cytoplasm. Overexpression of Staufen-1 resulted in a 5-fold enhancement in nuclear export and/or translation of unspliced HERV-K(HML-2) viral RNAs in the presence of Rec and its Rec-responsive element (RcRE) binding site together with a clear increase in virus production. Staufen-1 was previously shown to interact with the Gag protein of HIV-1, promoting Gag oligomerization and RNA encapsidation. We demonstrate here that Staufen-1 also binds to the Gag protein of HERV-K(HML-2). Under stress conditions, Rec colocalizes with Staufen-1 in stress granules in cells that express viral RNA but not in mRNA-decay-related processing bodies. Our results suggest a new role for Staufen-1 as a cellular Rec and HERV-K(HML-2) Gag cofactor.

The Rec protein of HERV-K(HML-2) upregulates androgen receptor activity by binding to the human small glutamine-rich tetratricopeptide repeat protein (hSGT).

The expression of endogenous retroviruses of the HERV-K(HML-2) family is strongly upregulated in germ cell tumors and several other cancers. Although the accessory Rec protein of HERV-K(HML-2) has been shown to induce carcinoma in situ in transgenic mice, to increase the activity of c-myc and to interact with the androgen receptor (AR), whether or not Rec expression is indeed implicated causally in the initiation or progression of any human malignancies remains unclear. We used the yeast two-hybrid system involving the Rec protein of a recently integrated HERV-K(HML-2) element in an effort to identify potential Rec-related oncogenic mechanisms. This revealed the human small glutamine-rich tetratricopeptide repeat (TPR)-containing protein (hSGT) to be a cellular binding partner. The interaction of Rec with this known negative regulator of the AR was confirmed by coimmunoprecipitation, pull-down assays and colocalization studies. The interaction involves the TPR motif of hSGT and takes place in the cytoplasm and in the nucleoli. Using an AR-responsive promoter and gene we could demonstrate that Rec interference with hSGT resulted in an up to five-fold increase in the activity of AR. Furthermore, in AR positive cells, Rec was shown to act as transactivator by enhancing AR-mediated activation of the HERV-K(HML-2) LTR promoter. This is in line with previous observations of elevated HERV-K(HML-2) expression in steroid-regulated tissues. On the basis of our findings we propose a "vicious cycle" model of Rec-driven hyperactivation of the AR leading to increased cell proliferation, inhibition of apoptosis and eventually to tumor induction or promotion.

Identification of late assembly domains of the human endogenous retrovirus-K(HML-2).

BACKGROUND: Late assembly (L)-domains are protein interaction motifs, whose dysfunction causes characteristic budding defects in enveloped viruses. Three different amino acid motifs, namely PT/SAP, PPXY and YPX(n)L have been shown to play a major role in the release of exogenous retroviruses. Although the L-domains of exogenous retroviruses have been studied comprehensively, little is known about these motifs in endogenous human retroviruses. RESULTS: Using a molecular clone of the human endogenous retrovirus K113 that had been engineered to reverse the presumed non-synonymous postinsertional mutations in the major genes, we identified three functional L-domains of the virus, all located in the Gag p15 protein. A consensus PTAP tetrapeptide serves as the core of a main L-domain for the virus and its inactivation reduces virus release in HEK 293T cells by over 80%. Electron microscopy of cells expressing the PTAP mutant revealed predominantly late budding structures and budding chains at the plasma membrane. The fact that this motif determines subcellular colocalization with Tsg101, an ESCRT-I complex protein known to bind to the core tetrapeptide, supports its role as an L-domain. Moreover, two YPX(n)L motifs providing additional L-domain function were identified in the p15 protein. One is adjacent to the PTAP sequence and the other is in the p15 N-terminus. Mutations in either motif diminishes virus release and induces an L-domain phenotype while inactivation of all three L-domains results in a complete loss of particle release in HEK 293T cells. The flexibility of the virus in the use of L-domains for gaining access to the ESCRT machinery is demonstrated by overexpression of Tsg101 which rescues the release of the YPX(n)L mutants. Similarly, overexpression of Alix not only enhances release of the PTAP mutant by a factor of four but also the release of a triple mutant, indicating that additional cryptic YPX(n)L domains with a low affinity for Alix may be present. No L-domain activity is provided by the proline-rich peptides at the Gag C-terminus. CONCLUSIONS: Our data demonstrate that HERV-K(HML-2) release is predominantly mediated through a consensus PTAP motif and two auxiliary YPX(n)L motifs in the p15 protein of the Gag precursor.

The influence of SaeRS and σ(B) on the expression of superantigens in different Staphylococcus aureus isolates.

Staphylococcus aureus is a major human pathogen. Superantigens (SAg) are important virulence factors in S. aureus, but the regulation of SAg gene expression is largely unknown. Using 2 sequenced S. aureus strains (COL and Newman) and 4 clinical isolates, regulation of gene expression was investigated in more detail for 12 SAgs. The SAg-encoding genes were expressed in a growth phase-dependent manner: while the egc operon was mainly transcribed at low optical densities, the transcription of seb was induced at high optical densities. The transcript levels of sea, sek, seq, sep, and tst-1 did not change significantly during growth. The T cell-mitogenic activity of supernatants correlated with the transcription data. SaeRS and σ(B) strongly influenced SAg gene transcription. σ(B) activated transcription of seh, tst-1, and of the egc operon. A possible σ(B)-dependent promoter was identified in front of the egc operon. In contrast, a loss of σ(B) enhanced the transcript level of seb, suggesting an indirect effect of the alternative sigma factor on the transcription of this gene. Transcriptional studies of an saeS mutant showed that the two-component system only activates transcription of seb. The influence of σ(B) and SaeRS on the expression of SAg genes was validated by T cell proliferation assays. For sigB mutants in different strains, different effects on the T cell-mitogenic potential were observed depending on the SAg gene repertoire of the isolates.

Reconstitution of the ancestral glycoprotein of human endogenous retrovirus k and modulation of its functional activity by truncation of the cytoplasmic domain.

Endogenous retroviruses present in the human genome provide a rich record of ancient infections. All presently recognized elements, including the youngest and most intact proviruses of the human endogenous retrovirus K(HML-2) [HERV-K(HML-2)] family, have suffered postinsertional mutations during their time of chromosomal residence, and genes encoding the envelope glycoprotein (Env) have not been spared these mutations. In this study, we have, for the first time, reconstituted an authentic Env of a HERV-K(HML-2) provirus by back mutation of putative postinsertional amino acid changes of the protein encoded by HERV-K113. Aided by codon-optimized expression, we demonstrate that the reconstituted Env regained its ability to be incorporated into retroviral particles and to mediate entry. The original ancient HERV-K113 Env was synthesized as a moderately glycosylated gp95 precursor protein cleaved into surface and transmembrane (TM) subunits. Of the nine N-linked oligosaccharides, four are part of the TM subunit, contributing 15 kDa to its apparent molecular mass of 41 kDa. The carbohydrates, as well as the cytoplasmic tail, are critical for efficient intracellular trafficking, processing, stability, and particle incorporation. Whereas deletions of the carboxy-terminal 6 residues completely abrogated cleavage and virion association, more extensive truncations slightly enhanced incorporation but dramatically increased the ability to mediate entry of pseudotyped lentiviruses. Although the first HERV-K(HML-2) elements infected human ancestors about 30 million years ago, our findings indicate that their glycoproteins are in most respects remarkably similar to those of classical contemporary retroviruses and can still mediate efficient entry into mammalian cells.

Fibrils of prostatic acid phosphatase fragments boost infections with XMRV (xenotropic murine leukemia virus-related virus), a human retrovirus associated with prostate cancer.

The xenotropic murine leukemia virus-related virus (XMRV) has recently been detected in prostate cancer tissues and may play a role in tumorigenesis. It is currently unclear how this virus is transmitted and which factors promote its spread in the prostate. We show that amyloidogenic fragments known as semen-derived enhancer of virus infection (SEVI) originating from prostatic acid phosphatase greatly increase XMRV infections of primary prostatic epithelial and stromal cells. Hybrid simian/human immunodeficiency chimeric virus particles pseudotyped with XMRV envelope protein were used to demonstrate that the enhancing effect of SEVI, or of human semen itself, was at the level of viral attachment and entry. SEVI enhanced XMRV infectivity but did not bypass the requirement for the xenotropic and polytropic retrovirus receptor 1. Furthermore, XMRV RNA was detected in prostatic secretions of some men with prostate cancer. The fact that the precursor of SEVI is produced in abundance by the prostate indicates that XMRV replication occurs in an environment that provides a natural enhancer of viral infection, and this may play a role in the spread of this virus in the human population.

A Recent Human Immunodeficiency Virus Outbreak Among People Who Inject Drugs in Munich, Germany, Is Associated With Consumption of Synthetic Cathinones.

BACKGROUND: Needle and syringe sharing among people who inject drugs (PWID) can result in a rapid regional spread of a human immunodeficiency virus (HIV) variant. Such outbreaks have been identified recently in several countries and have raised public health attention because of an association with new psychoactive substances (NPS). METHODS: Dried serum spots from approximately 60% of newly diagnosed HIV cases in Germany in 2013-2018 were received together with statutory notification data. Samples were sequenced in the pol-region, genotyped, and viral phylogenies were analyzed. For selected samples, the hepatitis C virus (HCV) status and the presence of NPS were determined. RESULTS: An outbreak of closely related 27 subtype C infections with a core of 11 cases with almost identical sequences was identified using phylogenetic analyses. The first case of the outbreak was diagnosed in 2015, and the last one was in 2018. With exception of 3 infections, all were reported from Munich, the capital of the federal state of Bavaria. Of 26 analyzed outbreak members, 24 (92.3%) had a resolved or viremic HCV coinfection. In 8 of 18 (44%) cases, α-pyrrolidinopentiothiophenone and/or the related substance α-pyrrolidinoheptiophenone was identified. CONCLUSIONS: Despite harm reduction services in place, HIV outbreaks of considerable size can occur in PWID. The establishment of a real-time molecular surveillance is advised to rapidly identify outbreaks and target prevention measures.

Evaluation of a BioRad Avidity assay for identification of recent HIV-1 infections using dried serum or plasma spots.

Serological methods to differentiate between recently acquired and established HIV-1 infections are a useful tool in the HIV-surveillance to characterize the epidemic, identify groups at risk and assess HIV-preventive interventions. Therefore, an avidity-based, modified BioRad Genscreen™ HIV-1/2 assay (BRAEUR) was evaluated according to the avidity-based, modified BioRad HIV-1/2 Plus O protocol (BRAUSA). Overall, 692 well defined samples (82.5% B and 17.5% non-B subtypes) from recent (<180 days, n = 239), intermediate (181-364 days, n = 35) or long term infections (≥365 days, n = 419) were used to determine a 'mean duration of recent infection' (MDRI), a 'median DRI' (MdDRI), the false recent rate (FRR), and concordance between the BRAs and the Sedia BED HIV-1 Capture enzyme immunoassay (BED). The optimal avidity index cut-off was determined to be 70% resulting in an MDRI of 233 days (95% IQR: 174-351) and an MdDRI of 171 days (95% IQR: 142-212). Concordance with the BRAUSA was high with 96.4%. The FRR of 6.0% as well as the MdDRI are similar to the BED (8.4%; 170 (139-214) days). Therefore, the BRAEUR is a suitable alternative to replace the BED and trend analysis will be feasible after minimal adjustments for the MdDRI and the MDRI.

Increasing proportions of HIV-1 non-B subtypes and of NNRTI resistance between 2013 and 2016 in Germany: Results from the national molecular surveillance of new HIV-diagnoses.

BACKGROUND: Molecular surveillance of newly diagnosed HIV-infections is important for tracking trends in circulating HIV-variants, including those with transmitted drug resistances (TDR) to sustain ART efficacy. METHODS: Dried serum spots (DSS) are received together with the statutory notification of a new diagnosis. 'Recent infections' (<155 days) classified by a 'recent infection test algorithm' (BED-CEIA and clinical data) are genotyped in HIV-protease (PR), reverse transcriptase (RT) and integrase (INT) to determine the HIV-1 subtype, to calculate prevalence and trends of TDR, to predict baseline susceptibility and to identify potential transmission clusters for resistant variants. RESULTS: Between January 2013 and December 2016, 1,885 recent infections were analysed regarding the PR/RT genomic region, with 43.5% of these also being subjected to the analysis of INT. The proportion of HIV-1 non-B viruses (31.3%; 591/1,885) increased from 21.6% to 36.0%, particularly the subtypes A (5.0% to 8.3%) and C (3.2% to 7.7%; all ptrends < 0.01). The subtype A increment is mainly due to transmissions within men who have sex with men (MSM) while subtype C transmissions are associated with heterosexuals and people who inject drugs. The prevalence of TDR was stable at 11.0% (208/1,885) over the study period. Resistances to nucleotide RT inhibitors (NRTI) and PR inhibitors (PI) were 4.5% and 3.2%, respectively, without identifiable trends. In contrast, resistances to non-NRTIs (NNRTI, 4.7%) doubled between 2014 and 2016 from 3.2% to 6.4% (ptrend = 0.02) mainly due to the K103N mutation (from 1.7% to 4.1%; ptrend = 0.03) predominantly detected in recently infected German MSM not linked to transmission clusters. Transmitted INSTI mutations were present in only one case (T66I) and resistance to dolutegravir was not identified at all. Reduced susceptibility to recommended first-line therapies was low with 1.0% for PIs, 1.3% for NRTIs and 0.7% for INSTIs, but high for the NNRTIs efavirence (4.9%) and rilpivirine (6.0%) due to the K103N mutation and the polymorphic mutation E138A. These trends in therapy-naïve individuals impact current first-line regimens and require awareness and vigilant surveillance.

Robust and sensitive subtype-generic HIV-1 pol genotyping for use with dried serum spots in epidemiological studies.

HIV-1 genotyping of larger pol-fragments from dried serum/plasma spots (DSS/DPS) is often hindered by RNA-degradation during transportation at ambient temperature. We evaluated an in-house HIV-1 DSS/DPS-genotyping assay designed in two shorter overlapping fragments covering all resistance mutations in protease and reverse transcriptase. Validation criteria such as specificity, detection limit, accuracy, reproducibility and storage conditions were assessed using reference plasma samples prepared as DPS and clinical DSS from the German molecular HIV-1 surveillance processed under real-life transportation conditions. The specificity was 100% for both samples types, and the experimental DPS detection limit of 1000 copies/ml yielded a 98.7% (3,329/3373) success rate for DSS (including all subtypes) above this detection limit. Accuracy for DPS compared to the gold standard was 99.1% and the reproducibility was 100% for DPS replicates and 99.9% for DSS pairs. Storage of DPS at room temperature was possible for 90 or 30 days and at -20 °C for at least 180 or 90 days at viral loads of 10,000 or 1000 copies/ml, respectively. The HIV-1 pol-genotyping assay presented here is a sensitive, robust and subtype generic tool for a large-scale population-based HIV-1 drug resistance surveillance for the use of DSS/DPS.

HERV-K(HML-2), a seemingly silent subtenant - but still waters run deep.

A large proportion of the human genome consists of endogenous retroviruses, some of which are well preserved, showing transcriptional activity, and expressing retroviral proteins. The HERV-K(HML-2) family represents the most intact members of these elements, with some having open and intact reading frames for viral proteins and the ability to form virus-like particles. Although generally suppressed in most healthy tissues by a variety of epigenetic processes and antiviral mechanisms, there is evidence that some members of this family are (at least partly) still active - particularly in certain stem cells and various tumors. This raises the possibility of their involvement in tumor induction or in developmental processes. In recent years, many new insights into this fascinating field have been attained, and this review focuses on new discoveries about coevolutionary events and intracellular defense mechanisms against HERV-K(HML-2) activity. We also describe what might occur when these mechanisms fail or become modulated by viral proteins or other viruses and discuss the new vistas opened up by the reconstitution of ancestral viral proteins and even complete HML-2 viruses.

The human endogenous retrovirus K(HML-2) has a broad envelope-mediated cellular tropism and is prone to inhibition at a post-entry, pre-integration step.

The HERV-K(HML-2) family is the most recent addition to the collection of human endogenous retroviruses. It comprises proviruses that encode functional proteins that can assemble into replication defective particles carrying the envelope protein. Using a reconstituted HERV-K113 envelope sequence, we have analyzed its ability to mediate entry into a set of 33 cell lines from 10 species. Of these, 30 were permissive, demonstrating an amphotropism consistent with a broad expression of receptor protein(s). In an initial effort to identify a receptor for HERV-K(HML-2) we investigated whether transferrin receptor 1 and hyaluronidase 2, known cellular receptors of the closely related betaretroviruses mouse mammary tumor virus (MMTV) and Jaagsiekte sheep retrovirus (JSRV), could facilitate HERV-K(HML-2) entry. However, neither of these proteins could serve as a receptor for HERV-K(HML-2). Moreover, during attempts to further characterize the tropism of HERV-K(HML-2), we identified a cellular activity that inhibits infection at a post-entry, pre-integration step.

CMV-promoter driven codon-optimized expression alters the assembly type and morphology of a reconstituted HERV-K(HML-2).

The HERV-K(HML-2) family contains the most recently integrated and best preserved endogenized proviral sequences in the human genome. All known elements have nevertheless been subjected to mutations or deletions that render expressed particles non-infectious. Moreover, these post-insertional mutations hamper the analysis of the general biological properties of this ancient virus family. The expression of consensus sequences and sequences of elements with reverted post-insertional mutations has therefore been very instrumental in overcoming this limitation. We investigated the particle morphology of a recently reconstituted HERV-K113 element termed oriHERV-K113 using thin-section electron microscopy (EM) and could demonstrate that strong overexpression by substitution of the 5'LTR for a CMV promoter and partial codon optimization altered the virus assembly type and morphology. This included a conversion from the regular C-type to an A-type morphology with a mass of cytoplasmic immature cores tethered to the cell membrane and the membranes of vesicles. Overexpression permitted the release and maturation of virions but reduced the envelope content. A weaker boost of virus expression by Staufen-1 was not sufficient to induce these morphological alterations.

HERV-K(HML-2), the Best Preserved Family of HERVs: Endogenization, Expression, and Implications in Health and Disease.

Retroviruses that have the ability to infect germ line cells can become an integral and inherited part of the host genome. About 8% of the human chromosomal DNA consists of sequences derived from infections by retroviruses that presumably circulated 2-40 millions of years ago, and some elements are actually much older. Post-insertional recombinations, deletions, and mutations have rendered all known human endogenous retroviruses (HERVs) non-infectious. However some, particularly the most recently acquired proviruses of the HERV-K(HML-2) family, can expresses viral proteins and produce viral particles. In this review we will first discuss the major aspects of the endogenization process and peculiarities of the different HERV-K families. We will then focus on the genes and proteins encoded by HERV-K(HML-2) as well as inactivation of these proviruses by postinsertional mutations and their inhibition by antiretroviral factors. After describing the evolutionary interplay between host and endogenous retrovirus we will delve deeper into the currently limited understanding of HERV-K and its possible association with disease, particularly tumorigenesis.